Pharmac. Ther. Vol. 51, pp. 71-95, 1991
0163-7258/91 $0.00+ 0.50 © 1991 PergamonPress pie
Printed in Great Britain.All rights reserved
Specialist Subject Editor: C. W. TAYLOR
PROTEIN KINASE C SILVIA STABEL* a n d PETER J. PARKER," * Max-Delbriick-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-Linn~- Weg 10, D- 5000 K61n 30, F.R.G. tlmperial Cancer Research Fund, Protein Phosphorylation Laboratory, Lincoln's Inn Fields, London WC2A 3PX, U.K.
A~tract--Based on the molecular structure of the individual members of the protein kinase C family, general properties and the mode of activation of this enzyme family are discussed. Examples are presented of how the investigation of protein kinase C function in vivo has been approached at the molecular level.
CONTENTS 1. Introduction 2. Structural Aspects of PKC Molecules 2.1. Organization of the PKC polypeptides 2.1.1. The pseudosubstrate prototope 2.1.2. The zinc fingers within the C, domain 2.1.3. The C2 domain and the kinase domain 2.2. Homologies to other proteins 2.3. PKC in lower eukaryotes 3. Functional Aspects of PKC In Vitro 3.1. General properties 3.2. Ca2+-dependencies 3.3. Diacylglycerol and phorbol ester responsiveness 3.4. Substrate specificities 4. Function and Regulation of PKC In Vivo 4.1. Sources of diacylglycerol 4.2. Activation, translocation and down-regulation of PKC 4.3. Analysis of the PKC signalling pathway Note Added in Proof Acknowledgements References
71 73 74 75 76 78 78 80 81 81 82 83 84 84 84 85 86 89 89 89
1977; Takai et aL, 1977). Soon after, however, it was realized that apart from proteolysis the enzyme could also be reversibly activated in vitro by unsaturated diacylglycerol (DAG) in the presence of acidic phospholipids such as phosphatidylserine (Takai et aL, 1979a,b). This observation provided the first possible link between receptor-induced phosphatidylinositol breakdown and protein phosphorylation (Kishimoto et al., 1980; Nishizuka, 1984). The activating effects of DAG on the enzyme can be mimicked by tumor-promoting phorbol esters in vitro (Castagna et al., 1982) suggesting that PKC plays some role in the process of neoplastic promotion of initiated tumor cells by phorbol esters (Asbendel, 1985; Kikkawa et al., 1983; Nishizuka, 1989). The binding of phorbol esters to the enzyme and the activation of the kinase activity of PKC by these compounds has been employed to purify PKC polypeptides from a number of sources (Ashendel et al., 1983; Kikkawa et al., 1982; Leach et al., 1983; Niedel et al., 1983; Parker et al., 1984; Wise et al.,
1. I N T R O D U C T I O N Today protein kinase C (PKC) covers a family of serine- and threonine-specific protein kinases which have been identified functionally by common enzymatic properties, like phorbol ester binding, phospholipid-dependent kinase activity or by common structural features (reviewed by Nishizuka, 1988; Parker et al., 1989; Stabel, 1990). In a growing number of cases cloned cDNAs are added to the PKC family according to the structural homologies they share with the prototypes of this kinase family, although the biochemical properties of the encoded protein products are not yet known. This is particularly true for PKC-type molecules isolated from lower eukaryotes as will be discussed below. The enzymatic activity of PKC was initially identified in rat brain by Nishizuka and coworkers as a novel type of cytoplasmic serine- and threoninespecific protein kinase which appeared to be proteolytically activated from a proenzyme (Inoue et al., 71
72
S. STABELand P. J. PARKER
1982). Based on peptide sequences from homogeneous purified protein, c D N A clones encoding P K C were isolated (Parker et al., 1986). During this process it became apparent that P K C is not specified by a single m R N A but that there exist multiple structurally highly related m R N A s in brain potentially coding for a family of enzymes with possibly identical or similar properties (Coussens et al., 1986; Housey et al., 1987; K n o p f et al., 1986; Makowske et al., 1986; O h n o et al., 1987; Ono et al., 1986b). Artificial expression of these first c D N A s in mammalian or insect expression systems proved that the c D N A s indeed all encoded proteins that were characterized by PKC-specific properties namely phorbol ester binding and calciumand phospholipid-dependent kinase activity which was activated by D A G or phorbol ester (Burns et al.,
1990; Kikkawa et al., 1987; K n o p f et al., 1986; Ono et al., 1987b; Patel and Stabel, 1989). By means of chromatography on hydroxyapatite and by means of antipeptide antibodies, polypeptides corresponding to the first isolated members of the P K C c D N A family have also been identified in vivo (Huang et al., 1986b, 1987; Jaken and Kiley, 1987; Kikkawa et al., 1987; Marais and Parker, 1989; Ono et al., 1987b; Sekiguchi et aL, 1988) proving that there exists a family of highly related P K C proteins which may all participate in the phenomena previously ascribed to a single P K C enzyme. In this review we will focus on the molecular and structural aspects of P K C and we will try to relate the molecular analysis of P K C to its proposed function and regulation in vivo.
TABLE 1. Cloned Mammalian Protein Kinase C Types Type*
Residues
MW:~
Source
Nomenclature~
Reference
alpha (~t)
672
76.8
bovine human rabbit rat mouse mouse human
ct ct ~ ~ ~ ct ~
Parker et al. (1986) Coussens et al. (1986) Ohno et al. (1987) Ono et al. (1988b) Rose-John et al. (1988) Megidish and Mazurek (1989) Finkenzeller et al. (1990)
betaI (ill)
671
76.8
rat rat human rabbit rat human
III I f12 fl RP41 fl
Knopf et al. (1986) Ono et al. (1986a) Coussens et al. (1987) Ohno et al. (1987) Housey et al. (1987) Kubo et al. (1987a,b)
betalI (fill)
673
76.9
bovine rat rat human rabbit human mouse
fl II II fl~ ~ fl fl
Coussens et al. (1986) Knopf et al. (1986) Ono et al. (1986a) Coussens et al. (1987) Ohno et al. (1987) Kubo et al. (1987a,b) Tang and Ashendel (1990)
gamma (~,)
697
78.4
bovine human rat rabbit rat
7 7 I 6 7
Coussens et al. (1986) Coussens et al. (1986) Knopf et al. (1986) Ohno et al. (1988b) Ono et al. (1988b)
delta O)
673
77.5
rat
6
Ono et al. (1987a, 1988a)
epsilon (E)
737
83.5
rat rabbit rat mouse
E n RP16 E
Ono et al. (1987a, 1988a) Ohno et al. (1988a) Housey et al. (1987) Schaap et al. (1989)
epsilon' (E')
497
56.1
rat
E'
Ono et al. (1988a)
zeta (()
592
67.7
rat
(
Ono et al. (1987a, 1988a, 1989a)
eta (q)
683 6801b
80 77.6
mouse human
q L
Osada et al. (1990) Bacher et al. (1991)
*Grouping into types according to homology of predicted amino acid sequence. tNumber of amino acid residues of the predicted proteins. :~Calculated molecular weight of the predicted protein. §Nomenclature used in the respective reference. liThe formal possibility that this is an alternative splice product of the same gene is discussed in the text.
Protein kinase C 2. S T R U C T U R A L ASPECTS OF PKC MOLECULES The PKC c D N A clones isolated first and the corresponding polypeptides are now called ~, fl~, f12 and y types, isoforms, isoenzymes or subspecies of PKC. Table 1 lists the origin of the mammalian PKC cDNAs isolated to date and their assignment to the different subspecies of PKC. Due to their structural organization PKC ~, ill, f12 and y define the 'group A' PKC enzymes in contrast to 'group B' enzymes 6, E, (, and ~/ which are distinguished from the former ones on the basis of their structure and enzymatic properties (see below). The genes for ~, fl and ~, have been localized on different human chromosomes: ~ is encoded on chromosome 17, fl on 16 and y on chromosome 19 (Coussens et al., 1986). The fll and f12 mRNAs are generated by alternative splicing of the most C-terminal exon of the fl-gene producing two polypeptides which only differ in the sequence of their 50 most C-terminal amino acids (Coussens et al., 1987; Kubo et al., 1987a; Ono et al., 1986a). Differential expression of both fl-mRNAs, fll and /~2, has been shown in hematopoietic cell lines and in brain tissue (Coussens et al., 1987) and use of specific antibodies has shown that both fl polypeptides are indeed expressed in brain tissue in vivo (Hosoda et al., 1989; Saito et al., 1989) thus raising the tantalizing question of whether there will be specific cellular processes to be discovered which are catalyzed by two proteins which only differ in their 50 C-terminal amino acids or whether the two PKCfl polypeptides are the result of some evolutionary redundancy. An analogous situation may turn out to apply for the E type of PKC where two different cDNA clones have been isolated which differ in their 5'-sequence potentially giving rise to two polypeptides, one of 737 amino acids and a predicted molecular weight of 83.474 kDa, termed e, and one of 497 amino acids and a theoretical molecular weight of 56.059 kDa, which for consistency we will term E2 (Housey et al., 1987; Ono et al., 1988a). PKC E2 differs from E by the absence of the 240 N-terminal amino acids of E and substitution of this sequence by a sequence carrying a stop codon in front of methionine 241 of PKC E. Translation of this m R N A would give rise to an N-terminally truncated PKC E2 product, starting at methionine 241 of E (Ono et al., 1988a). Preliminary evidence suggests that these two potential protein products of the E-gene may be generated by alternative splicing (Ono et al., 1988a). If indeed expressed as a protein product in vivo, E2would only contain the second zinc finger of the full-length E protein product (cf. Fig. 1A). Another potential case for alternative splicing within the PKC family might be represented by the subspecies. The ( c D N A encodes a polypeptide of 592 amino acids and a predicted molecular weight of 67.740 kDa. Another c D N A clone lacking the first 112 amino acids of the body of the ( polypeptide was repeatedly isolated but the biological significance of these m R N A s is unclear (Ono et al., 1988a; Liyanage and Stabel, submitted). As far as the genomic organization of the PKC genes is concerned the exon/intron structure of a
73
PKC gene has only been elucidated for one of the three so far identified PKC species in Drosophila, dPKC (Rosenthal et al., 1987b). The dPKC gene spans about 20 kb and consists of 13 coding exons and at least one untranslated exon at its 5'-end. It has been noted that the functional domains of the dPKC polypeptide are not reflected in the exon/intron structure of the gene as the second zinc finger structure and the ATP binding domain are disrupted by introns (Rosenthal et al., 1987b). Partial genomic mapping of the human fl gene shows identical intron/exon organization to that of dPKC (Coussens et al., 1987; Haley, King and Parker, in preparation). The regulation of tissue-specific expression of the PKC enzymes presents another puzzling problem. The PKC~ enzyme for instance is exclusively expressed in brain and spinal cord (Nishizuka, 1988) whereas most of the other types occur in brain as well as in other tissues. This regulation obviously occurs at the transcriptional level and so far nothing is known about the factors governing expression of these enzymes. The promoter region of the rat and the mouse PKC y gene has been isolated (Chen et al., 1990; Leitges, Proikas and Stabel, manuscript in preparation) and it shows features characteristic of many housekeeping genes like the absence of TATA or CAAT boxes from their usual positions, but in contrast to a number of such genes it seems to have only one major transcriptional start site located 243 bp upstream of the ATG. Although consensus sequences for several trans-activating factors like AP2, Spl and CREB have been identified in the 5'-flanking region of the PKC Y gene, the mechanism by which brain-specific expression of this isozyme is achieved is still unclear. Genomic sequence data so far available, suggest that the 3'-boundary of the first exon of this gene and the 5'and 3'-boundary of the second exon are identical between Drosophila and the rat/mouse PKC y gene (Chen et al., 1990; Leitges, Proikas and Stabel, manuscript in preparation). Soon after the discovery of the three closely related cDNAs coding for PKC ct, fl, and y, additional members of the PKC family, PKC 6, e, and ~, were identified by low stringency screening of brain cDNA libraries (Ohno et al., 1988a; Ono et al., 1987a, 1988a; Schaap et al., 1989). The chromosomal location of the respective genes has not been determined yet, although it is evident from their sequences that they too are products of distinct genes. Like the ct, fl, group of PKC enzymes the members 6, E, ( are highly expressed in brain and in additional tissues (Nishizuka, 1988). The preferential isolation of PKC members highly expressed in brain has been due to the fact that most of the approaches chose brain cDNA libraries as a source for screening. Screening of two epidermis cDNA libraries under reduced stringency conditions with ct, fl, y, E, and ( probes identified yet another PKC isoenzyme, PKCr/ or PKC-L, with a tissue distribution distinct from the thus far identified enzymes. The m R N A for this PKC is strongly expressed in skin and lung and only weakly in brain (Bacher et al., 1991; Osada et al., 1990). These findings indicate the possibility that the family of PKC polypeptides might still increase in
74
S. STABELand P. J. PARKER
A
REGULATORY
CATALYTIC
!Pseudo6ubetrate alpha beta 1/11 gamma
672 671/673 897 m
_ _
apsllon
m
.
zeta
..... J
delta
m
.
.
.
.
673
.~1
737
~
592
eta
epsilon" ATP binding site REGULATORY
B
CATALYTIC |¢
rat
I alpha beta VII gamma
VI
Cl
V2
C2
V3
i
C3 v
C4
V5
672 671/673 697 676
I X-PKC I Xenopus L X-PKC II
671
I 53E(ey)
700
°-[7
639
C.ele0art~
tpa-1 *
526"
yeast
PKC1
634
1151
¢ ATP binding site
FIG. 1. Domain structure of PKC molecules from mammals (A) and lower eukaryotes (B). Conserved (c) and variable (v) domains are indicated by C1-4 and V1-5, respectively. Patterned boxes within the C1 domain represent location of the conserved zinc finger motif CX2CXI3/14CX2CXTCX7C.Numbers on the right give amino acids of the polypeptide predicted from the cDNA sequences. See text for details and Tables 1 and 2 for references. Epsilon' is a truncated version of the , cDNA which may represent an alternatively spliced product of the E gene (Ono et al., 1988a). number through the identification of members which are preferentially or exclusively expressed in other tissues than the ones used for screening so far. The obvious question arising from these observations is whether each of these different distinct enzymes plays a specific role in cell type-specific processes like endocytosis, secretion, transmission of electric potentials, growth or differentiation. It is also not clear why some types like ct or ~/ are apparently expressed ubiquitously albeit at low levels, with very high expression only in certain cell types. It is conceivable that some types, e.g. the ubiquitously expressed type ~, may serve some very general functions whereas other types fulfil more specialized cell type-specific tasks.
2.1. ORGANIZATIONOF THE P K C POLYPEPTIDES
Comparison of the amino acid sequences of PKCs predicted from the cDNAs highlighted the presence of regions with a high degree of homology between PKC types; the conserved regions C1 to C4, interspersed with regions of lower homology variable regions VI to V5 (Coussens et al., 1986). Figure 1A shows a schematic representation of the structural organization of the putative mammalian PKC polypeptides for which cDNA sequence data is available. It has been speculated that the conserved regions represent areas of the molecule involved in functions common to all PKC enzymes which is obviously true
Protein kinase C for the kinase domain. The less well conserved, variable blocks of sequences inbetween the conserved regions may either represent spacer sequences that allow for flexibility of the polypeptides or domains responsible for specific properties of the enzymes (Carpenter et al., 1987). To date there is no evidence to support the idea that the V-regions are important for the biochemical characteristics of a particular enzyme. Figure 1B compares the structural features of the mammalian enzymes to those of putative PKC molecules identified in lower eukaryotes which are discussed in more detail below. The N-terminal (approximately) half of the polypeptide represents the regulatory domain since it carries binding sites for the activator phorbol esters (Huang et al., 1986b; Kaibuchi et al., 1989; Lee and Bell, 1986; Ono et al., 1989b) and a pseudosubstrate prototope. The removal of this domain by mild proteolysis leads to constitutive and activator-independent kinase activity of the enzyme (Inoue et al., 1977; Mochly-Rosen et al., 1990; Parker et al., 1986). The kinase domain is located in the C-terminal part of the polypeptide. All members of the PKC family identified to date carry at least one zinc-finger structure (in most cases two) in the N-terminal regulatory half of the molecule (see Fig. 1A). Upstream of the finger structures, at a fixed distance from the first finger lies a block of amino acids exhibiting the features of a pseudosubstrate prototope supposed to be essential for regulating the kinase activity (see below). The C1 domain is preceded by an N-terminal extension of varying length with the types E and ~/ having the longest extensions among the mammalian PKCs identified so far. The most recently identified type r/is most closely related to PKC E as reflected in homologies between the conserved domains: homology in the C1 region is 48% with ~t, 48.6% with fl, 48% with ~,, 54.2% with 6, 34.9% with ( and 74.8% with E. Also the kinase domain is most homologous to PKC E with 74.4% identity, whereas homology to the other types in this domain is around 60% (Osada et aL, 1990). Remarkably, the degree of homology in the N-terminal variable extensions of PKC r/and PKC E is also rather high at 53.3% (Osada et al., 1990). A cDNA encoding PKC-L would appear to be the human homolog of PKC r/ (Bacher et al., 1991). However in PKC-L the second cysteine repeat is interrupted and the sequences of PKC-L and r/ are divergent between residues 278 and 310. Interestingly the divergence starts at the junction of exons 4 and 5 in the dPKC gene. Whether this difference in fact reflects alternative splicing or a frameshift incurred during c D N A isolation has yet to be determined. In the following sections the functionally relevant structures and regions of the PKC molecules will be discussed in more detail. 2.1.1. The Pseudosubstrate Prototope The activity of a number of protein kinases has been recognized to be regulated or controlled by the presence of so-called pseudosubstrate prototopes which occur as autoinhibitory domains or sites in the kinase molecule or associated regulatory subunits
75
(reviewed in Hardie, 1988; Kemp et al., 1989; Kennelly et al., 1987; Soderling, 1990). The pseudosubstrate prototope contains a consensus phosphorylation sequence for the respective kinase but no phosphorylatable residue and is thought to control kinase activity by blocking access of the substrate through occupation of the catalytic site. The sequence of a number of in vitro substrates of PKC has been determined and the use of synthetic peptide substrates has indicated that PKC requires basic residues surrounding the target serine or threonine (House et al., 1987; Marais et al., 1990), a feature common to a number of serine-/threonine-specific protein kinases. A sequence fulfilling the requirements for a PKC phosphorylation site without a phosphorylatable residue (alanine instead of serine) was first identified in the 'group A' PKCs (House et al., 1987). Inclusion of a synthetic peptide of this sequence in an in vitro kinase assay either using a mixture of PKC enzymes or individual PKC types inhibits the activation of PKC in vitro in a competitive manner, whereas the same peptide carrying a serine in place of the alanine behaves as a good substrate for PKC (House et al., 1987; Pears et al., 1990; Schaap et al., 1989). From these data a molecular model has been derived where the pseudosubstrate sequence blocks the catalytic site of PKC. Upon binding of activators to the regulatory domain a conformational change may release the pseudosubstrate sequence from the catalytic site, allowing access of the substrate (Parker et al., 1989). An analogous stretch of amino acids is found in all PKC molecules suggesting that the basic sequence requirements for phosphorylation by PKC subtypes are similar. Figure 2 shows a compilation of the putative pseudosubstrate sequences for all cloned PKC types. Common to these sequences is the basic, positively charged environment of the pseudosubstrate residue alanine, although different PKC types show variations in the number and spacing of basic residues surrounding the alanine, e.g. in PKC r/(and PKC-L) the alanine is immediately preceded by a basic residue (arginine) whereas in all other enzymes the alanine is preceded by a glycine (Fig. 2). This feature may hint to unique substrate requirements of PKC r/which shows a different tissue distribution in comparison with the rest of the family. These variations in the pseudosubstrate sequences may reflect subtle differences in target sequence requirements for phosphorylation by the different PKC enzymes in vivo which may be difficult to assess in vitro by the use of synthetic substrate peptides (see Marais et al., 1990). The pseudosubstrate prototope is followed by the zinc finger structures with the distance between the alanine residue and the first finger being highly conserved in all PKCs, this distance is 24 amino acids for PKC ct, fl, y, 6, and ~, and 23 amino acids for PKC E and r/. Although studies on T lymphocytes, fibroblasts and sea urchin eggs have successfully employed peptides corresponding to the pseudosubstrate prototope sequence to inhibit PKC activation in vivo (Alexander et al., 1989; Eicholtz et al., 1990; Shen and Buck, 1990) the use of such peptides as specific inhibitors has been questioned since the activity of cAMP-
76
S. STABELand P, J. PARKER yPKCI tpa-I
dPKC53E(br) dPKC53E(ey) dPKC98F XPKCI XPKCII rat alpha beta gsmm. delta epsilon
zeta mouse
eta
395 ? 28 54 55 24 16 19 19 18 141 153 113 155
G I S N F I% R R R L T P S T
L H I% H Q R R R R R N F F F F F M R I R
Y. L R A A A A C N K Y K
R R R R • R R R R R R R
K K R K K
K K K R Q R Q
~!xxxaxR G G G G G G G G G G R
A L K A M K A M R A L R A L R A L R A L R A L R A I K A V R ARRW ~ M R
K R R Q Q Q Q Q Q R
K K R K K K K K A R
N G N N N N V K RK R R -
s s z r s Q VHEZRG VFNVKD L E M V N G V H Q V N G V H E V K N V H E V K E V H E V K D V H E V K N V H E V K S I H Y I K N V H Q V N G L Y RAN G VH QVN G
FIG. 2. Pseudosubstrate prototope sequences from PKC cDNAs. Alignment of amino acid sequences of PKC pseudosubstrate prototopes from yeast (yPKC1; Levin et al., 1990), C. elegans (tpa-l; Tabuse et al., 1989), three different Drosophila enzymes (dPKC; Rosenthal et al., 1987b; Schaeffer et al., 1989), two Xenopus enzymes (XPKC; Chen et al., 1989) and mammalian species (see Table 1 for references). Highly conserved basic residues are marked by an asterisk, the serine to alanine substitution is boxed.
dependent kinase is also affected by peptides mimicking the pseudosubstrate site of PKC (Smith et al., 1990; Soderling, 1990). The reason for this cross-reactivity lies probably in the fact that the primary sequence specificity between a number of kinases appears to be rather similar, i.e. basic residues Nterminal to the substrate serine. The validity of the molecular model of PKC activation, however, whereby the pseudosubstrate prototope inhibits kinase function in the intact molecule has been supported by the observation that PKC can be activated in vitro by an antibody directed against the pseudosubstrate prototype (Makowske and Rosen, 1989) and by site-directed mutagenesis of the pseudosubstrate prototope (Pears et al., 1990). 2.1.2. The Zinc Fingers within the C~ Domain Zinc-binding finger structures have first been identified in the Xenopus transcription factor T F I I I A where they are required for sequence-specific binding of the factor to D N A (Berg, 1990). Subsequently, the finger motif was discovered in a number of other proteins mostly but not exclusively nucleic acid binding proteins. Although for the majority of these proteins with zinc-finger structures direct interaction with D N A or R N A has been reported, this primary sequence motif with its resulting secondary structure is also certainly used for protein-protein interactions as suggested by the dimerization and cooperative binding of the bacteriophage T4 helix-destabilizing protein (gene 32) (Pan et al., 1989) and the adenovirus E1A protein which does not bind directly to D N A but mediates transcriptional induction of viral and cellular genes probably by interacting with other proteins (Chatterjee et al., 1988). All members of the PKC family carry two so-called zinc-fingers within the C 1 domain, except the ( type which only has one such structure (cf. Fig. 1A). The zinc-finger sequence in the PKC molecules would be of the 'Cys-Cys' type as represented by the steroid receptor superfamily and the adenovirus E1A protein. The 'Cysq2ys' type differs from other zinc-binding fingers in that coordination of the zinc ion would solely be effected by cysteine residues and not by a
combination of cysteine and histidine residues (Berg, 1990). While the zinc-binding structure itself as identified in the steroid receptors and the E1A protein is only made up of four cysteine residues corresponding to cysteine number 1-4 in Fig. 3 it is worth noting that the finger structure in all PKC molecules is followed by two additional cysteines the spacing of which is also highly conserved (cysteines 5 and 6 in Fig. 3). Thus this structure in PKC can be viewed as an 'extended' zinc-finger structure which shows a spacing of cysteines identical to the steroid receptors and the E1A protein but extended by two cysteines of unknown function. The fact that this type of 'extended' finger is also found in unrelated molecules like D A G kinase (Sakane et al., 1990; Schaap et al., 1990), a phorbol ester-binding protein n-chimaerin (Hall et al., 1990) and the Raf family of protein kinases (Rapp et al., 1988), suggests that either the zinc-finger structure per se (Cysl-Cys4) can serve a different function in the context of the PKC motif Cys-X2-Cys-Xla-Cys-X2-Cys-X7-CyS-XT-Cys, where X is any amino acid, or that the PKC motif combines two structural functions; the first four cysteins coordinate zinc like the analogous structures in other proteins and the fifth and sixth cysteine serve some unknown function. The spacing of cysteines within each of the two repeats is highly conserved and follows the pattern Cys-X2-Cys-Xl3-Cys-X2-Cys for both fingers in PKC ct, fl, ~, 3, ( and the second finger of E and C y s - X : ~ y s - X 1 4 - C y s - X 2 ~ y s for the first finger in E, r/ and PKC-L (Fig. 3). Embedded in the extended finger structure is a highly conserved amino acid triplet VHK, I H K or VHR, which has been suggested to be involved in interactions with phospholipid since this triplet is also found in the catalytic site of phospholipase A2 (see below and Maraganore, 1987). The VHK motif follows all PKC finger structures three amino acids downstream of the last cysteine of the finger (Fig. 3); it is absent from the incomplete second finger in PKC-L. Although bound zinc has been directly demonstrated by atomic absorption spectroscopy or nuclear magnetic resonance for many zinc-finger-containing proteins either of the 'Cys-His' or the 'Cys-Cys' type
Protein kinaseC 2
1
A
t,, c PKC PKC PKC PKC PKC • PKC X.i. X.i. dPKC dPKC dPKC C.e.
alpha beta gm.~R delta epsilon eta zeta PKCI PKCII 98F 5 3 E (ey) 53E (br) tpa-i
yPKCl DAG kinase • n-chimaerin • c-raf • A-raf • B-raf
77 3
C 0. C •. C .V C .. C •. C GQ C .. C .o C .. C G. C .. C .L C AY C NL C EY C DI C DF C DF
C
C C C C C C C
C C CK C C C C C C C
. . . . . .
- . o 0
. . . .
...... -I .... L. KE.V..-LN...YK R ..... VI .... Y. RE .... V ..... Y. SER...LA-R..YR ...... -L ...... ......
-..°....
R E .... I - . . . . Y . K ..... - ....... ..... - ....... S..M..-LN...YQ G..LR .... YT... E S S I - . - L .... L S AN.M..LI-A..VK QKFL ..... LN..R LKFL ..... FH..R RKFL ..... FQ..R
C C C C C C C C C C C C C C C C C
c .T~C *
m
PKC alpha PKC beta P K C g....1 PKC delta PKC epsilon PKC eta ePKC zeta X. 1. P K C I X.i. PKCII dPKC 98F d P K C 5 3 E (ey) d P K C 5 3 E (b=) C.e. tpa-i yPKCI DAG kinase • n-chimaerin • c-raf • A-tar aB-raf
~DH C .. C C .. C C .. C C .. C C .. C CGQC C .. C C .. C C .. C C .E C C .. C C .. C CC. C C .R C C .. C C .IC C .F C
c .FC
GSLLYGLI-
HQGMK
C
o..oo...-..o°o
C
. . . . . . . V - . .... .T..W..V-K..L. .... W . . L - R . . L Q .... W.IM-R..LQ SERIW..A-R..YR ...... M.- .....
C C C C C C
.....
o°o-...o.
........
-K.
.L..H.VA-...V. ...... IY-...L. . .M .... F-K..LR .YI.PW.R-. KVR. QKKIRIYHSLV. LH ANFI~4...-A..V. QKFL ..... LN. FR LKFL ..... FH.FR RKFL ..... F..FR
DT .. .. .. KV KI IN E. E. E. EN
6
rsl e7
Cl C S .... R.I C ........ Cl N A A I . . K I C I D K I I G R ICl T C . . . . . IC . . L I I T K I Cl T C . . . . . 1C .H L I V T A ICl K L L .... iC . V L . P L T ICI Cl ,.°o... ,C Cl .o..... ,C T L .... KI C . L S . V S K I Cl R . N Z .QKI C C K . . V . K I Cl S Y . . . . . IC . . . . . . . . Cl S A A . . . K I C . .K.IblQI Cl K . L C . . K I C Y T N . V T K ICl K Y T . . DQI C A M K - A L P ICl GLN.. -QI C S X M . P N D ICl G Y K F . EH! C S T K . P T M ICl GYKF. Q"I C S S K . P T V ICl GYKF. QR C STE.PLMI c l *
U C I D~WUKQ CIM ..... R CIE .... RR C l G .... H K C l K .... R R C l K .... I R CIKLL...R Cl...I..V CIM ..... R ClG ..... R ClNL...HA
-C C C C C C C C C C C
m
V I N V P S L !C .M ..... C .RS .... C REK. AN. C ET..APN C OA..APN C HVL..LT C o.o.ooo
.M ..... QK..ANT C QET.. PM C AR C K E . . . . . C SA Cl . . . . . C INVAC. BK C E R L M S N . C C S ~ Cl G I M C . A Q C AHL.. DF C LP~gHE C VN ClHLEI.DD C A D C I G L . . . . . C S l ~ I . . ND C S T K . . TM C Q. C l G Y K F . E H C SSK..TV C Q. C I G Y K F . Q H C_ s T ¢ . . ~ C Q. C l G Y K F . Q R C
°LQ
E
...C ...C RQIC ...C ...C INIC ...C ...C .. C EEIC C .LIC .DIC NLIC ADIC .T',C .TIC
*
B
5
4
Qvl
SH C TDFIWG-FGKQGFQ
C
C C C C C C
C C C C
_c *
FIG. 3. The 'extended' zinc-finger structures of PKC and unrelated proteins. Alignment of the first (A) and second (B) zinc finger structures of PKC sequences and unrelated enzymes is shown as indicated. Molecules that contain only one finger structure are marked with a filled circle. In these cases this one finger sequence is aligned with both the first (A) and the second (B) fingers of molecules which contain two zinc-finger structures. Conserved cysteine residues are numbered from 1 to 6. Identical residues in all sequences are marked by an asterisk. Dots represent amino acids identical to the top sequence of PKC ~, and dashes represent gaps introduced to allow for better alignment. The ValHisLys (VHK) triplet is marked by a shaded box.
(Berg, 1990), it is not known whether the PKC proteins contain zinc. Effects of zinc, however, on the activity of PKC have been reported (Csermely et al., 1988; Speizer et al., 1989; Waiters and Johnson, 1990). In addition, phorbol ester binding to a novel phorbol ester binding protein (n-chimaerin) which shares the extended zinc-finger structure with the PKC family (see Fig. 3 and below) has been shown to be dependent on zinc (Ahmed et al., 1990). Unlike the case of the steroid receptors there is no evidence that the zinc-finger in PKC can mediate specific or even nonspecific DNA binding, rather most of the evidence available to date points to a role of this structure in phospholipid-dependent phorbol ester binding (Ono et al., 1989b). Studies addressing the function of the C t domain in PKC have so far used deletion mutagenesis and substitution of indi-
vidual cysteine residues in the zinc finger. Expression of the C1 domain of PKC 7 in E. coli yielded a polypeptide able to bind phorbol ester in a phospholipid-dependent manner (Ono et al., 1989b). Deletion of either of the units containing the conserved six cysteine residues does not interfere with the binding of phorbol ester by the E. coli cell extract, whereas mutation of the third and fourth cysteine residue of either finger abolishes binding (Ono et al., 1989b). These studies show that one of the zinc-finger-containing units is sufficient to confer phospholipid-dependent phorbol ester binding to the C1 domain of PKC 7 expressed in E. coli. Studies using deletion mutants of PKC ct and PKC fl cDNA and hybrids between the regulatory domain of cAMP-dependent protein kinase (PKA) and the catalytic domain of PKC and vice versa came to
78
S. STABELand P. J. PARKER
slightly different conclusions after transient expression of the cDNAs in mammalian COS cells. The C 1 domain of the PKC polypeptide is necessary and sufficient for phorbol ester binding to the transfected cells. However, mutants of PKC a lacking the second zinc finger did not bind phorbol ester (Kaibuchi et aL, 1989). By contrast deletion of the first PKC 7 finger does not abolish phorbol ester binding (Cazaubon et al., 1990). Surprisingly, deletion of only 30 amino acids near the N-terminus including the pseudosubstrate prototope also abolished phorboi ester binding. However, it was argued that in this case this might constitute an indirect effect due to an altered conformation of the mutant polypeptide (Kaibuchi et al., 1989). Deletion of the C2 domain had no effect on phorbol ester binding on whole cells compared with wild-type PKC ~. Mutations which remove the kinase domain of PKC ~t ( P K C - P K A hybrids) or PKC fl increased the binding capacity about two- to three-fold (Kaibuchi et al., 1989; Muramatsu et al., 1989). Enhanced phorbol ester binding has also been noted in kinasenegative mutants of PKC a and PKC ~, carrying a mutation in the ATP-binding site (Freisewinkel et al., 1991; Ohno et al., 1990). So it appears that the 'extended' zinc-finger structure in the PKC molecules through binding of the lipid activators is somehow involved in the activation process by binding of phospholipid and phorbol ester activators and probably thereby mediating a conformational change that activates the kinase function. That such changes occur is evident from the loss of monoclonal antibody binding (Cazaubon et al., 1990). How exactly binding of the lipid activators is mediated by this structure is not known and it will be of interest to determine whether and which common features can be identified which justify the similarity between structures that mediate binding of nucleic acids and phospholipids/phorbol esters. 2.1.3. The C2 Domain and the Kinase Domain The C 2 region is defined by its presence as a conserved region in all four 'group A' PKC members ~,/~, 32, and ~ and is part of the regulatory domain of these enzymes. The C2 domain is missing in the 'group B' PKC members b, E, ~ and r/(Fig. 1A). After transient expression of the respective cDNAs in mammalian or insect cells 'B-group' members of PKC apparently do not require Ca z + for either autophosphorylation in vitro (Bacher et aL, 1991) or phosphorylation of histone (Ono et al., 1988a) or peptide substrates (Schaap and Parker, 1990; Schaap et al., 1989). Also binding of phorbol ester and phorbol ester-induced translocation of the E and & enzymes to the particulate fraction is independent of Ca 2÷ (Akita et al., 1990; Kiley et al., 1990; Olivier and Parker, 1991). This behavior is in contrast to the 'A-group' enzymes ~t,/~ and 7, which all require Ca z ÷ for their kinase activity as well as for phorbol ester binding and translocation (Burns et al., 1990; Huang et al., 1988; Kiley et al., 1990; Marais and Parker, 1989; Sekiguchi et al., 1988). Therefore it seems likely that the C 2 domain provides for the Ca 2÷-dependence of the enzymatic and lipid-interacting properties of the
'group A' enzymes. However none of the known Ca2+-binding motifs has been identified in this domain. A sequence resembling a potential Ca 2+binding site of the 'EF hand' type has been noted in PKC ~ just downstream of the C 2 domain (Parker et al., 1986). A similar array of amino acids is found in PKC fl but not in PKC 7. The absence of this sequence in PKC ~ raises the question about potential Ca 2+ binding structures in this enzyme since all three types ct, fl and 7 have been shown to be dependent on Ca 2÷ in vitro. Mutagenesis experiments support a function of the C2 domain in mediating Ca 2÷-dependence since deletion of this region renders the kinase activity as well as phorbol ester binding activity independent of Ca 2÷ (Kaibuchi et al., 1989; Ono et al., 1989b) whereas expression of the C2 d o m a i n p e r se in E. coli does not support phorbol ester binding (Ono et al., 1989b). Early studies showed that the PKC polypeptide can be cleaved in vitro by trypsin or calpain into two fragments, one exhibiting phorbol ester binding and the other activator-independent kinase activity (Inoue et al., 1977; Lee and Bell, 1986; Parker et al., 1986). Obviously the kinase domain of PKC is believed to play a crucial role in mediating the effects of phorbol esters as well as D A G on cellular processes by catalyzing the phosphorylation of substrates. Where and in which context the kinase function of the various PKC enzymes becomes effective may be subject to a variety of intracellular parameters like the intracellular Ca 2+ level, the type and temporal features of D A G production, the subcellular localization and substrate specificity and availability of the particular PKC. In addition, autophosphorylation of PKC has been well documented (Flint et al., 1990; Huang et al., 1986a; Mitchell et al., 1989) although the effects of autophosphorylation on the enzymatic properties of PKC are not clear yet. Autophosphorylation has been claimed to decrease the affinity of PKC for Ca 2+ and increase its affinity for phorbol ester (Huang et al., 1986a). On the other hand, engineered PKC mutants either completely lacking the kinase domain or inactivated by a point mutation in the ATP-binding site also show increased phorbol ester binding suggesting in contrast to the above study that the affinity for these agents is negatively modulated by autophosphorylation (Freisewinkel et al., 1991; Kaibuchi et al., 1989; Ohno et al., 1990). 2.2. HOMOLOGIESTO OTHER PROTEINS The presence of conserved domains in the PKC molecules suggests that some of them at least could serve some general functions which might be reflected in similar structures found in these domains and in other proteins. This is obviously true for the kinase domain which shows a number of features found in all kinases and in particular in serine/threoninespecific kinases (Hanks et al., 1988). Additionally the conserved clusters in the N-terminal regulatory domain exhibit features apparently shared by other proteins. The C~ domain containing a putative zinc-finger structure has been discussed in detail above and indeed this structure has attracted a lot of interest because of its well investigated function
Protein kinase C in other proteins which includes nucleic acid binding and protein-protein interactions (see Berg, 1990; Evans and Hollenberg, 1988). The first part of the cysteine-rich structure of PKC is shared by the steroid receptor superfamily and the adenovirus E1A protein and follows the pattern CyS-XE~Cys-Xl3/14Cys-Xz42ys. More extensive homology, however, spanning the whole pattern of cysteine spacing C y s - X 2 ~ y s - X 13/i4~ys-X2~ys-XT-Cys-X7-Cys is found with individual proteins like the c-Raf kinase family (Ishikawa et al., 1986), the diacylglycerol kinase (Sakane et al., 1990; Schaap et al., 1990) and n-chimaerin, a brain-specific sequence of unknown function (Hall et al., 1990). Figure 3 shows an alignment of the cysteine-rich structures of PKC and these unrelated proteins. The presence of a zinc-finger pattern in the N-terminal presumed regulatory region of the c-Raf kinase family (Rapp et al., 1988) has led to the suggestion that by analogy to PKC this structure in c-Raf could also be involved in regulation of the kinase activity of c-Raf by soluble second messengers (Li et al., 1991). How a regulation via this domain of the c-Raf protein could be effected is unclear; this structure does not mediate phorbol ester binding in the c-Raf polypeptide (Srzeri and Stabel, unpublished). Diacylglycerol (DAG) kinase represents another protein with two zinc-fingers where this structure could be associated with lipid binding. DAG kinase contains two zinc-finger structures of which the second one has the same spacing of cysteine residues as the corresponding structure in PKC (Sakane et al., 1990; Schaap et aL, 1990). Within the first finger of D A G kinase is embedded the amino acid pattern characteristic for an ATP binding site, Gly-X-GlyX-X-Gly-XI4-Lys (Hanks et al., 1988), which might reflect the fact that this protein is a lipid kinase, although the significance of this 'double feature' is not clear and it is not known whether this pattern indeed represents an ATP binding site in this case. At a similar location in the loop of the first but not the second finger in PKC ~, fl and 7 the sequence Gly-X~31y-X-X-Gly is also present, however, this is not followed by a lysine indicative for an ATP binding site, suggesting that the glycines in the finger loop might in both cases serve some function other than nucleotide binding. The idea that the zinc-finger structure of PKC might mediate lipid binding had been voiced earlier from sequence comparison with another lipid metabolizing enzyme phospholipase A 2 (Maraganore, 1987; Stabel, 1990). Pancreatic phospholipase A 2 catalyzes the release of fatty acids from the sn2 position of 1,2-diacyl-sn-glycero-phosphocholines and the active site residues are highly conserved between mammalian and snake venom phospholipases (Seilhamer et al., 1986). His48 in the active site is believed to be involved in the catalytic mechanism and Lys49 or Asp49 have been shown to function in the formation of the catalytic complex (Maraganore and Heinrikson, 1986). At the appropriate position relative to *Interestingly this same region of the bcr protein is also conserved in two regulatory subunits of the type I phosphatidylinositol kinase (Otsu et al., 1991); the function in this context remains unknown. JPT 51/I--F
79
conserved cysteine residues in PKC and in nchimaerin (see below) the pair His48Lys49 can be found, whereas DAG kinase carries His-Asp at these positions. The most intriguing and perhaps significant homology found between PKC and other proteins is the one to the recently identified brain-specific product n-chimaerin (Ahmed et al., 1990; Hall et al., 1990). n-Chimaerin was identified as a relatively abundant brain-specific mRNA encoding a 34kDa protein product of unknown function which shows a high sequence homology in its N-terminal part to the C 1 region of PKC and in its C-terminal part to bcr, the 'breakpoint cluster region' involved in Philadelphia chromosome translocation which encodes a 160 kDa protein of unknown function (Hall et al., 1990).* The homology between the N-terminal region of n-chimaerin and the C1 region of PKC is about 50% and is the highest homology found for this region to a protein outside the PKC family. Expression of the N-terminal part of n-chimaerin in bacteria proved that this structure is able to bind phorbol esters in the absence of Ca 2+ which supports the idea that the C2 and not the C1 region of PKC mediates Ca 2+ dependence (see above). Phorbol ester binding by nchimaerin is dependent on phosphatidylserine; other phospholipids like phosphatidylinositol, phosphatidylcholine or phosphatidylethanolamine were not effective in supporting binding (Ahmed et al., 1990). Also, bacterially expressed protein which was denatured and refolded in the absence of zinc ions did not bind phorbol ester suggesting that phorbol ester binding requires a zinc-dependent structure. Besides the novelty of identifying a phorbol ester-binding sequence outside the PKC family this finding has significant implications for all studies using phorbol esters to probe the role of PKC. Worth noting from these studies is that apparently only some fingers of the general structure Cys-X2-Cys-X13/14-Cys-X2Cys-XT-Cys-X7-Cys can support phorbol ester binding and that this property might be determined by specific amino acids within this structure which could be identified by gene technology methods. Sequence homology to the C 2 region within the regulatory domain of the 'A-group' of PKC proteins has been noted for two proteins, p65 and phospholipase C. p65 is a t r a n s - m e m b r a n e protein specific for synaptic vesicles which binds calmodulin and might be involved in the process of neurotransmitter release (Perin et al., 1990). This protein contains within the cytoplasmic domain two copies of a 116 amino acids repeat which share about 40% sequence identity with each other and the conserved region C 2 in 'A-group' PKCs (Perin et al., 1990). No homology is found between p65 and the 'B-group' PKC members suggesting that the C 2 region represents a separate functional module shared only by 'A-group' PKCs and p65. Bacterially expressed p65 strongly binds phosphatidylserine and sphingolipids including gangliosides, but not phosphatidylcholine, sphingosine, phorbol esters, DAG or calmodulin (Perin et al., 1990). The homology spans nearly the whole C2 region. This same region, namely C2, shows some weaker homology to a stretch of sequence in phospholipase C (Baker, 1989). A region of about 100 amino acids
80
S. STABELand P. J. PARKER
which shows weak but significant homology to the C2 region of 'A-group' PKCs has been noted n e a r the C-terminus of phospholipase C-y (Stahl et al., 1988) and is less well conserved in PLC-fl (Katan et al., 1988). The presence of this homology in these two enzymes has led to the speculation that in both cases these sequences might participate in D A G or lipid binding (Baker, 1989). 2.3. PKC IN LOWER EUKARYOTES The search and isolation of PKC-like enzymes from lower eukaryotes has experienced a boom in the past two years or so (Table 2 and Fig. 1B). A PKC-like enzyme in lower eukaryotes was first identified in a Drosophila genomic D N A library (Rosenthal et al., 1987b). To date three PKC types have been identified in Drosophila which are encoded by different genes and are differentially expressed. The first PKC cDNA from Drosophila, dPKC, was isolated by low stringency screening with a mammalian probe (Rosenthal et al., 1987b). The same gene and cDNA was reisolated subsequently together with two new members of the Drosophila PKC gene family also by low stringency screening with mammalian PKC probes (Schaeffer et al., 1989). Within 50 kb of the first isolated gene, dPKC, which maps on the second chromosome at position 53E4-7 (Schaeffer et al., 1989) is located another PKC gene encoding a 700 amino acid protein which differs throughout its length from dPKC. Since both genes map at cytogenetic position 53E the nomenclature of 53E(br) for dPKC which is expressed in adult brain (Rosenthal et al., 1987b) and 53E(ey) for the newly identified homologous protein which shows exclusive expression in the Drosophila eye was proposed. The two 53E enzymes are most likely not differentially spliced products of one gene since their longest stretch of identity is 12 nucleotides. Clustering of homologous genes is not an uncommon phenomenon in Drosophila although the reasons for this are unclear. It is possible that this feature results from duplication of an ancestral gene. Both predicted proteins have a structural organization like the ~ and fl type of mammalian PKCs (cf. Fig. 1B), i.e. they possess a C2 region and they lack the insertion in region V4 which is typical for PKC 7-
At cytogenetic position 98F on the third Drosophila chromosome another PKC gene was identified (Schaeffer et al., 1989); this resembles the 6 type of mammalian PKCs in that it lacks a C2-1ike region. The 98F enzyme is expressed throughout development whereas both 53E enzymes are mainly expressed in adults. Transcripts of 98F and 53E(br) are found predominantly in brain, the 53E(ey) enzyme is solely expressed in photoreceptor cells. As expected from their homology with different types of the mammalian enzymes the 53E enzymes are more closely related to each other than to the 98F enzyme. Transcripts of all three thus far identified Drosophila PKC genes have only been identified in adult head tissues, not in the body. Therefore the possibility exists that there are additional PKC isozymes to be discovered which are expressed in the body structures of the fly (Schaeffer et al., 1989). The exclusive expression of the 53E(ey) enzyme in photoreceptor cells suggests a very specific role for this PKC enzyme in the photoreception process in the Drosophila eye. Generation of Drosophila mutants deficient or mutated at the PKC locus could provide a valuable means to elucidate this role in the invertebrate eye. In this respect it is of interest that the NorpA gene product shows clear structural homology to mammalian phosphatidylinositol-specific phospholipase C (Bloomquist et al., 1988); deletion of both copies of this gene abolishes photoreception. The fact that the Drosophila enzyme 98F resembles more closely the 6 type of mammalian PKCs lacking the C2 region whereas the 53E enzymes show a structural organization like the mammalian 7, fl, and 6 polypeptides, suggests that these two groups of PKC proteins are ancient. If'A-group' and 'B-group' PKCs have common ancestors the divergence of the two groups must have occurred before the ancestors of mammalian and arthropods diverged (Schaeffer et al., 1989). In contrast to prokaryotes various eukaryotic microorganisms either produce compounds with similarities to vertebrate signal molecules or, alternatively, are able to react to authentic signal molecules from vertebrates (Janssens, 1987). For two eukaryotic microbes it has been reported that extracellular signals evoke the accumulation of inositol phosphates: glucose in Saccharomyces cells arrested at the Go/G I
TABLE2. Cloned Protein Kinase C Types from Lower Eukaryotes Closest homology to mammalian type
Species
Nomenclature*
Amino acidst
Xenopus
X-PKCI X-PKCII
676 671
~t/fl
d 53E(br) 53E(ey) 98F
639
~/fl
700 634
~/fl
tpa-I
526:~
?
Tabuse et al. (1989)
fl
Levin et al. (1990)
Drosophila
C. elegans
PKC1 1151 *Nomenclature used in the reference cited. tNumber of amino acids of the predicted proteins. :~Partial eDNA sequence.
S. cerevisiae
6
Reference Chen et aL (1989) Chen et al. (1989) Rosenthal et Schaeffer et Schaeffer et Schaeffer et
al. al. al. al.
(1987a) (1989) (1989) (1989)
Protein kinase C cell cycle boundary by starvation (Kaibuchi et al., 1986), and cAMP in the amoeba Dictyostelium (Europe-Finner and Newell, 1987). The clear demonstration of an enzyme with structural characteristics of a PKC polypeptide in Drosophila is contrasted by the failure as yet to clone a related enzyme from Dictyostelium, although protein kinase activity of a 140kDa protein whose phosphorylation was enhanced by phosphatidylserine but not by Ca 2+ or D A G has been described (Jimenez et al., 1989). Also, using an epidermal growth factor-receptor-derived peptide substrate a protein kinase activity has been described in the same organism which is stimulated by PS and PMA or D A G in a synergistic manner and independently of calcium (Luderus et al., 1989). It remains to be seen whether these activities indeed represent PKC homologs. Screening of a Saccharomyces cerevisiae genomic library with probes derived from mammalian PKC ), and /3 with reduced stringency, detected a D N A clone with the coding capacity for a protein of 1151 amino acids and a calculated molecular weight of 131,524Da (Levin et al., 1990). The gene, PKC1, consists of a single large open reading frame without putative introns. Sequence comparison revealed that the putative yeast enzyme is most closely related to the mammalian PKC members ct and/31 with 51 and 53% amino acid identity in the catalytic domain, respectively (Levin et al., 1990). Although the regulatory domain is less well conserved, two zinc-fingers, a C1 domain and a pseudosubstrate prototope are readily identified in the sequence from yeast. The putative yeast PKC protein carries an N-terminal extension of 375 amino acids similar to the mammalian members 6, E, ~ and ~/, however the sequence of this extension bears no resemblance to any other protein or to the N-terminal extensions of PKC 6, E, or ~. From genomic hybridization data it appears that the PKC1 gene is unique in the S. cerevisiae genome and it maps 20.6 cM distal to CDC27 on chromosome II (Levin et al., 1990). Genetic analysis shows that the gene is essential for completing the yeast cell cycle. Absence of PKC1 results in recessive lethality with nonviable spores which still germinate but then are arrested. Conditional PKC1 deletion mutants are arrested after D N A synthesis and are halted in growth as well as in division giving rise to a 'terminal arrest' phenotype (Levin et al., 1990). None of the other known cell division cycle mutants in S. cerevisiae with a similar phenotype (cdc49 and cdc50) appear to be allelic to PKC1. In contrast to the conclusion that there is only a single PKC gene in the yeast S. cerevisiae, Nishizuka and coworkers have identified a Ca 2÷-, phosphatidylserine- and diacylglycerol-stimulated protein kinase activity in S. cerevisiae extracts which may be different from the above enzyme (Ogita et al., 1990). The yeast enzyme appears to have a molecular weight of about 90 kDa and its preferred substrate is myelin basic protein in contrast to the mammalian ~ enzyme which preferentially phosphorylates histone HI. Phosphorylation by the yeast enzyme occurs preferentially on threonine residues; mammalian PKC prefers serine residues in myelin basic protein and in histone. A remarkable feature of the yeast enzyme is that despite its response to diacylglycerol it cannot be
81
activated by phorbol esters. This would be the first case where activation of a PKC enzyme in vitro by diacylglycerol cannot be mimicked by phorbol ester. The nematode Caenorhabditis elegans has become a model organism to study developmental processes. Exposure of this soil organism to tumor-promoting phorbol esters leads to severe disturbances in the behavior and growth of the animals. Biologically inactive phorbol esters do not cause any symptoms. This observation was used by Miwa and coworkers to clone the gene that mediates the effects of phorbol esters by transposon tagging (Tabuse et al., 1989). The gene, tpa-1, was used to isolate a partial cDNA clone encoding a polypeptide of 562 amino acids with the Y-end or N-terminus still unidentified. A computer search for homologies identified the highest homology of this putative protein to the PKC family. 45% identical residues are shared between the Cl region of tpa-1 and mammalian PKC ct, the C, region is followed by a stretch of 72 amino acids with less than 10% homology to PKC ct and the kinase domain shows a homology of 53% to the respective domain in PKC ~. A pseudosubstrate prototope is also found at the appropriate location. Mutations in the tpa-1 gene abolish the effects of phorbol ester on the nematode (Tabuse and Miwa, 1983) and it appears that the tpa-1 gene represents a phorbol ester receptor in C. elegans. It is not known, however, whether this PKC homolog in C. elegans plays a regulatory role in the growth and/or development of this organism. Two types of PKC cDNA clones have been isolated from a Xenopus laevis oocyte cDNA library by low stringency screening with a rat/3 cDNA probe (Chen et al., 1989). They encode putative polypeptides of 676 (XPKCI) and 671 (XPKCII) amino acids. X-PKCI and II show 93% amino acid identity in the C, region, 80% in the C2 region and 85% in the conserved catalytic domain. When compared to mammalian PKC types, both Xenopus enzymes show stronger structural homology to the ~ and /3 types than to any of the other mammalian isozymes. The identification of two closely related members points to the existence of a family of these proteins also in amphibia.
3. F U N C T I O N A L ASPECTS OF PKC I N VITRO For the purposes of discussion, a consideration of the properties of PKC proteins can be divided into general properties that define this class of proteins and specific properties that indicate functional distinctions between the members of the PKC family. 3.1. GENERALPROPERTIES
The ability of PKCs to interact with and be activated by membranes and D A G presents a functional definition of these proteins. As such the binding to lipids is a critical and indeed well studied phenomenon (recently reviewed in Epand and Lester, 1990). This interaction which has been most clearly documented for the ct//3/~ enzymes appears to be minimally a two-step process. The first step
82
S, STABELand P. J. PARKER
is the formation of a ternary enzyme + C a 2 + + phospholipid complex; this is a nonproductive association* from which the enzyme can be removed by addition of Ca 2÷-chelators. Evidence has been provided to indicate that a number of Ca 2÷ ions are involved in each event and furthermore that the apparent affinity of the enzyme for Ca 2+ is greatly enhanced by the presence of phospholipids (Bazzi and Nelsestuen, 1990). This scenario is reminiscent of the lipocortin/calelectrin group of proteins for which a consensus sequence has been suggested as playing a critical role in Ca 2÷ -phospholipid association, however no such sequence is present in P K C (Geisow, 1986). The region most likely to be involved in Ca 2+-dependent-phospholipid interactions is the C2 domain which is only present in the 7 , / / a n d ? gene products (see Section 2 and below). Formal, direct p r o o f for this has yet to be obtained but the circumstantial evidence is compelling. The second step in association is the binding of D A G (or phorbol ester) which through conformational changes leads to activation. The nature of the conformational changes have not been precisely defined but are clearly evidenced through for example, assessment of alterations in surface availability (antibody binding, Cazaubon et al., 1990) and protease accessibility (Kishimoto et al., 1989). The active complex containing D A G (or phorbol ester) is only slowly dissociable by removal of calcium by chelating agents and this is largely the basis of the 'translocation' assay employed for monitoring activation of P K C in vivo (see Fig. 4).t However it is evident that there is also a component of P K C that is only extractable with detergents~: implying a more penetrative interaction of P K C with the membrane. This phenomenon of 'insertion' has also been documented in vitro and is associated with constitutive activation of the enzyme (Bazzi and Nelsestuen, 1988). The extent to which this membrane 'inserted' pool is functional in vivo remains to be defined, although the in vitro analysis would suggest that it is active. This compartment of P K C has been proposed to play a role in memory, since it would be expected that chronic/repeated stimulation of P K C
*It is possible to drive PKC activation in vitro only in the presence of phospholipid + Ca 2+. However in mixed lipid or lipid/detergent micelles there is a strong dependence on DAG (or phorbol ester) for activation (Hannun et al., 1985). tOn extraction of cells in the presence of chelating agents, PKC (~,fl,y) is removed from its transient Ca 2+dependent association with membranes and is monitored as a 'soluble' enzyme; on extraction in the presence of Ca 2÷ these PKCs will be detected as membrane associated. However the complex formed with DAG is relatively stable and even in the nominal absence of Ca 2+, PKC is seen to remain on the membrane fraction. This phenomenon has been used as a measure of activation status in many situations, the so called 'translocation' assay. This assay would more accurately be described as a 'membrane stabilization' assay. :~There is also in many cell types a variable content of detergent nonextractable PKC that is made up of cytoskeleton associated and nucleus/nuclear matrix associated enzyme. The role and indeed activation status of this compartment of PKC is as yet unclear.
NO PRETREATMENT
+ TPA
EXTRACTION +Ca 2+
-Ca 2+
®® ®® ®
®
® ® ® ® ®
®
®
® ®
® ® ®
-Ca 2+
®
®
® ®
®
FIG. 4. Translocation of PKC enzymes. Depicted is a scheme illustrating the dependence of PKC ~ and E interactions with membranes on the extraction procedure and activation status. For PKC ~, at nominally zero Ca: ÷ concentrations and in the absence of TPA (or DAG) the enzyme is in the soluble fraction. However in the presence of Ca 2+, PKC shows a particulate association. By contrast PKC E is in the soluble fraction regardless of the presence or absence of Ca 2+. Prior treatment of cells with TPA leads to complexing (and activation) of both PKC ~ and PKC ¢ and under these circumstances both enzymes show particulate association.
would proportionately increase the membrane 'inserted' pool in a stochastic fashion, leading to a constitutive elevation of P K C activity (Burgoyne, 1989). A model of the general interactions described above is illustrated in Fig. 5. The physiological distribution of P K C between each of these compartments is difficult to assess precisely largely because of the dynamics of the initial phase of membrane association. However an assessment of the activation status is more accessible through the membrane stabilization assay and specific examples are described further in Section 4.
3.2.
C A 2 + -DEPENDENCIES
The apparent dependence of P K C (~, // and 7) activity on Ca 2+ is complex. Under conditions where P K C ~ , / / a n d ? show Ca 2+ -dependence the activation constants are in the physiological range i.e. 0.1-1 #M (Huang et al., 1988; Jaken and Kiley, 1987; Marais and Parker, 1989). However partial or complete attenuation of Ca2+-dependence can be achieved with certain substrates (or even different preparations of a particular substrate). In view of this variation it is expedient to determine interaction with Ca 2+ by
Protein kinase C C2
83
CI
>
Proteolysis
(EOH
t
Ca/Plipid
DAG
00OH
O30H
FIG. 5. Model of PKC interactions. Shown is a schematic representation of PKC ~/[3/~ containing both amino terminal conserved domains C t and C2, In the absence of effectors the pseudosubstrate site (PSS) interacts with the carboxy terminal kinase domain to maintain an inactive state. This form of the enzyme can interact with Ca2+ and phospholipid (Ca/PLipid) to form inactive membrane associated complexes (see also Fig. 4 and legend). On binding DAG conformational changes in the regulatory domain lead to the release of the pseudosubstrate site inhibitor region with consequent activation. In this activated form, the enzyme is more susceptible to proteolysis and shows a reduced half-life. In some cell types activation also leads to an increase in the cytoskeleton (CYT) association of PKC.
some other parameter; this has been achieved most simply for the interaction with membranes (whether the effect of Ca2 + in enhancing membrane association is the primary role of Ca 2+ in stimulating activity is as yet unresolved). It was demonstrated elegantly by Wolf and colleagues (Wolf et al., 1985) that purified PKC (consisting of a mixture of a, t , y) would interact with plasma membranes (inverted erythrocyte ghosts) in a Ca2+-dependent manner. Thus extraction of cells in the presence or absence of Ca 2+ leads to a differential distribution of PKC activity that reflects the Ca 2+-driven association of Ca2÷-dependent PKC activities with membranes. The quite distinct behavior of other PKC forms like e (Kiley et al., 1990) and c~(Olivier and Parker, 1991) highlights the Ca 2+-independence of these activities (Fig. 4). The possible physiological consequences of Ca 2+ -dependent and -independent forms of PKC are further discussed in Section 4.
3.3. DIACYLGLYCEROLAND PHORBOLESTER RESPONSIVENESS The activation of PKC by DAG (in the presence of phospholipids) is the common feature of this group of kinases. Structure/function analyses of DAG stereoisomers and analogs have highlighted the specificity for 1,2 sn DAG and the need for a free 3-OH (e.g. Bonser et al., 1988; Ganong et al., 1986; Rando and Young, 1984). Replacement of acyl with alkyl linkages has also been shown to abolish activation suggesting importance for the carboxyl groups (Bonser et al., 1988). However others have observed activation by ether and alkyl linked diradylglycerols (Ford et al., 1989; Heymans et al., 1987). This would appear to be an unresolved issue, however one of some importance given the potential source of DAGs (see below). To date no substantial differences in activation constants for DAGs have been noted for
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the different PKC isotypes; this is consistent with the high degree of conservation of the putative D A G binding site. The ability of phorbol esters to activate PKC through binding at the D A G site has aroused much interest. Structure/function analysis for this class of activators has largely been carried out on mixtures of PKC ~, fl and 7 enzymes. However more recently highly purified individual PKC enzymes have been assessed for their phorbol ester-dependence and furthermore for their response to various related structures. This has provided evidence for a distinct pattern of responsiveness to different phorbols that themselves have individual functional profiles in biological systems (Evans et al., 1991). One practical conclusion from these observations is that the nature of the biological response to these agents may be defined by the particular set of expressed PKC enzymes that can respond to the particular phorbol; this would provide some rationale for the diverse effects of these agents. That the phorbol esters can apparently distinguish particular PKC enzymes is also an encouragement to those seeking to obtain selective activation/inhibition of these enzymes. One further aspect of such studies is that they may reveal the existence of novel PKC activities as appears to be the case for resiniferatoxin (Evans et al., 1990).
substrate for recombinant PKC E* (Schaap and Parker, 1990), PKC r//L (Bacher et al., 1991; Osada et al., 1990) and PKC 6 (Olivier and Parker, 1991) (although it has been shown that a 'natural' PKC activity immunoreactive with a 6-selective antibody will phosphorylate histones very efficiently (Leibersperger et al., 1990); the basis for this discrepancy has yet to be resolved but may suggest the role of some stable association/modification in the regulation of PKC 6 activity/specificity). A more systematic approach has been made in the case of PKC ct, fll and 7 by addressing the role of N- and C-terminal basic residues in defining primary structural requirements for substrates. These studies indicate that while PKC ~ and fl show a very similar pattern of specificity, PKC 7 exhibits a strong preference for substrates with a C-terminal basic amino acid (Marais et al., 1990). The studies outlined above indicate that although there is as yet limited information on physiological targets, there are relative and absolute distinctions in substrate specificity between PKCs. It may be concluded that the expression of particular PKC enzymes will have a significant impact on the cellular consequences of triggering this signalling pathway.
4. F U N C T I O N A N D R E G U L A T I O N OF PKC 3.4. SUBSTRATESPECIFICITIES
IN VIVO
In contrast to the wealth of information accumulated on the activation of this signalling pathway and the structures of the components within it, there is a dearth of information on physiological substrates for PKC and in particular those whose phosphorylation has a defined effect, As a consequence there are few data on the specificity of different PKC isotypes for physiological substrates. Two that have been addressed are the E G F receptor which is phosphorylated on Thr654 and the fl protein phosphorylated on Ser32. In the case of the E G F receptor PKC • and fl have been shown to be significantly better catalysts than ~ in this phosphorylation (Ido et al., 1987); the relative activities being ~t > fl > 7, whilst for fl the rank order of effectiveness is fll > ~t > ~ (Sheu et al., 1990). These differences between isotypes although modest are likely to be significant in vivo where through the action of phosphatases steady state as opposed to initial rate considerations apply. Further distinctions in specificity have been documented through the use of nonphysiological and synthetic PKC substrates. The major (probably) nonphysiological substrate employed for PKC assays is the lysine-rich histone fraction (HI). Interestingly in addressing the function of members of the PKC family unearthed by homology screening with cDNA probes, it has been noted that histone is a poor
This section is subdivided for consideration of intracellular messenger sources (Section 4.1), the means available for assessing PKC activation and consequences to PKC itself (Section 4.2) and to the cell (Section 4.3).
*Although a poor substrate for PKC-E, histone is readily phosphorylated if PKC-E is proteolytically activated. Thus in this instance the specificity is not an intrinsic property of the catalytic domain but is in part imposed by the regulatory domain. Formal proof of this has been obtained through the construction ofa PKC ~/~,chimeric polypeptide (Pears et al., 1991).
4.1. SOURCES OF DIACYLGLYCEROL The trigger for the activation of the signal transduction pathway in which PKC lies, is the production of diacylglycerol. Historically this has been considered to arise from agonist induced activation of phospholipase Cs which act upon inositol lipids, in particular phosphatidylinositol 4,5-bisphosphate (Ptdlns4,5P2) (reviewed in Berridge, 1987). The delineation of this pathway and the control of the relevant phospholipases have recently been reviewed elsewhere (Meldrum et al., 1991). Suffice it to say that there is compelling circumstantial evidence for agonist-induced breakdown of Ptdlns4,5P2 with the consequent activation of PKC through the D A G produced. The precursor role of Ptdlns4,5P 2 is of particular significance in that the inositol 1,4,5 trisphosphate coincidently produced evokes a rise in cytosolic Ca 2÷ (Streb et al., 1983). This clearly will have an impact upon PKC ~, fl~/2 and 7 which display a Ca2+-dependence in their interaction with membranes (see Section 3.2); by increasing the membrane occupancy of these particular PKCs, it is anticipated that they will be more effectively activated by the limiting D A G produced. Evidence that this is the case, is suggested by the observations that elevated Ca 2+ appears to prime PKC responses and also to increase the apparent affinity for PDBu binding in vivo (for example see Akers and Routtenberg, 1987).
Protein kinase C Interestingly recent studies on the time course of D A G production in a number of distinct cell types has revealed a second prolonged phase of agonistinduced production. This has been readily observed through the use of postextraction conversion of D A G to riP-labelled phosphatidic acid (PA) using bacterial D A G kinase (Walsh and Bell, 1986). This provides an extremely effective procedure for analysis of D A G production without the need to label a particular (unknown) lipid pool in vivo. Analysis of the types of acylation present in this second phase DAG, has led to the conclusion that it is derived predominantly from phosphatidylcholine (PtdCho) (e.g. Pessin et al., 1990). While D A G can of course be produced by PLC hydrolysis of PtdCho, there is accumulating evidence that D A G is also produced via the combined action of a PLD (recently reviewed in Billah and Canthes, 1990) and phosphatidic acid phosphohydrolase (reviewed in Brindley, 1984). This latter enzyme is a microsomal enzyme (in its membrane bound state) suggesting that not only is Ptd-Cho-derived D A G temporally shifted relative to Ptdlns-derived D A G but also may be spatially distinct. As such it is not clear whether the 'translocation' assay successfully employed for plasma membrane D A G activation of PKC will necessarily yield information on PKC activation by microsomal D A G (the distinct properties ofmicrosomal membranes may alter the stability of the putative lipid/DAG/PKC complex). Thus although 'second phase translocation' has not been observed, the conclusion that PKC is not activated has yet to be corroborated. Indeed quite the opposite, since evidence has been presented to indicate that during the second phase response to TRH stimulation in GH4C 1 cells, PKC E becomes down-regulated (Akita et al., 1990; Kiley et al., in preparation). This would appear to be a typical down-regulation that is a consequence of chronic stimulation. Interestingly in this instance
85
it is a selective down-regulation since PKC ~ and/~ which are also expressed in these cells are not downregulated. This suggests that either PKC E is more susceptible to down-regulation or that in the absence of an elevation of cytoplasmic Ca 2+ PKC ~ and 13are not effectively activated by this second phase DAG. There is another de novo route for production of D A G for which there is evidence for agonist stimulation. The extent to which this represents a metabolic as opposed to an intracellular messenger response is not clear although it is pertinent to note that D A G is likely to be free to 'flip' within the membrane and as such may become available to cytoplasmic PKC during for example microsomal triglyceride synthesis/breakdown. An ability to monitor PKC activation in this compartment is evidently an important objective. The degree to which different PKCs respond selectively to the production of D A G in a contextual (Ca 2÷ or no Ca 2+) and spatial (plasma membrane, microsomal, etc.) manner remains to be determined. However it is now well established that Ptdlns4,5P2 is not the only (lipid) precursor for D A G production (as indicated in Fig. 6). As such there is no doubt that a number of regulatory mechanisms remain to be elucidated concerning the precise means by which agonists induce the PLC/PLD directed breakdown of the noninositol lipids. 4.2. ACTIVATION,TRANSLOCATIONAND DOWN-REGULATIONOF PKC The two most commonly used procedures for assessing the activation of PKC in vivo are (i) assessment of the phosphorylation of PKC specific substrates and (ii) measurement of the translocation of PKC. The first of these strategies has been best documented for the '80 k' protein originally identified
PtdCho
PtdIns 4,5 P2 PLC Ins 1,4,5 P3
A
ChoP ~
I
Cho
PA
c~ ÷
Glycerol
DAG
~
PKC
DAG
FIG. 6. Sources of DAG. Shown are possible sources of DAG including phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate 0atdIns4,5Pz) and phospholipase C and D-catalyzed hydrolysis of phosphatidylcholine (PtdCho). A de novo route is also indicated.
86
S. STABELand P. J. PARKER
by Rozengurt and colleagues in Swiss 3T3 cells* (Rozengurt et al., 1983). This protein appears to be a selective PKC substrate and as a relatively abundant protein with a characteristic pL it has served as a useful marker (see Section 4.3 for further discussion). A major shortcoming of this general strategy of using PKC substrates is that in the absence of selective physiological substrates for the different PKCs it is not possible to distinguish whether one or more become activated at particular times. The translocation assay is based upon the phenomenon illustrated above in Fig. 4. In essence, the production of an active complex of PKC-lipid-DAG stabilizes the membrane associated form to extraction in the presence of Ca2+-chelators. Thus in 'PKCactivated' cells there is observed an increase in the membrane associated PKC relative to control cells, with a parallel decrease in cytosolic activity.t As mentioned above there are no specific substrates for different PKC isotypes that have been described that would permit a selective assessment of activation at a functional level, but selectivity can be introduced in this assay through the use of monospecific antisera; by immunoblot it is possible to monitor the behavior of individual PKC isotypes (e.g. Kiley et al., 1990). A consequence of chronic activation of PKC in vivo is down-regulation. This has been shown to be due to increased proteolysis (Ballester and Rosen, 1985; Woodgett and Hunter, 1987; Young et al., 1987) and for PKC ct appears to be due to cleavage in the V3 domain (Young et al., 1988; see Section 2). The mechanism involved in the increased proteolysis has not been elucidated, however the increased susceptibility of the active conformation of PKC to proteases in vitro (Kishimoto et al., 1989) suggests that in this passive manner increased proteolysis occurs in vivo. In contrast to this interpretation it has been reported that a mutant form of PKC ct which is inactive as a kinase does not down-regulate suggesting that substrate phosphorylation or some autophosphorylation event is involved (Ohno et al., 1990). However we have shown that in distinct cellular contexts both mutant (kinase negative) PKC ~ (Pears and Parker, 1991) and PKC y (Freisewinkel et al., 1991) will down-regulate. Thus it is clear that in these cases intramolecular autophosphorylation is not a prerequisite for down-regulation. It is certainly the case that different cellular contexts yield distinct patterns of induced down-regulation and it may be concluded that perhaps distinct pathways are involved for the different PKCs. The extent to which this 'pharmacological' induction of down-regulation reflects a universal physiological process is unclear. However it has been noted that in situations where there is chronic diacylglycerol *The bovine brain homologue of 80 k has been cloned and has been coined the MARCKS protein--myristylated, alanine rich, c-kinase substrate (Stumpo et al., 1989). tit should be noted that this is the simplest scenario. Due to down-regulation of the activated PKC the loss of cytosolic PKC may exceed the increase in membrane associated enzyme. Furthermore there are a number of reports describing acute losses of PKC activity (but not protein) suggesting a distinct plane of regulation (e.g. Cochet et al., 1986).
production, e.g. ras transformation and TRH stimulation, down-regulation has been observed (Akita et al., 1990; Wolfman and Macara, 1987). It may be concluded that the 'normal' production of endogenous DAG has a finite effect on PKC proteolysis. Beyond the acute membrane association of PKC there are a number of reports demonstrating that PKC also becomes associated with the Triton X-100 insoluble fraction of cells. This includes both association with the cytoskeleton (e.g. Kiley and Jaken, 1990; Mochly-Rosen et al., 1990; Zalewski et al., 1990) and the nucleus (e.g. Masmoudi et al., 1989). In the case of the cytoskeleton interaction we have noted that activated mutant forms of PKC ct are increased in their association (C. Pears and P. J. Parker, unpublished) suggesting that activation may be primarily responsible for sequestration in this compartment. Whether these associations reflect a necessary event that is responsible for some element of the physiological response is not yet known. 4.3. ANALYSISOF THE PKC SIGNALLING PATHWAY
There is little information as yet about the biochemical events mediating the proposed connection between PKC activation and cellular responses. One important goal therefore has been to identify and characterize physiological substrates for PKC. A detailed review of in vitro and potential in vivo substrates for PKC can be found in Woodgett et al. (1987). An acidic protein of 87 kDa (the MARCKS protein; see above) with multiple p l species within the range of 4.0-4.9 was shown to be a substrate for PKC in cell lysates from rat brain or 3T3 cells and became an early candidate for a specific PKC substrate (Isacke et al., 1986; Rodriguez-Pena and Rozengurt, 1986; Wu et al., 1982). The protein showed a widespread occurrence in different species and tissues with the highest levels in brain, spleen and lung and lowest levels in heart and skeletal muscle (Albert et al., 1986). Its distribution follows the distribution of PKC (Kuo et al., 1980; Minakuchi et al., 1981) and therefore made this protein a likely mediator of at least certain PKC functions. Phosphorylation of the 80 kDa protein was also seen in intact fibroblasts (Rozengurt et al., 1983); its rapid increase in phosphorylation was believed to reflect activation of PKC. Evidence for this was (1) that addition of biologically active phorbol esters to quiescent cells induces phosphorylation within 15 sec, (2) stimulation of lipid breakdown by PDGF or exogenous PLC or addition of the synthetic diacylglycerol OAG also led to rapid 80 k phosphorylation in quiescent cells. These phosphorylations can be prevented by down-regulation of PKC by prolonged treatment of cells with biologically active phorbol esters (Rodriguez-Pena and Rozengurt, 1986). The 80 k protein was found in all rat tissues examined and was most abundant in brain (Blackshear et al., 1986). Phosphorylation of 80 k could not be detected after addition of insulin or dibutyryl-cAMP. Comparison of the peptide maps of 80 k labelled in vivo after TPA treatment and of 80 k phosphorylated in vitro by PKC showed nearly identical patterns of phosphopeptides suggesting that 80 k is indeed a PKC substrate in vivo (Blackshear et al., 1986) and underlining the potential usefulness of 80 k
Protein kinase C phosphorylation as an apparently specific and sensitive marker of PKC activation in a wide variety of cell types (Isacke et al., 1986). Purification of this protein from bovine brain and amino acid composition analysis showed that it contained 28.6 mol% alanine and from the axial and frictional ratios it was concluded that the 87 k protein is an extremely elongated monomer and that phosphorylation of the purified protein by PKC in vitro occurred exclusively on serines (Albert et al., 1987; Patel and Kligman, 1987). A related 80 k protein was purified from rat brain which had 17.8 mol% alanine and may be related to the 87 k from bovine brain (Morris and Rozengurt, 1988). While phosphorylation of an 80 k protein has been observed within seconds of PDBu addition to quiescent Swiss 3T3 cells, phosphorylation of other proteins including one of 87 k has been reported after minutes of treatment (Rozengurt et al., 1983). Addition of PDBu or bombesin to quiescent Swiss 3T3 cells induces phosphorylation of 80 k and 87 k within 5 rain (Morris and Rozengurt, 1988). Sequence analysis of peptides of 80 k purified from rat brain indicated that there may exist a family of these PKC substrates since the two sequences are not identical (Erusalimsky et al., 1989). The 80 k protein is myristoylated cotranslationally (James and Olson, 1989). A cDNA clone for the 80-87 k bovine protein has been isolated (Stumpo et al., 1989) which codes for an open reading frame of 335 amino acids with a predicted molecular weight of 32 kDa. Despite its predicted molecular weight the protein migrates with an apparent molecular weight of 80-87 k following expression from the cDNA in mammalian cells lacking the protein. Upon addition of TPA to the cells this protein was phosphorylated (Stumpo et al., 1989). The unusual behavior upon gel electrophoresis is probably due to the acidic p I and general hydrophilicity. Striking features of this protein are its high content of alanine residues (28.4 mol%) and the relative abundance of glycine, proline and glutamate. Apart from the initiator methionine it contains no other methionine residues and it carries an N-terminal myristoylation consensus sequence. The phosphorylation sites for PKC on the 80 k protein have been determined (Graft et al., 1989); two of them are found in an unusual highly basic stretch of 25 amino acids K K K K K R F S F K K S F K L S G F S F K K N K K which carries a repeat of the sequence F S F K K . Data from the purified protein indicate that all 4 serines in this basic domain are phosphorylated by PKC in vitro and that the protein does not contain major phosphorylation sites outside this domain after phosphorylation in vitro or in vivo. The basic stretch which contains the PKC phosphorylation sites is perfectly conserved in the M A R C K S protein from beef, human and chicken (Stumpo et al., 1989). The sequence of the 80 k protein has revealed no similarity to known protein sequences. Two other major endogenous PKC substrates have been examined in detail. One, a 40-47 k protein in platelets (Nishizuka, 1984), has been purified (Imaoka et al., 1983) and was tentatively identified as Ins(145)P 3 5'-phosphomonoesterase (Connolly et al., 1986). Immunological screening of a eDNA expression library from HL-60 cells yielded a cDNA which codes for a cytosolic 40k protein with a
87
potential CaZ+-binding 'EF-hand' structure at the C-terminus, a potential PKC phosphorylation sequence and with no homology to any known protein (Tyers et al., 1988). Peptide sequences from the 47 k substrate in platelets are found in this cDNA. 1047 is enriched in HL-60 cells upon differentiation with retinoic acid and it may have a common function in platelets and leukocytes since it is found in hemopoietic cell lines of myeloid and lymphoid origin but not in cell lines from liver, fibroblasts, skin, lens or nerve tissue. The gene appears to be well conserved in vertebrates but is not detected in insects or yeast suggesting an important role for p47 in highly developed hemopoietic systems. Interestingly, p47 has several potential PKC phosphorylation sites one of which ( Q K F A R K STRRSIRL) closely resembles the (pseudo)substrate sequence of PKC ( R F A R K G S L R Q ) . The sequence of lO47 does not yield any further direct clues as to its possible functions and it appears that the Ins(145)P3 5'-phosphomonoesterase activity can be partially separated from the platelet p47 (Tyers et al., 1988). In the absence of any known function and enzymatic characteristics the name PLECKSTRIN has been proposed for p47 (platelet and leukocyte C kinase substrate with the most probable phosphorylation site K F A R K S T R R S I R ) (Tyers et al., 1988). A second brain protein most often referred to as B-50, fl or GAP-43 has been purified and has been hypothesized to modulate the activity of phosphatidylinositol-4-phosphate kinase (Benowitz et al., 1987; Chan et al., 1986; Van Dongen et al., 1985). While there is debate about the precise function of the fl protein, the phosphorylation of this synaptosomal protein is circumstantially associated with long term potentiation (Routtenberg, 1986). The structure of the protein has been elucidated by cDNA cloning (Rosenthal et al., 1987a). As indicated above, in vitro there is some relative specificity for phosphorylation of fl by PKC ct, fl, V but it is not known whether such specificity operates in vivo. With the availability of eDNA clones coding for the individual enzymes another approach has been used to investigate the role of PKC in cellular regulation. By overexpressing individual PKC enzymes through transfection of their cDNAs into mammalian cells their contribution to defined cellular events can be examined. When PKC fl~ is overexpressed 10- to 50-fold as judged by activity assays in rat or mouse fibroblasts the addition of phorbol ester results in exaggerated responses compared to control cells (altered morphology and increased growth rate). Some of the cell lines but not all show formation of small colonies in soft agar (Housey et al., 1988; Krauss et al., 1989). These effects are also visible without the addition of activators of PKC probably due to the production of intracellular D A G through the presence of growth factors in the culture medium. Introduction and overexpression of the 7-type of PKC in NIH 3T3 mouse fibroblasts had similar consequences with alterations in saturation density and reduced dependence on serum for growth (Persons et al., 1988). Selection of cell lines which express about 3-fold higher levels of this enzyme did not lead to morphological alterations under normal culture conditions.
88
S. STABELand P. J. PARKER
The mitogenic responses to TPA and cardiolipin, claimed to specifically activate PKC ~ in vitro (Huang et al., 1988) were enhanced as was anchorage-independent growth, but only in the presence of activators (Cuadrado et al., 1990). In no case, however, has expression of wild-type PKC sequences and activation by activators led to a fully transformed phenotype emphasizing the nononcogenic action of unmutated PKC. It is not known whether mutated, e.g. constitutively activated PKC types could show a fully transforming activity, although isolation of a transforming mutant of PKC from a murine fibrosarcoma has been reported (Megidish and Mazurek, 1989). Introduction of normal PKC ~ into Swiss 3T3 cells and up to 10-fold overexpression did not lead to a transformed phenotype of the cells, not even in the presence of TPA (Eldar et al., 1990), but as with PKC 13overexpressing cells exhibited an enhanced growth rate and reduced serum requirement. Overproducing cells had less EGF receptors on their surface due to decreased EGF receptor mRNA levels; there was no change in the affinity for EGF. This study therefore concluded that PKC ~ is involved in cellular mechanisms regulating the expression of EGF receptor molecules in Swiss 3T3 cells. A loss of EGF-binding sites on the cell surface had been described earlier as one of the properties of certain virally or chemically transformed fibroblasts (Moses et al., 1981; Todaro et al., 1976). Whether these early observations are related to the observed reduction here in at least three independent cell lines overexpressing PKC ~ is not known. Involvement of individual PKC enzymes in celltype-specific reactions can also be excluded by this approach, e.g. through the introduction of PKC flj into IL-3-dependent haematopoietic cells. Although selected cell lines have increased PKC fl~ levels the cells were not more sensitive to IL-3 than control cells nor did they grow in a factor-independent fashion in soft agar (Kraft et al., 1990). Interestingly, however, cells overproducing PKC/?~ showed a 10-fold increase in the formation of transformed colonies when cotransfected with the c - H - r a s oncogene (Hsiao et al., 1989), suggesting a synergistic action between ras oncogenes and PKC. Synergism between ras and PKC has been demonstrated by a number of reports (Fleischman et al., 1986; Hsiao et al., 1984; Lacal et al., 1987; Wolfman and Macara, 1987) the mechanism, however, by which ras transformation and PKC activation could be linked is unclear. It has been speculated that this synergistic effect could be due to the increased levels of DAG reported for ras-transformed cells (Fleischman et al., 1986; Preiss et al., 1986) which may be at least partially responsible for this effect. This does, however, not discriminate between the possibilities that ras acts downstream of PKC (Yu et al., 1988) or upstream of PKC (Lacal et al., 1987). In contrast to this speculation which assumes the synergism between ras and PKC to occur at the polypeptide level, more recently it has been reported that ras transformation can alter the relative expression PKC types present in a cell (Borner et al., 1990). In rat fibroblasts and liver epithelial cells which express the types ~ and c transformation by the ras oncogene or inducible
expression of ras from an introduced construct leads to increased expression of PKC c~ and decreased expression of PKC E at the protein and mRNA level, thereby offering a novel interpretation of ras/PKC cooperation namely by alterations in the set of PKC enzymes within the cell. It will be interesting to determine whether this reflects differential roles of and E in cell growth with high levels of ~ and low levels of E being required for growth. Such a situation could be similar to the one described for the cellular rrg gene product, low expression of which has been shown to be crucial for ras transformation (Contente et al., 1990). A quite suggestive function for PKC has recently been described in T-lymphocytes (reviewed by Berry and Nishizuka, 1990). Evidence was presented that activation of the T-cell receptor by phytohaemagglutinin or a CD3-specific monoclonal antibody leads to activation of cellular p21ras measured as a roughly 10-fold increase of ras in the GTP-bound, i.e. activated, state (Downward et al., 1990). This activated state is the result of decreased hydrolysis of the GTP bound to the ras protein and not of altered nucleotide exchange rates. The evidence is compelling that the GTP hydrolysis which is normally effected by the GTPase activating protein GAP is decreased due to inhibition of the GAP protein. All available evidence suggests that PKC either directly or indirectly is responsible for this inhibition. In this context it is interesting to note that activation of the T-cell receptor has also recently been claimed to lead to phosphorylation and activation of another cytoplasmic serine-/threonine-specific kinasc c-Raf through an apparently PKC-dependent pathway (Siegel et al., 1990). The physiological function of the Raf kinase is not known although it seems to be involved in growth control and intracellular signal transduction pathways which finally lead to gene activation (Jamal and Ziff, 1990; Kolch et al., 1991: Li et al., 1991). Activation of PKC can, itself have effects on transcription factor transactivation function. Evidence has been presented that in resting epithelial cells and fibroblasts activation of PKC results in site-specific dephosphorylation of the c-Jun protein which coincides with increased AP-I binding activity and transactivation by c-Jun (Boyle et aL, 1991). The data suggest that induction of TPA-induciblc promoter element (TRE) function by PKC activators (Angel et al., 1987) is mediated by enhanced binding and transcriptional activity of a dephosphorylated AP-1 complex. The region identified, adjacen~ lo the DNA-binding domain on the c-Jun protein, coincides with a region previously identified to be essential for activation of c-Jun in vitro and in vit, o and had been termed activation domain A2 (Baichwal and 7jian, 1990). The transcriptional activator NF-kB presents another intriguing example where a clearer picture of the role of PKC in the induction of gene expression may be emerging. NF-kB was detected as a prc,~ein that could complex to a 10 base pair site in the k immunoglobulin light chain enhancer called kB (Sen and Baltimore, 1986). Because it was constitutively present only in those B cells of the appropriate stage for light chain expression and was crucial for k
Protein kinase C enhancer function, NF-kB first appeared to be a tissue-restricted transcription factor (Atchison and Perry, 1987). It was, however, soon found to be present in a covert cytoplasmic form in a number of other cells where phorbol esters can induce the factor to move to the nucleus and to exhibit specific DNA binding (Baeuerle et al., 1988) NF-kB is now considered a rather ubiquitous intracellular messenger that mediates inducible and tissue-specific gene expression in many systems (reviewed in Lenardo and Baltimore, 1989). A protein of about 35 kDa which appears as 70 kDa on gel filtration and hence may form a dimer, is able to specifically inhibit the DNA binding activity of NF-kB and has therefore been named inhibitor-kB (I-kB) (Baeuerle and Baltimore, 1988). The purified inactive complex NF-kB/I-kB can be induced to show DNA binding in vitro by phosphorylation with PKC in vitro which phosphorylates sites on the inhibitor protein rather than on NF-kB itself (Ghosh and Baltimore, 1990). The fully active DNA-binding complex of NF-kB appears to be a heterotetramer consisting of two molecules of p50 and two molecules of p65. Cloning of the p50 and the p65 subunits of this complex has revealed that there exists a family of NF-kB-like proteins, the rel family of polypeptides, including KBF1, a factor that binds to a class I major histocompatibility complex enhancer, the v-re! oncogene of the avian retrovirus Rev-T and the Drosophila morphogen dorsal (Ghosh et al., 1990; Kieran et al., 1990; Meyer et al., 1991; Nolan et al., 1991). Whereas both the p50 and the p65 subunits appear to participate in the DNA binding of the active NF-kB complex, only the p65 subunit mediates inhibition by the inhibitor protein I-kB, probably by direct interaction (Nolan et al., 1991). Dissociation of the inactive cytoplasmic NF-kB/I-kB complex by direct phosphorylation of I-kB by activated PKC provides a simple, direct strategy by which PKC activation at the cell surface affects nuclear events. The activation of PKC at the cell surface and the subsequent regulation of many cytoplasmic functions, has also led to the idea that a number of such responses may be effected through kinase cascades. This is typified by the phosphorylation of the ribosomal subunit $6 which can be stimulated by TPA treatment of cells but is effected by a specific $6 kinase that is itself regulated by phosphorylation (albeit not a direct substrate for PKC) (Ballou et al., 1988). It appears then that at some level PKC feeds into a pathway involving an $6 kinase-kinase and $6 kinase. A similar situation exists for the activation of the so-called mitogen-activated protein kinase (MAP kinase) (Ray and Sturgill, 1987) which can be stimulated following TPA (or mitogen) treatment of cells and is itself activated by a MAP kinase-kinase which is functionally TPA inducible (Adams and Parker, 1991). These kinase cascades presumably serve to permit (i) amplification (ii) phosphorylation events distal to PKC activation and (iii) integration of the 'PKC pathway' with other signalling systems. So it appears that the pleiotropic effects that are ascribed to PKC activation may be due to the fact that this enzyme family may impinge on or directly control a number of important intracellular control circuits which are only now beginning to emerge.
89 NOTE ADDED IN PROOF
Resequencing of the PKC-L eDNA confirmed that PKC-L also has two complete zinc-fingers and is indeed the human homolog of PKC-~/ (Bacher et al., correction submitted). gratefully acknowledge Marek Liyanage (Max-Delbriick-Labor) for help with the sequence alignments. We should also like to thank Amanda Wilkinson for preparation of the manuscript.
Acknowledgements--We
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Protein kinase C kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J. biol. Chem. 252: 7603-7609. TAKAI,Y., KISHIMOTO,A., IWASA,Y., KAWAHARA,Y., MORI, T. and NISHIZUKA,Y. (1979a) Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J. biol. Chem. 254: 3692-3695. TAKA1, Y., KISHIMOTO, A., KIKKAWA, U., MORI, T. and NISHIZUKA, Y. (1979b) Unsaturated diacylglycerol as a possible messenger for the activation of calciumactivated, phospholipid-dependent protein kinase system. Biochem. biophys. Res. Commun. 91: 1218-1224. TANG, Y.-M. and ASHENDEL,C. L. (1990) Isolation of cloned mouse protein kinase C beta-II cDNA and its sequence. Nucl. Acids Res. 18: 5310-5310. TODARO, G. J., DELARCO, J. E. and COHEN, S. (1976) Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells. Nature 264:26-3 I. TYERS, M., RACHUBINSKI,R. A., STEWART,M. I., VARICCHIO, A. M., SHORR, R. G. L., HASLAM,R. J. and HARLEY,C. B. (1988) Molecular cloning and expression of the major protein kinase C substrate in platelets. Nature 333: 470-473. VAN DONGEN, C. J., ZWIERS, H., DE GRAAN, P. N. E. and GISPEN, W. H. (1985) Modulation of the activity of purified phosphatidylinositol 4-phosphate kinase by phosphorylated and dephosphorylated B-50 protein. Biochem. biophys. Res. Commun. 128: 1219-1227. WALSH, J. P. and BELL, R. M. (1986) sn-l,2-Diacylglycerol kinase of Escherichia-Coli-structural and kinetic analysis of the lipid cofactor dependence. J. biol. Chem. 261: 5062-5068. WALTERS,J. D. and JOHNSON,J. D. (1990) Terbium as a luminescent probe of metal-binding sites in protein kinase C. J. biol. Chem. 265: 4223~,226.
JPT
51/1~
95
WISE, B. C., RAYNOR,R. L. and Kuo, J. F. (1982) Phospholipid-sensitive Ca 2+ -dependent protein kinase from heart. J. biol. Chem. 257: 8481-8488. WOLF,M., CUATRECASAS,P. and SAHYOUN,N. (1985) Interaction of protein kinase C with membranes is regulated by Ca 2+, phorbol esters and ATP. J. biol. Chem. 260: 15718-15722. WOLFMAN, A. and MACARA,I. G. (1987) Elevated levels of diacylglycerol and decreased phorbol ester sensitivity in ras-transformed fibroblasts. Nature 325: 359-361. WOODGETT, J. R. and HUNTER, T. (1987) Immunological evidence for two physiological forms of protein kinase C. Molec. cell. Biol. 7: 85-96. WOODGET J. R., HUNTER, T. and GOULD, K. L. (1987) Protein kinase C and its role in cell growth. In: Cell Membranes--Methods and Reviews, Vol. 3, pp. 215-340, ELSON, E., FRAZlER, W. and GLASER, L. (eds) Plenum Press, New York. Wu, W. C.-S., WALAAS,S. I., NAmN, A . C. and GKEENGARD, P. (1982) Calcium/phospholipid regulates phosphorylation of a Mr '87k' substrate protein in brain synaptosomes. Proc. natn. Acad. Sci. U.S.A. 79: 5248-5253. YOUNG, S., PARKER, P. J., ULLRICH, A. and STABEL, S. (1987) Down-regulation of protein kinase C is due to an increased rate of degradation. Biochem. J. 244: 775-779. YOUNG, S., ROTHBARD, J. and PARKER, P. (1988) A monoclonal antibody recognising the site of limited proteolysis of protein kinase C. Eur. J. Biochem. 173: 247-252. Yu, C.-L., TSAI, M.-H. and STACY,D. W. (1988) Cellular ras activity and phospholipid metabolism. Cell 52: 63-71. ZALEWSKI, P. n., FORBES,I. J., GIANNAKIS,C., COWLED, A. and BUTTS, W. H. (1990) Synergy between zinc and phorbol ester in translocation of protein kinase C to cytoskeleton. FEBS Lett. 273: 131-134.
0163-7258/91 $0.00+ 0.50 © 1991 PergamonPress pie
Printed in Great Britain.All rights reserved
Specialist Subject Editor: C. W. TAYLOR
PROTEIN KINASE C SILVIA STABEL* a n d PETER J. PARKER," * Max-Delbriick-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-Linn~- Weg 10, D- 5000 K61n 30, F.R.G. tlmperial Cancer Research Fund, Protein Phosphorylation Laboratory, Lincoln's Inn Fields, London WC2A 3PX, U.K.
A~tract--Based on the molecular structure of the individual members of the protein kinase C family, general properties and the mode of activation of this enzyme family are discussed. Examples are presented of how the investigation of protein kinase C function in vivo has been approached at the molecular level.
CONTENTS 1. Introduction 2. Structural Aspects of PKC Molecules 2.1. Organization of the PKC polypeptides 2.1.1. The pseudosubstrate prototope 2.1.2. The zinc fingers within the C, domain 2.1.3. The C2 domain and the kinase domain 2.2. Homologies to other proteins 2.3. PKC in lower eukaryotes 3. Functional Aspects of PKC In Vitro 3.1. General properties 3.2. Ca2+-dependencies 3.3. Diacylglycerol and phorbol ester responsiveness 3.4. Substrate specificities 4. Function and Regulation of PKC In Vivo 4.1. Sources of diacylglycerol 4.2. Activation, translocation and down-regulation of PKC 4.3. Analysis of the PKC signalling pathway Note Added in Proof Acknowledgements References
71 73 74 75 76 78 78 80 81 81 82 83 84 84 84 85 86 89 89 89
1977; Takai et aL, 1977). Soon after, however, it was realized that apart from proteolysis the enzyme could also be reversibly activated in vitro by unsaturated diacylglycerol (DAG) in the presence of acidic phospholipids such as phosphatidylserine (Takai et aL, 1979a,b). This observation provided the first possible link between receptor-induced phosphatidylinositol breakdown and protein phosphorylation (Kishimoto et al., 1980; Nishizuka, 1984). The activating effects of DAG on the enzyme can be mimicked by tumor-promoting phorbol esters in vitro (Castagna et al., 1982) suggesting that PKC plays some role in the process of neoplastic promotion of initiated tumor cells by phorbol esters (Asbendel, 1985; Kikkawa et al., 1983; Nishizuka, 1989). The binding of phorbol esters to the enzyme and the activation of the kinase activity of PKC by these compounds has been employed to purify PKC polypeptides from a number of sources (Ashendel et al., 1983; Kikkawa et al., 1982; Leach et al., 1983; Niedel et al., 1983; Parker et al., 1984; Wise et al.,
1. I N T R O D U C T I O N Today protein kinase C (PKC) covers a family of serine- and threonine-specific protein kinases which have been identified functionally by common enzymatic properties, like phorbol ester binding, phospholipid-dependent kinase activity or by common structural features (reviewed by Nishizuka, 1988; Parker et al., 1989; Stabel, 1990). In a growing number of cases cloned cDNAs are added to the PKC family according to the structural homologies they share with the prototypes of this kinase family, although the biochemical properties of the encoded protein products are not yet known. This is particularly true for PKC-type molecules isolated from lower eukaryotes as will be discussed below. The enzymatic activity of PKC was initially identified in rat brain by Nishizuka and coworkers as a novel type of cytoplasmic serine- and threoninespecific protein kinase which appeared to be proteolytically activated from a proenzyme (Inoue et al., 71
72
S. STABELand P. J. PARKER
1982). Based on peptide sequences from homogeneous purified protein, c D N A clones encoding P K C were isolated (Parker et al., 1986). During this process it became apparent that P K C is not specified by a single m R N A but that there exist multiple structurally highly related m R N A s in brain potentially coding for a family of enzymes with possibly identical or similar properties (Coussens et al., 1986; Housey et al., 1987; K n o p f et al., 1986; Makowske et al., 1986; O h n o et al., 1987; Ono et al., 1986b). Artificial expression of these first c D N A s in mammalian or insect expression systems proved that the c D N A s indeed all encoded proteins that were characterized by PKC-specific properties namely phorbol ester binding and calciumand phospholipid-dependent kinase activity which was activated by D A G or phorbol ester (Burns et al.,
1990; Kikkawa et al., 1987; K n o p f et al., 1986; Ono et al., 1987b; Patel and Stabel, 1989). By means of chromatography on hydroxyapatite and by means of antipeptide antibodies, polypeptides corresponding to the first isolated members of the P K C c D N A family have also been identified in vivo (Huang et al., 1986b, 1987; Jaken and Kiley, 1987; Kikkawa et al., 1987; Marais and Parker, 1989; Ono et al., 1987b; Sekiguchi et aL, 1988) proving that there exists a family of highly related P K C proteins which may all participate in the phenomena previously ascribed to a single P K C enzyme. In this review we will focus on the molecular and structural aspects of P K C and we will try to relate the molecular analysis of P K C to its proposed function and regulation in vivo.
TABLE 1. Cloned Mammalian Protein Kinase C Types Type*
Residues
MW:~
Source
Nomenclature~
Reference
alpha (~t)
672
76.8
bovine human rabbit rat mouse mouse human
ct ct ~ ~ ~ ct ~
Parker et al. (1986) Coussens et al. (1986) Ohno et al. (1987) Ono et al. (1988b) Rose-John et al. (1988) Megidish and Mazurek (1989) Finkenzeller et al. (1990)
betaI (ill)
671
76.8
rat rat human rabbit rat human
III I f12 fl RP41 fl
Knopf et al. (1986) Ono et al. (1986a) Coussens et al. (1987) Ohno et al. (1987) Housey et al. (1987) Kubo et al. (1987a,b)
betalI (fill)
673
76.9
bovine rat rat human rabbit human mouse
fl II II fl~ ~ fl fl
Coussens et al. (1986) Knopf et al. (1986) Ono et al. (1986a) Coussens et al. (1987) Ohno et al. (1987) Kubo et al. (1987a,b) Tang and Ashendel (1990)
gamma (~,)
697
78.4
bovine human rat rabbit rat
7 7 I 6 7
Coussens et al. (1986) Coussens et al. (1986) Knopf et al. (1986) Ohno et al. (1988b) Ono et al. (1988b)
delta O)
673
77.5
rat
6
Ono et al. (1987a, 1988a)
epsilon (E)
737
83.5
rat rabbit rat mouse
E n RP16 E
Ono et al. (1987a, 1988a) Ohno et al. (1988a) Housey et al. (1987) Schaap et al. (1989)
epsilon' (E')
497
56.1
rat
E'
Ono et al. (1988a)
zeta (()
592
67.7
rat
(
Ono et al. (1987a, 1988a, 1989a)
eta (q)
683 6801b
80 77.6
mouse human
q L
Osada et al. (1990) Bacher et al. (1991)
*Grouping into types according to homology of predicted amino acid sequence. tNumber of amino acid residues of the predicted proteins. :~Calculated molecular weight of the predicted protein. §Nomenclature used in the respective reference. liThe formal possibility that this is an alternative splice product of the same gene is discussed in the text.
Protein kinase C 2. S T R U C T U R A L ASPECTS OF PKC MOLECULES The PKC c D N A clones isolated first and the corresponding polypeptides are now called ~, fl~, f12 and y types, isoforms, isoenzymes or subspecies of PKC. Table 1 lists the origin of the mammalian PKC cDNAs isolated to date and their assignment to the different subspecies of PKC. Due to their structural organization PKC ~, ill, f12 and y define the 'group A' PKC enzymes in contrast to 'group B' enzymes 6, E, (, and ~/ which are distinguished from the former ones on the basis of their structure and enzymatic properties (see below). The genes for ~, fl and ~, have been localized on different human chromosomes: ~ is encoded on chromosome 17, fl on 16 and y on chromosome 19 (Coussens et al., 1986). The fll and f12 mRNAs are generated by alternative splicing of the most C-terminal exon of the fl-gene producing two polypeptides which only differ in the sequence of their 50 most C-terminal amino acids (Coussens et al., 1987; Kubo et al., 1987a; Ono et al., 1986a). Differential expression of both fl-mRNAs, fll and /~2, has been shown in hematopoietic cell lines and in brain tissue (Coussens et al., 1987) and use of specific antibodies has shown that both fl polypeptides are indeed expressed in brain tissue in vivo (Hosoda et al., 1989; Saito et al., 1989) thus raising the tantalizing question of whether there will be specific cellular processes to be discovered which are catalyzed by two proteins which only differ in their 50 C-terminal amino acids or whether the two PKCfl polypeptides are the result of some evolutionary redundancy. An analogous situation may turn out to apply for the E type of PKC where two different cDNA clones have been isolated which differ in their 5'-sequence potentially giving rise to two polypeptides, one of 737 amino acids and a predicted molecular weight of 83.474 kDa, termed e, and one of 497 amino acids and a theoretical molecular weight of 56.059 kDa, which for consistency we will term E2 (Housey et al., 1987; Ono et al., 1988a). PKC E2 differs from E by the absence of the 240 N-terminal amino acids of E and substitution of this sequence by a sequence carrying a stop codon in front of methionine 241 of PKC E. Translation of this m R N A would give rise to an N-terminally truncated PKC E2 product, starting at methionine 241 of E (Ono et al., 1988a). Preliminary evidence suggests that these two potential protein products of the E-gene may be generated by alternative splicing (Ono et al., 1988a). If indeed expressed as a protein product in vivo, E2would only contain the second zinc finger of the full-length E protein product (cf. Fig. 1A). Another potential case for alternative splicing within the PKC family might be represented by the subspecies. The ( c D N A encodes a polypeptide of 592 amino acids and a predicted molecular weight of 67.740 kDa. Another c D N A clone lacking the first 112 amino acids of the body of the ( polypeptide was repeatedly isolated but the biological significance of these m R N A s is unclear (Ono et al., 1988a; Liyanage and Stabel, submitted). As far as the genomic organization of the PKC genes is concerned the exon/intron structure of a
73
PKC gene has only been elucidated for one of the three so far identified PKC species in Drosophila, dPKC (Rosenthal et al., 1987b). The dPKC gene spans about 20 kb and consists of 13 coding exons and at least one untranslated exon at its 5'-end. It has been noted that the functional domains of the dPKC polypeptide are not reflected in the exon/intron structure of the gene as the second zinc finger structure and the ATP binding domain are disrupted by introns (Rosenthal et al., 1987b). Partial genomic mapping of the human fl gene shows identical intron/exon organization to that of dPKC (Coussens et al., 1987; Haley, King and Parker, in preparation). The regulation of tissue-specific expression of the PKC enzymes presents another puzzling problem. The PKC~ enzyme for instance is exclusively expressed in brain and spinal cord (Nishizuka, 1988) whereas most of the other types occur in brain as well as in other tissues. This regulation obviously occurs at the transcriptional level and so far nothing is known about the factors governing expression of these enzymes. The promoter region of the rat and the mouse PKC y gene has been isolated (Chen et al., 1990; Leitges, Proikas and Stabel, manuscript in preparation) and it shows features characteristic of many housekeeping genes like the absence of TATA or CAAT boxes from their usual positions, but in contrast to a number of such genes it seems to have only one major transcriptional start site located 243 bp upstream of the ATG. Although consensus sequences for several trans-activating factors like AP2, Spl and CREB have been identified in the 5'-flanking region of the PKC Y gene, the mechanism by which brain-specific expression of this isozyme is achieved is still unclear. Genomic sequence data so far available, suggest that the 3'-boundary of the first exon of this gene and the 5'and 3'-boundary of the second exon are identical between Drosophila and the rat/mouse PKC y gene (Chen et al., 1990; Leitges, Proikas and Stabel, manuscript in preparation). Soon after the discovery of the three closely related cDNAs coding for PKC ct, fl, and y, additional members of the PKC family, PKC 6, e, and ~, were identified by low stringency screening of brain cDNA libraries (Ohno et al., 1988a; Ono et al., 1987a, 1988a; Schaap et al., 1989). The chromosomal location of the respective genes has not been determined yet, although it is evident from their sequences that they too are products of distinct genes. Like the ct, fl, group of PKC enzymes the members 6, E, ( are highly expressed in brain and in additional tissues (Nishizuka, 1988). The preferential isolation of PKC members highly expressed in brain has been due to the fact that most of the approaches chose brain cDNA libraries as a source for screening. Screening of two epidermis cDNA libraries under reduced stringency conditions with ct, fl, y, E, and ( probes identified yet another PKC isoenzyme, PKCr/ or PKC-L, with a tissue distribution distinct from the thus far identified enzymes. The m R N A for this PKC is strongly expressed in skin and lung and only weakly in brain (Bacher et al., 1991; Osada et al., 1990). These findings indicate the possibility that the family of PKC polypeptides might still increase in
74
S. STABELand P. J. PARKER
A
REGULATORY
CATALYTIC
!Pseudo6ubetrate alpha beta 1/11 gamma
672 671/673 897 m
_ _
apsllon
m
.
zeta
..... J
delta
m
.
.
.
.
673
.~1
737
~
592
eta
epsilon" ATP binding site REGULATORY
B
CATALYTIC |¢
rat
I alpha beta VII gamma
VI
Cl
V2
C2
V3
i
C3 v
C4
V5
672 671/673 697 676
I X-PKC I Xenopus L X-PKC II
671
I 53E(ey)
700
°-[7
639
C.ele0art~
tpa-1 *
526"
yeast
PKC1
634
1151
¢ ATP binding site
FIG. 1. Domain structure of PKC molecules from mammals (A) and lower eukaryotes (B). Conserved (c) and variable (v) domains are indicated by C1-4 and V1-5, respectively. Patterned boxes within the C1 domain represent location of the conserved zinc finger motif CX2CXI3/14CX2CXTCX7C.Numbers on the right give amino acids of the polypeptide predicted from the cDNA sequences. See text for details and Tables 1 and 2 for references. Epsilon' is a truncated version of the , cDNA which may represent an alternatively spliced product of the E gene (Ono et al., 1988a). number through the identification of members which are preferentially or exclusively expressed in other tissues than the ones used for screening so far. The obvious question arising from these observations is whether each of these different distinct enzymes plays a specific role in cell type-specific processes like endocytosis, secretion, transmission of electric potentials, growth or differentiation. It is also not clear why some types like ct or ~/ are apparently expressed ubiquitously albeit at low levels, with very high expression only in certain cell types. It is conceivable that some types, e.g. the ubiquitously expressed type ~, may serve some very general functions whereas other types fulfil more specialized cell type-specific tasks.
2.1. ORGANIZATIONOF THE P K C POLYPEPTIDES
Comparison of the amino acid sequences of PKCs predicted from the cDNAs highlighted the presence of regions with a high degree of homology between PKC types; the conserved regions C1 to C4, interspersed with regions of lower homology variable regions VI to V5 (Coussens et al., 1986). Figure 1A shows a schematic representation of the structural organization of the putative mammalian PKC polypeptides for which cDNA sequence data is available. It has been speculated that the conserved regions represent areas of the molecule involved in functions common to all PKC enzymes which is obviously true
Protein kinase C for the kinase domain. The less well conserved, variable blocks of sequences inbetween the conserved regions may either represent spacer sequences that allow for flexibility of the polypeptides or domains responsible for specific properties of the enzymes (Carpenter et al., 1987). To date there is no evidence to support the idea that the V-regions are important for the biochemical characteristics of a particular enzyme. Figure 1B compares the structural features of the mammalian enzymes to those of putative PKC molecules identified in lower eukaryotes which are discussed in more detail below. The N-terminal (approximately) half of the polypeptide represents the regulatory domain since it carries binding sites for the activator phorbol esters (Huang et al., 1986b; Kaibuchi et al., 1989; Lee and Bell, 1986; Ono et al., 1989b) and a pseudosubstrate prototope. The removal of this domain by mild proteolysis leads to constitutive and activator-independent kinase activity of the enzyme (Inoue et al., 1977; Mochly-Rosen et al., 1990; Parker et al., 1986). The kinase domain is located in the C-terminal part of the polypeptide. All members of the PKC family identified to date carry at least one zinc-finger structure (in most cases two) in the N-terminal regulatory half of the molecule (see Fig. 1A). Upstream of the finger structures, at a fixed distance from the first finger lies a block of amino acids exhibiting the features of a pseudosubstrate prototope supposed to be essential for regulating the kinase activity (see below). The C1 domain is preceded by an N-terminal extension of varying length with the types E and ~/ having the longest extensions among the mammalian PKCs identified so far. The most recently identified type r/is most closely related to PKC E as reflected in homologies between the conserved domains: homology in the C1 region is 48% with ~t, 48.6% with fl, 48% with ~,, 54.2% with 6, 34.9% with ( and 74.8% with E. Also the kinase domain is most homologous to PKC E with 74.4% identity, whereas homology to the other types in this domain is around 60% (Osada et aL, 1990). Remarkably, the degree of homology in the N-terminal variable extensions of PKC r/and PKC E is also rather high at 53.3% (Osada et al., 1990). A cDNA encoding PKC-L would appear to be the human homolog of PKC r/ (Bacher et al., 1991). However in PKC-L the second cysteine repeat is interrupted and the sequences of PKC-L and r/ are divergent between residues 278 and 310. Interestingly the divergence starts at the junction of exons 4 and 5 in the dPKC gene. Whether this difference in fact reflects alternative splicing or a frameshift incurred during c D N A isolation has yet to be determined. In the following sections the functionally relevant structures and regions of the PKC molecules will be discussed in more detail. 2.1.1. The Pseudosubstrate Prototope The activity of a number of protein kinases has been recognized to be regulated or controlled by the presence of so-called pseudosubstrate prototopes which occur as autoinhibitory domains or sites in the kinase molecule or associated regulatory subunits
75
(reviewed in Hardie, 1988; Kemp et al., 1989; Kennelly et al., 1987; Soderling, 1990). The pseudosubstrate prototope contains a consensus phosphorylation sequence for the respective kinase but no phosphorylatable residue and is thought to control kinase activity by blocking access of the substrate through occupation of the catalytic site. The sequence of a number of in vitro substrates of PKC has been determined and the use of synthetic peptide substrates has indicated that PKC requires basic residues surrounding the target serine or threonine (House et al., 1987; Marais et al., 1990), a feature common to a number of serine-/threonine-specific protein kinases. A sequence fulfilling the requirements for a PKC phosphorylation site without a phosphorylatable residue (alanine instead of serine) was first identified in the 'group A' PKCs (House et al., 1987). Inclusion of a synthetic peptide of this sequence in an in vitro kinase assay either using a mixture of PKC enzymes or individual PKC types inhibits the activation of PKC in vitro in a competitive manner, whereas the same peptide carrying a serine in place of the alanine behaves as a good substrate for PKC (House et al., 1987; Pears et al., 1990; Schaap et al., 1989). From these data a molecular model has been derived where the pseudosubstrate sequence blocks the catalytic site of PKC. Upon binding of activators to the regulatory domain a conformational change may release the pseudosubstrate sequence from the catalytic site, allowing access of the substrate (Parker et al., 1989). An analogous stretch of amino acids is found in all PKC molecules suggesting that the basic sequence requirements for phosphorylation by PKC subtypes are similar. Figure 2 shows a compilation of the putative pseudosubstrate sequences for all cloned PKC types. Common to these sequences is the basic, positively charged environment of the pseudosubstrate residue alanine, although different PKC types show variations in the number and spacing of basic residues surrounding the alanine, e.g. in PKC r/(and PKC-L) the alanine is immediately preceded by a basic residue (arginine) whereas in all other enzymes the alanine is preceded by a glycine (Fig. 2). This feature may hint to unique substrate requirements of PKC r/which shows a different tissue distribution in comparison with the rest of the family. These variations in the pseudosubstrate sequences may reflect subtle differences in target sequence requirements for phosphorylation by the different PKC enzymes in vivo which may be difficult to assess in vitro by the use of synthetic substrate peptides (see Marais et al., 1990). The pseudosubstrate prototope is followed by the zinc finger structures with the distance between the alanine residue and the first finger being highly conserved in all PKCs, this distance is 24 amino acids for PKC ct, fl, y, 6, and ~, and 23 amino acids for PKC E and r/. Although studies on T lymphocytes, fibroblasts and sea urchin eggs have successfully employed peptides corresponding to the pseudosubstrate prototope sequence to inhibit PKC activation in vivo (Alexander et al., 1989; Eicholtz et al., 1990; Shen and Buck, 1990) the use of such peptides as specific inhibitors has been questioned since the activity of cAMP-
76
S. STABELand P, J. PARKER yPKCI tpa-I
dPKC53E(br) dPKC53E(ey) dPKC98F XPKCI XPKCII rat alpha beta gsmm. delta epsilon
zeta mouse
eta
395 ? 28 54 55 24 16 19 19 18 141 153 113 155
G I S N F I% R R R L T P S T
L H I% H Q R R R R R N F F F F F M R I R
Y. L R A A A A C N K Y K
R R R R • R R R R R R R
K K R K K
K K K R Q R Q
~!xxxaxR G G G G G G G G G G R
A L K A M K A M R A L R A L R A L R A L R A L R A I K A V R ARRW ~ M R
K R R Q Q Q Q Q Q R
K K R K K K K K A R
N G N N N N V K RK R R -
s s z r s Q VHEZRG VFNVKD L E M V N G V H Q V N G V H E V K N V H E V K E V H E V K D V H E V K N V H E V K S I H Y I K N V H Q V N G L Y RAN G VH QVN G
FIG. 2. Pseudosubstrate prototope sequences from PKC cDNAs. Alignment of amino acid sequences of PKC pseudosubstrate prototopes from yeast (yPKC1; Levin et al., 1990), C. elegans (tpa-l; Tabuse et al., 1989), three different Drosophila enzymes (dPKC; Rosenthal et al., 1987b; Schaeffer et al., 1989), two Xenopus enzymes (XPKC; Chen et al., 1989) and mammalian species (see Table 1 for references). Highly conserved basic residues are marked by an asterisk, the serine to alanine substitution is boxed.
dependent kinase is also affected by peptides mimicking the pseudosubstrate site of PKC (Smith et al., 1990; Soderling, 1990). The reason for this cross-reactivity lies probably in the fact that the primary sequence specificity between a number of kinases appears to be rather similar, i.e. basic residues Nterminal to the substrate serine. The validity of the molecular model of PKC activation, however, whereby the pseudosubstrate prototope inhibits kinase function in the intact molecule has been supported by the observation that PKC can be activated in vitro by an antibody directed against the pseudosubstrate prototype (Makowske and Rosen, 1989) and by site-directed mutagenesis of the pseudosubstrate prototope (Pears et al., 1990). 2.1.2. The Zinc Fingers within the C~ Domain Zinc-binding finger structures have first been identified in the Xenopus transcription factor T F I I I A where they are required for sequence-specific binding of the factor to D N A (Berg, 1990). Subsequently, the finger motif was discovered in a number of other proteins mostly but not exclusively nucleic acid binding proteins. Although for the majority of these proteins with zinc-finger structures direct interaction with D N A or R N A has been reported, this primary sequence motif with its resulting secondary structure is also certainly used for protein-protein interactions as suggested by the dimerization and cooperative binding of the bacteriophage T4 helix-destabilizing protein (gene 32) (Pan et al., 1989) and the adenovirus E1A protein which does not bind directly to D N A but mediates transcriptional induction of viral and cellular genes probably by interacting with other proteins (Chatterjee et al., 1988). All members of the PKC family carry two so-called zinc-fingers within the C 1 domain, except the ( type which only has one such structure (cf. Fig. 1A). The zinc-finger sequence in the PKC molecules would be of the 'Cys-Cys' type as represented by the steroid receptor superfamily and the adenovirus E1A protein. The 'Cysq2ys' type differs from other zinc-binding fingers in that coordination of the zinc ion would solely be effected by cysteine residues and not by a
combination of cysteine and histidine residues (Berg, 1990). While the zinc-binding structure itself as identified in the steroid receptors and the E1A protein is only made up of four cysteine residues corresponding to cysteine number 1-4 in Fig. 3 it is worth noting that the finger structure in all PKC molecules is followed by two additional cysteines the spacing of which is also highly conserved (cysteines 5 and 6 in Fig. 3). Thus this structure in PKC can be viewed as an 'extended' zinc-finger structure which shows a spacing of cysteines identical to the steroid receptors and the E1A protein but extended by two cysteines of unknown function. The fact that this type of 'extended' finger is also found in unrelated molecules like D A G kinase (Sakane et al., 1990; Schaap et al., 1990), a phorbol ester-binding protein n-chimaerin (Hall et al., 1990) and the Raf family of protein kinases (Rapp et al., 1988), suggests that either the zinc-finger structure per se (Cysl-Cys4) can serve a different function in the context of the PKC motif Cys-X2-Cys-Xla-Cys-X2-Cys-X7-CyS-XT-Cys, where X is any amino acid, or that the PKC motif combines two structural functions; the first four cysteins coordinate zinc like the analogous structures in other proteins and the fifth and sixth cysteine serve some unknown function. The spacing of cysteines within each of the two repeats is highly conserved and follows the pattern Cys-X2-Cys-Xl3-Cys-X2-Cys for both fingers in PKC ct, fl, ~, 3, ( and the second finger of E and C y s - X : ~ y s - X 1 4 - C y s - X 2 ~ y s for the first finger in E, r/ and PKC-L (Fig. 3). Embedded in the extended finger structure is a highly conserved amino acid triplet VHK, I H K or VHR, which has been suggested to be involved in interactions with phospholipid since this triplet is also found in the catalytic site of phospholipase A2 (see below and Maraganore, 1987). The VHK motif follows all PKC finger structures three amino acids downstream of the last cysteine of the finger (Fig. 3); it is absent from the incomplete second finger in PKC-L. Although bound zinc has been directly demonstrated by atomic absorption spectroscopy or nuclear magnetic resonance for many zinc-finger-containing proteins either of the 'Cys-His' or the 'Cys-Cys' type
Protein kinaseC 2
1
A
t,, c PKC PKC PKC PKC PKC • PKC X.i. X.i. dPKC dPKC dPKC C.e.
alpha beta gm.~R delta epsilon eta zeta PKCI PKCII 98F 5 3 E (ey) 53E (br) tpa-i
yPKCl DAG kinase • n-chimaerin • c-raf • A-raf • B-raf
77 3
C 0. C •. C .V C .. C •. C GQ C .. C .o C .. C G. C .. C .L C AY C NL C EY C DI C DF C DF
C
C C C C C C C
C C CK C C C C C C C
. . . . . .
- . o 0
. . . .
...... -I .... L. KE.V..-LN...YK R ..... VI .... Y. RE .... V ..... Y. SER...LA-R..YR ...... -L ...... ......
-..°....
R E .... I - . . . . Y . K ..... - ....... ..... - ....... S..M..-LN...YQ G..LR .... YT... E S S I - . - L .... L S AN.M..LI-A..VK QKFL ..... LN..R LKFL ..... FH..R RKFL ..... FQ..R
C C C C C C C C C C C C C C C C C
c .T~C *
m
PKC alpha PKC beta P K C g....1 PKC delta PKC epsilon PKC eta ePKC zeta X. 1. P K C I X.i. PKCII dPKC 98F d P K C 5 3 E (ey) d P K C 5 3 E (b=) C.e. tpa-i yPKCI DAG kinase • n-chimaerin • c-raf • A-tar aB-raf
~DH C .. C C .. C C .. C C .. C C .. C CGQC C .. C C .. C C .. C C .E C C .. C C .. C CC. C C .R C C .. C C .IC C .F C
c .FC
GSLLYGLI-
HQGMK
C
o..oo...-..o°o
C
. . . . . . . V - . .... .T..W..V-K..L. .... W . . L - R . . L Q .... W.IM-R..LQ SERIW..A-R..YR ...... M.- .....
C C C C C C
.....
o°o-...o.
........
-K.
.L..H.VA-...V. ...... IY-...L. . .M .... F-K..LR .YI.PW.R-. KVR. QKKIRIYHSLV. LH ANFI~4...-A..V. QKFL ..... LN. FR LKFL ..... FH.FR RKFL ..... F..FR
DT .. .. .. KV KI IN E. E. E. EN
6
rsl e7
Cl C S .... R.I C ........ Cl N A A I . . K I C I D K I I G R ICl T C . . . . . IC . . L I I T K I Cl T C . . . . . 1C .H L I V T A ICl K L L .... iC . V L . P L T ICI Cl ,.°o... ,C Cl .o..... ,C T L .... KI C . L S . V S K I Cl R . N Z .QKI C C K . . V . K I Cl S Y . . . . . IC . . . . . . . . Cl S A A . . . K I C . .K.IblQI Cl K . L C . . K I C Y T N . V T K ICl K Y T . . DQI C A M K - A L P ICl GLN.. -QI C S X M . P N D ICl G Y K F . EH! C S T K . P T M ICl GYKF. Q"I C S S K . P T V ICl GYKF. QR C STE.PLMI c l *
U C I D~WUKQ CIM ..... R CIE .... RR C l G .... H K C l K .... R R C l K .... I R CIKLL...R Cl...I..V CIM ..... R ClG ..... R ClNL...HA
-C C C C C C C C C C C
m
V I N V P S L !C .M ..... C .RS .... C REK. AN. C ET..APN C OA..APN C HVL..LT C o.o.ooo
.M ..... QK..ANT C QET.. PM C AR C K E . . . . . C SA Cl . . . . . C INVAC. BK C E R L M S N . C C S ~ Cl G I M C . A Q C AHL.. DF C LP~gHE C VN ClHLEI.DD C A D C I G L . . . . . C S l ~ I . . ND C S T K . . TM C Q. C l G Y K F . E H C SSK..TV C Q. C I G Y K F . Q H C_ s T ¢ . . ~ C Q. C l G Y K F . Q R C
°LQ
E
...C ...C RQIC ...C ...C INIC ...C ...C .. C EEIC C .LIC .DIC NLIC ADIC .T',C .TIC
*
B
5
4
Qvl
SH C TDFIWG-FGKQGFQ
C
C C C C C C
C C C C
_c *
FIG. 3. The 'extended' zinc-finger structures of PKC and unrelated proteins. Alignment of the first (A) and second (B) zinc finger structures of PKC sequences and unrelated enzymes is shown as indicated. Molecules that contain only one finger structure are marked with a filled circle. In these cases this one finger sequence is aligned with both the first (A) and the second (B) fingers of molecules which contain two zinc-finger structures. Conserved cysteine residues are numbered from 1 to 6. Identical residues in all sequences are marked by an asterisk. Dots represent amino acids identical to the top sequence of PKC ~, and dashes represent gaps introduced to allow for better alignment. The ValHisLys (VHK) triplet is marked by a shaded box.
(Berg, 1990), it is not known whether the PKC proteins contain zinc. Effects of zinc, however, on the activity of PKC have been reported (Csermely et al., 1988; Speizer et al., 1989; Waiters and Johnson, 1990). In addition, phorbol ester binding to a novel phorbol ester binding protein (n-chimaerin) which shares the extended zinc-finger structure with the PKC family (see Fig. 3 and below) has been shown to be dependent on zinc (Ahmed et al., 1990). Unlike the case of the steroid receptors there is no evidence that the zinc-finger in PKC can mediate specific or even nonspecific DNA binding, rather most of the evidence available to date points to a role of this structure in phospholipid-dependent phorbol ester binding (Ono et al., 1989b). Studies addressing the function of the C t domain in PKC have so far used deletion mutagenesis and substitution of indi-
vidual cysteine residues in the zinc finger. Expression of the C1 domain of PKC 7 in E. coli yielded a polypeptide able to bind phorbol ester in a phospholipid-dependent manner (Ono et al., 1989b). Deletion of either of the units containing the conserved six cysteine residues does not interfere with the binding of phorbol ester by the E. coli cell extract, whereas mutation of the third and fourth cysteine residue of either finger abolishes binding (Ono et al., 1989b). These studies show that one of the zinc-finger-containing units is sufficient to confer phospholipid-dependent phorbol ester binding to the C1 domain of PKC 7 expressed in E. coli. Studies using deletion mutants of PKC ct and PKC fl cDNA and hybrids between the regulatory domain of cAMP-dependent protein kinase (PKA) and the catalytic domain of PKC and vice versa came to
78
S. STABELand P. J. PARKER
slightly different conclusions after transient expression of the cDNAs in mammalian COS cells. The C 1 domain of the PKC polypeptide is necessary and sufficient for phorbol ester binding to the transfected cells. However, mutants of PKC a lacking the second zinc finger did not bind phorbol ester (Kaibuchi et aL, 1989). By contrast deletion of the first PKC 7 finger does not abolish phorbol ester binding (Cazaubon et al., 1990). Surprisingly, deletion of only 30 amino acids near the N-terminus including the pseudosubstrate prototope also abolished phorboi ester binding. However, it was argued that in this case this might constitute an indirect effect due to an altered conformation of the mutant polypeptide (Kaibuchi et al., 1989). Deletion of the C2 domain had no effect on phorbol ester binding on whole cells compared with wild-type PKC ~. Mutations which remove the kinase domain of PKC ~t ( P K C - P K A hybrids) or PKC fl increased the binding capacity about two- to three-fold (Kaibuchi et al., 1989; Muramatsu et al., 1989). Enhanced phorbol ester binding has also been noted in kinasenegative mutants of PKC a and PKC ~, carrying a mutation in the ATP-binding site (Freisewinkel et al., 1991; Ohno et al., 1990). So it appears that the 'extended' zinc-finger structure in the PKC molecules through binding of the lipid activators is somehow involved in the activation process by binding of phospholipid and phorbol ester activators and probably thereby mediating a conformational change that activates the kinase function. That such changes occur is evident from the loss of monoclonal antibody binding (Cazaubon et al., 1990). How exactly binding of the lipid activators is mediated by this structure is not known and it will be of interest to determine whether and which common features can be identified which justify the similarity between structures that mediate binding of nucleic acids and phospholipids/phorbol esters. 2.1.3. The C2 Domain and the Kinase Domain The C 2 region is defined by its presence as a conserved region in all four 'group A' PKC members ~,/~, 32, and ~ and is part of the regulatory domain of these enzymes. The C2 domain is missing in the 'group B' PKC members b, E, ~ and r/(Fig. 1A). After transient expression of the respective cDNAs in mammalian or insect cells 'B-group' members of PKC apparently do not require Ca z + for either autophosphorylation in vitro (Bacher et aL, 1991) or phosphorylation of histone (Ono et al., 1988a) or peptide substrates (Schaap and Parker, 1990; Schaap et al., 1989). Also binding of phorbol ester and phorbol ester-induced translocation of the E and & enzymes to the particulate fraction is independent of Ca 2÷ (Akita et al., 1990; Kiley et al., 1990; Olivier and Parker, 1991). This behavior is in contrast to the 'A-group' enzymes ~t,/~ and 7, which all require Ca z ÷ for their kinase activity as well as for phorbol ester binding and translocation (Burns et al., 1990; Huang et al., 1988; Kiley et al., 1990; Marais and Parker, 1989; Sekiguchi et al., 1988). Therefore it seems likely that the C 2 domain provides for the Ca 2÷-dependence of the enzymatic and lipid-interacting properties of the
'group A' enzymes. However none of the known Ca2+-binding motifs has been identified in this domain. A sequence resembling a potential Ca 2+binding site of the 'EF hand' type has been noted in PKC ~ just downstream of the C 2 domain (Parker et al., 1986). A similar array of amino acids is found in PKC fl but not in PKC 7. The absence of this sequence in PKC ~ raises the question about potential Ca 2+ binding structures in this enzyme since all three types ct, fl and 7 have been shown to be dependent on Ca 2÷ in vitro. Mutagenesis experiments support a function of the C2 domain in mediating Ca 2÷-dependence since deletion of this region renders the kinase activity as well as phorbol ester binding activity independent of Ca 2÷ (Kaibuchi et al., 1989; Ono et al., 1989b) whereas expression of the C2 d o m a i n p e r se in E. coli does not support phorbol ester binding (Ono et al., 1989b). Early studies showed that the PKC polypeptide can be cleaved in vitro by trypsin or calpain into two fragments, one exhibiting phorbol ester binding and the other activator-independent kinase activity (Inoue et al., 1977; Lee and Bell, 1986; Parker et al., 1986). Obviously the kinase domain of PKC is believed to play a crucial role in mediating the effects of phorbol esters as well as D A G on cellular processes by catalyzing the phosphorylation of substrates. Where and in which context the kinase function of the various PKC enzymes becomes effective may be subject to a variety of intracellular parameters like the intracellular Ca 2+ level, the type and temporal features of D A G production, the subcellular localization and substrate specificity and availability of the particular PKC. In addition, autophosphorylation of PKC has been well documented (Flint et al., 1990; Huang et al., 1986a; Mitchell et al., 1989) although the effects of autophosphorylation on the enzymatic properties of PKC are not clear yet. Autophosphorylation has been claimed to decrease the affinity of PKC for Ca 2+ and increase its affinity for phorbol ester (Huang et al., 1986a). On the other hand, engineered PKC mutants either completely lacking the kinase domain or inactivated by a point mutation in the ATP-binding site also show increased phorbol ester binding suggesting in contrast to the above study that the affinity for these agents is negatively modulated by autophosphorylation (Freisewinkel et al., 1991; Kaibuchi et al., 1989; Ohno et al., 1990). 2.2. HOMOLOGIESTO OTHER PROTEINS The presence of conserved domains in the PKC molecules suggests that some of them at least could serve some general functions which might be reflected in similar structures found in these domains and in other proteins. This is obviously true for the kinase domain which shows a number of features found in all kinases and in particular in serine/threoninespecific kinases (Hanks et al., 1988). Additionally the conserved clusters in the N-terminal regulatory domain exhibit features apparently shared by other proteins. The C~ domain containing a putative zinc-finger structure has been discussed in detail above and indeed this structure has attracted a lot of interest because of its well investigated function
Protein kinase C in other proteins which includes nucleic acid binding and protein-protein interactions (see Berg, 1990; Evans and Hollenberg, 1988). The first part of the cysteine-rich structure of PKC is shared by the steroid receptor superfamily and the adenovirus E1A protein and follows the pattern CyS-XE~Cys-Xl3/14Cys-Xz42ys. More extensive homology, however, spanning the whole pattern of cysteine spacing C y s - X 2 ~ y s - X 13/i4~ys-X2~ys-XT-Cys-X7-Cys is found with individual proteins like the c-Raf kinase family (Ishikawa et al., 1986), the diacylglycerol kinase (Sakane et al., 1990; Schaap et al., 1990) and n-chimaerin, a brain-specific sequence of unknown function (Hall et al., 1990). Figure 3 shows an alignment of the cysteine-rich structures of PKC and these unrelated proteins. The presence of a zinc-finger pattern in the N-terminal presumed regulatory region of the c-Raf kinase family (Rapp et al., 1988) has led to the suggestion that by analogy to PKC this structure in c-Raf could also be involved in regulation of the kinase activity of c-Raf by soluble second messengers (Li et al., 1991). How a regulation via this domain of the c-Raf protein could be effected is unclear; this structure does not mediate phorbol ester binding in the c-Raf polypeptide (Srzeri and Stabel, unpublished). Diacylglycerol (DAG) kinase represents another protein with two zinc-fingers where this structure could be associated with lipid binding. DAG kinase contains two zinc-finger structures of which the second one has the same spacing of cysteine residues as the corresponding structure in PKC (Sakane et al., 1990; Schaap et aL, 1990). Within the first finger of D A G kinase is embedded the amino acid pattern characteristic for an ATP binding site, Gly-X-GlyX-X-Gly-XI4-Lys (Hanks et al., 1988), which might reflect the fact that this protein is a lipid kinase, although the significance of this 'double feature' is not clear and it is not known whether this pattern indeed represents an ATP binding site in this case. At a similar location in the loop of the first but not the second finger in PKC ~, fl and 7 the sequence Gly-X~31y-X-X-Gly is also present, however, this is not followed by a lysine indicative for an ATP binding site, suggesting that the glycines in the finger loop might in both cases serve some function other than nucleotide binding. The idea that the zinc-finger structure of PKC might mediate lipid binding had been voiced earlier from sequence comparison with another lipid metabolizing enzyme phospholipase A 2 (Maraganore, 1987; Stabel, 1990). Pancreatic phospholipase A 2 catalyzes the release of fatty acids from the sn2 position of 1,2-diacyl-sn-glycero-phosphocholines and the active site residues are highly conserved between mammalian and snake venom phospholipases (Seilhamer et al., 1986). His48 in the active site is believed to be involved in the catalytic mechanism and Lys49 or Asp49 have been shown to function in the formation of the catalytic complex (Maraganore and Heinrikson, 1986). At the appropriate position relative to *Interestingly this same region of the bcr protein is also conserved in two regulatory subunits of the type I phosphatidylinositol kinase (Otsu et al., 1991); the function in this context remains unknown. JPT 51/I--F
79
conserved cysteine residues in PKC and in nchimaerin (see below) the pair His48Lys49 can be found, whereas DAG kinase carries His-Asp at these positions. The most intriguing and perhaps significant homology found between PKC and other proteins is the one to the recently identified brain-specific product n-chimaerin (Ahmed et al., 1990; Hall et al., 1990). n-Chimaerin was identified as a relatively abundant brain-specific mRNA encoding a 34kDa protein product of unknown function which shows a high sequence homology in its N-terminal part to the C 1 region of PKC and in its C-terminal part to bcr, the 'breakpoint cluster region' involved in Philadelphia chromosome translocation which encodes a 160 kDa protein of unknown function (Hall et al., 1990).* The homology between the N-terminal region of n-chimaerin and the C1 region of PKC is about 50% and is the highest homology found for this region to a protein outside the PKC family. Expression of the N-terminal part of n-chimaerin in bacteria proved that this structure is able to bind phorbol esters in the absence of Ca 2+ which supports the idea that the C2 and not the C1 region of PKC mediates Ca 2+ dependence (see above). Phorbol ester binding by nchimaerin is dependent on phosphatidylserine; other phospholipids like phosphatidylinositol, phosphatidylcholine or phosphatidylethanolamine were not effective in supporting binding (Ahmed et al., 1990). Also, bacterially expressed protein which was denatured and refolded in the absence of zinc ions did not bind phorbol ester suggesting that phorbol ester binding requires a zinc-dependent structure. Besides the novelty of identifying a phorbol ester-binding sequence outside the PKC family this finding has significant implications for all studies using phorbol esters to probe the role of PKC. Worth noting from these studies is that apparently only some fingers of the general structure Cys-X2-Cys-X13/14-Cys-X2Cys-XT-Cys-X7-Cys can support phorbol ester binding and that this property might be determined by specific amino acids within this structure which could be identified by gene technology methods. Sequence homology to the C 2 region within the regulatory domain of the 'A-group' of PKC proteins has been noted for two proteins, p65 and phospholipase C. p65 is a t r a n s - m e m b r a n e protein specific for synaptic vesicles which binds calmodulin and might be involved in the process of neurotransmitter release (Perin et al., 1990). This protein contains within the cytoplasmic domain two copies of a 116 amino acids repeat which share about 40% sequence identity with each other and the conserved region C 2 in 'A-group' PKCs (Perin et al., 1990). No homology is found between p65 and the 'B-group' PKC members suggesting that the C 2 region represents a separate functional module shared only by 'A-group' PKCs and p65. Bacterially expressed p65 strongly binds phosphatidylserine and sphingolipids including gangliosides, but not phosphatidylcholine, sphingosine, phorbol esters, DAG or calmodulin (Perin et al., 1990). The homology spans nearly the whole C2 region. This same region, namely C2, shows some weaker homology to a stretch of sequence in phospholipase C (Baker, 1989). A region of about 100 amino acids
80
S. STABELand P. J. PARKER
which shows weak but significant homology to the C2 region of 'A-group' PKCs has been noted n e a r the C-terminus of phospholipase C-y (Stahl et al., 1988) and is less well conserved in PLC-fl (Katan et al., 1988). The presence of this homology in these two enzymes has led to the speculation that in both cases these sequences might participate in D A G or lipid binding (Baker, 1989). 2.3. PKC IN LOWER EUKARYOTES The search and isolation of PKC-like enzymes from lower eukaryotes has experienced a boom in the past two years or so (Table 2 and Fig. 1B). A PKC-like enzyme in lower eukaryotes was first identified in a Drosophila genomic D N A library (Rosenthal et al., 1987b). To date three PKC types have been identified in Drosophila which are encoded by different genes and are differentially expressed. The first PKC cDNA from Drosophila, dPKC, was isolated by low stringency screening with a mammalian probe (Rosenthal et al., 1987b). The same gene and cDNA was reisolated subsequently together with two new members of the Drosophila PKC gene family also by low stringency screening with mammalian PKC probes (Schaeffer et al., 1989). Within 50 kb of the first isolated gene, dPKC, which maps on the second chromosome at position 53E4-7 (Schaeffer et al., 1989) is located another PKC gene encoding a 700 amino acid protein which differs throughout its length from dPKC. Since both genes map at cytogenetic position 53E the nomenclature of 53E(br) for dPKC which is expressed in adult brain (Rosenthal et al., 1987b) and 53E(ey) for the newly identified homologous protein which shows exclusive expression in the Drosophila eye was proposed. The two 53E enzymes are most likely not differentially spliced products of one gene since their longest stretch of identity is 12 nucleotides. Clustering of homologous genes is not an uncommon phenomenon in Drosophila although the reasons for this are unclear. It is possible that this feature results from duplication of an ancestral gene. Both predicted proteins have a structural organization like the ~ and fl type of mammalian PKCs (cf. Fig. 1B), i.e. they possess a C2 region and they lack the insertion in region V4 which is typical for PKC 7-
At cytogenetic position 98F on the third Drosophila chromosome another PKC gene was identified (Schaeffer et al., 1989); this resembles the 6 type of mammalian PKCs in that it lacks a C2-1ike region. The 98F enzyme is expressed throughout development whereas both 53E enzymes are mainly expressed in adults. Transcripts of 98F and 53E(br) are found predominantly in brain, the 53E(ey) enzyme is solely expressed in photoreceptor cells. As expected from their homology with different types of the mammalian enzymes the 53E enzymes are more closely related to each other than to the 98F enzyme. Transcripts of all three thus far identified Drosophila PKC genes have only been identified in adult head tissues, not in the body. Therefore the possibility exists that there are additional PKC isozymes to be discovered which are expressed in the body structures of the fly (Schaeffer et al., 1989). The exclusive expression of the 53E(ey) enzyme in photoreceptor cells suggests a very specific role for this PKC enzyme in the photoreception process in the Drosophila eye. Generation of Drosophila mutants deficient or mutated at the PKC locus could provide a valuable means to elucidate this role in the invertebrate eye. In this respect it is of interest that the NorpA gene product shows clear structural homology to mammalian phosphatidylinositol-specific phospholipase C (Bloomquist et al., 1988); deletion of both copies of this gene abolishes photoreception. The fact that the Drosophila enzyme 98F resembles more closely the 6 type of mammalian PKCs lacking the C2 region whereas the 53E enzymes show a structural organization like the mammalian 7, fl, and 6 polypeptides, suggests that these two groups of PKC proteins are ancient. If'A-group' and 'B-group' PKCs have common ancestors the divergence of the two groups must have occurred before the ancestors of mammalian and arthropods diverged (Schaeffer et al., 1989). In contrast to prokaryotes various eukaryotic microorganisms either produce compounds with similarities to vertebrate signal molecules or, alternatively, are able to react to authentic signal molecules from vertebrates (Janssens, 1987). For two eukaryotic microbes it has been reported that extracellular signals evoke the accumulation of inositol phosphates: glucose in Saccharomyces cells arrested at the Go/G I
TABLE2. Cloned Protein Kinase C Types from Lower Eukaryotes Closest homology to mammalian type
Species
Nomenclature*
Amino acidst
Xenopus
X-PKCI X-PKCII
676 671
~t/fl
d 53E(br) 53E(ey) 98F
639
~/fl
700 634
~/fl
tpa-I
526:~
?
Tabuse et al. (1989)
fl
Levin et al. (1990)
Drosophila
C. elegans
PKC1 1151 *Nomenclature used in the reference cited. tNumber of amino acids of the predicted proteins. :~Partial eDNA sequence.
S. cerevisiae
6
Reference Chen et aL (1989) Chen et al. (1989) Rosenthal et Schaeffer et Schaeffer et Schaeffer et
al. al. al. al.
(1987a) (1989) (1989) (1989)
Protein kinase C cell cycle boundary by starvation (Kaibuchi et al., 1986), and cAMP in the amoeba Dictyostelium (Europe-Finner and Newell, 1987). The clear demonstration of an enzyme with structural characteristics of a PKC polypeptide in Drosophila is contrasted by the failure as yet to clone a related enzyme from Dictyostelium, although protein kinase activity of a 140kDa protein whose phosphorylation was enhanced by phosphatidylserine but not by Ca 2+ or D A G has been described (Jimenez et al., 1989). Also, using an epidermal growth factor-receptor-derived peptide substrate a protein kinase activity has been described in the same organism which is stimulated by PS and PMA or D A G in a synergistic manner and independently of calcium (Luderus et al., 1989). It remains to be seen whether these activities indeed represent PKC homologs. Screening of a Saccharomyces cerevisiae genomic library with probes derived from mammalian PKC ), and /3 with reduced stringency, detected a D N A clone with the coding capacity for a protein of 1151 amino acids and a calculated molecular weight of 131,524Da (Levin et al., 1990). The gene, PKC1, consists of a single large open reading frame without putative introns. Sequence comparison revealed that the putative yeast enzyme is most closely related to the mammalian PKC members ct and/31 with 51 and 53% amino acid identity in the catalytic domain, respectively (Levin et al., 1990). Although the regulatory domain is less well conserved, two zinc-fingers, a C1 domain and a pseudosubstrate prototope are readily identified in the sequence from yeast. The putative yeast PKC protein carries an N-terminal extension of 375 amino acids similar to the mammalian members 6, E, ~ and ~/, however the sequence of this extension bears no resemblance to any other protein or to the N-terminal extensions of PKC 6, E, or ~. From genomic hybridization data it appears that the PKC1 gene is unique in the S. cerevisiae genome and it maps 20.6 cM distal to CDC27 on chromosome II (Levin et al., 1990). Genetic analysis shows that the gene is essential for completing the yeast cell cycle. Absence of PKC1 results in recessive lethality with nonviable spores which still germinate but then are arrested. Conditional PKC1 deletion mutants are arrested after D N A synthesis and are halted in growth as well as in division giving rise to a 'terminal arrest' phenotype (Levin et al., 1990). None of the other known cell division cycle mutants in S. cerevisiae with a similar phenotype (cdc49 and cdc50) appear to be allelic to PKC1. In contrast to the conclusion that there is only a single PKC gene in the yeast S. cerevisiae, Nishizuka and coworkers have identified a Ca 2÷-, phosphatidylserine- and diacylglycerol-stimulated protein kinase activity in S. cerevisiae extracts which may be different from the above enzyme (Ogita et al., 1990). The yeast enzyme appears to have a molecular weight of about 90 kDa and its preferred substrate is myelin basic protein in contrast to the mammalian ~ enzyme which preferentially phosphorylates histone HI. Phosphorylation by the yeast enzyme occurs preferentially on threonine residues; mammalian PKC prefers serine residues in myelin basic protein and in histone. A remarkable feature of the yeast enzyme is that despite its response to diacylglycerol it cannot be
81
activated by phorbol esters. This would be the first case where activation of a PKC enzyme in vitro by diacylglycerol cannot be mimicked by phorbol ester. The nematode Caenorhabditis elegans has become a model organism to study developmental processes. Exposure of this soil organism to tumor-promoting phorbol esters leads to severe disturbances in the behavior and growth of the animals. Biologically inactive phorbol esters do not cause any symptoms. This observation was used by Miwa and coworkers to clone the gene that mediates the effects of phorbol esters by transposon tagging (Tabuse et al., 1989). The gene, tpa-1, was used to isolate a partial cDNA clone encoding a polypeptide of 562 amino acids with the Y-end or N-terminus still unidentified. A computer search for homologies identified the highest homology of this putative protein to the PKC family. 45% identical residues are shared between the Cl region of tpa-1 and mammalian PKC ct, the C, region is followed by a stretch of 72 amino acids with less than 10% homology to PKC ct and the kinase domain shows a homology of 53% to the respective domain in PKC ~. A pseudosubstrate prototope is also found at the appropriate location. Mutations in the tpa-1 gene abolish the effects of phorbol ester on the nematode (Tabuse and Miwa, 1983) and it appears that the tpa-1 gene represents a phorbol ester receptor in C. elegans. It is not known, however, whether this PKC homolog in C. elegans plays a regulatory role in the growth and/or development of this organism. Two types of PKC cDNA clones have been isolated from a Xenopus laevis oocyte cDNA library by low stringency screening with a rat/3 cDNA probe (Chen et al., 1989). They encode putative polypeptides of 676 (XPKCI) and 671 (XPKCII) amino acids. X-PKCI and II show 93% amino acid identity in the C, region, 80% in the C2 region and 85% in the conserved catalytic domain. When compared to mammalian PKC types, both Xenopus enzymes show stronger structural homology to the ~ and /3 types than to any of the other mammalian isozymes. The identification of two closely related members points to the existence of a family of these proteins also in amphibia.
3. F U N C T I O N A L ASPECTS OF PKC I N VITRO For the purposes of discussion, a consideration of the properties of PKC proteins can be divided into general properties that define this class of proteins and specific properties that indicate functional distinctions between the members of the PKC family. 3.1. GENERALPROPERTIES
The ability of PKCs to interact with and be activated by membranes and D A G presents a functional definition of these proteins. As such the binding to lipids is a critical and indeed well studied phenomenon (recently reviewed in Epand and Lester, 1990). This interaction which has been most clearly documented for the ct//3/~ enzymes appears to be minimally a two-step process. The first step
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is the formation of a ternary enzyme + C a 2 + + phospholipid complex; this is a nonproductive association* from which the enzyme can be removed by addition of Ca 2÷-chelators. Evidence has been provided to indicate that a number of Ca 2÷ ions are involved in each event and furthermore that the apparent affinity of the enzyme for Ca 2+ is greatly enhanced by the presence of phospholipids (Bazzi and Nelsestuen, 1990). This scenario is reminiscent of the lipocortin/calelectrin group of proteins for which a consensus sequence has been suggested as playing a critical role in Ca 2÷ -phospholipid association, however no such sequence is present in P K C (Geisow, 1986). The region most likely to be involved in Ca 2+-dependent-phospholipid interactions is the C2 domain which is only present in the 7 , / / a n d ? gene products (see Section 2 and below). Formal, direct p r o o f for this has yet to be obtained but the circumstantial evidence is compelling. The second step in association is the binding of D A G (or phorbol ester) which through conformational changes leads to activation. The nature of the conformational changes have not been precisely defined but are clearly evidenced through for example, assessment of alterations in surface availability (antibody binding, Cazaubon et al., 1990) and protease accessibility (Kishimoto et al., 1989). The active complex containing D A G (or phorbol ester) is only slowly dissociable by removal of calcium by chelating agents and this is largely the basis of the 'translocation' assay employed for monitoring activation of P K C in vivo (see Fig. 4).t However it is evident that there is also a component of P K C that is only extractable with detergents~: implying a more penetrative interaction of P K C with the membrane. This phenomenon of 'insertion' has also been documented in vitro and is associated with constitutive activation of the enzyme (Bazzi and Nelsestuen, 1988). The extent to which this membrane 'inserted' pool is functional in vivo remains to be defined, although the in vitro analysis would suggest that it is active. This compartment of P K C has been proposed to play a role in memory, since it would be expected that chronic/repeated stimulation of P K C
*It is possible to drive PKC activation in vitro only in the presence of phospholipid + Ca 2+. However in mixed lipid or lipid/detergent micelles there is a strong dependence on DAG (or phorbol ester) for activation (Hannun et al., 1985). tOn extraction of cells in the presence of chelating agents, PKC (~,fl,y) is removed from its transient Ca 2+dependent association with membranes and is monitored as a 'soluble' enzyme; on extraction in the presence of Ca 2÷ these PKCs will be detected as membrane associated. However the complex formed with DAG is relatively stable and even in the nominal absence of Ca 2+, PKC is seen to remain on the membrane fraction. This phenomenon has been used as a measure of activation status in many situations, the so called 'translocation' assay. This assay would more accurately be described as a 'membrane stabilization' assay. :~There is also in many cell types a variable content of detergent nonextractable PKC that is made up of cytoskeleton associated and nucleus/nuclear matrix associated enzyme. The role and indeed activation status of this compartment of PKC is as yet unclear.
NO PRETREATMENT
+ TPA
EXTRACTION +Ca 2+
-Ca 2+
®® ®® ®
®
® ® ® ® ®
®
®
® ®
® ® ®
-Ca 2+
®
®
® ®
®
FIG. 4. Translocation of PKC enzymes. Depicted is a scheme illustrating the dependence of PKC ~ and E interactions with membranes on the extraction procedure and activation status. For PKC ~, at nominally zero Ca: ÷ concentrations and in the absence of TPA (or DAG) the enzyme is in the soluble fraction. However in the presence of Ca 2+, PKC shows a particulate association. By contrast PKC E is in the soluble fraction regardless of the presence or absence of Ca 2+. Prior treatment of cells with TPA leads to complexing (and activation) of both PKC ~ and PKC ¢ and under these circumstances both enzymes show particulate association.
would proportionately increase the membrane 'inserted' pool in a stochastic fashion, leading to a constitutive elevation of P K C activity (Burgoyne, 1989). A model of the general interactions described above is illustrated in Fig. 5. The physiological distribution of P K C between each of these compartments is difficult to assess precisely largely because of the dynamics of the initial phase of membrane association. However an assessment of the activation status is more accessible through the membrane stabilization assay and specific examples are described further in Section 4.
3.2.
C A 2 + -DEPENDENCIES
The apparent dependence of P K C (~, // and 7) activity on Ca 2+ is complex. Under conditions where P K C ~ , / / a n d ? show Ca 2+ -dependence the activation constants are in the physiological range i.e. 0.1-1 #M (Huang et al., 1988; Jaken and Kiley, 1987; Marais and Parker, 1989). However partial or complete attenuation of Ca2+-dependence can be achieved with certain substrates (or even different preparations of a particular substrate). In view of this variation it is expedient to determine interaction with Ca 2+ by
Protein kinase C C2
83
CI
>
Proteolysis
(EOH
t
Ca/Plipid
DAG
00OH
O30H
FIG. 5. Model of PKC interactions. Shown is a schematic representation of PKC ~/[3/~ containing both amino terminal conserved domains C t and C2, In the absence of effectors the pseudosubstrate site (PSS) interacts with the carboxy terminal kinase domain to maintain an inactive state. This form of the enzyme can interact with Ca2+ and phospholipid (Ca/PLipid) to form inactive membrane associated complexes (see also Fig. 4 and legend). On binding DAG conformational changes in the regulatory domain lead to the release of the pseudosubstrate site inhibitor region with consequent activation. In this activated form, the enzyme is more susceptible to proteolysis and shows a reduced half-life. In some cell types activation also leads to an increase in the cytoskeleton (CYT) association of PKC.
some other parameter; this has been achieved most simply for the interaction with membranes (whether the effect of Ca2 + in enhancing membrane association is the primary role of Ca 2+ in stimulating activity is as yet unresolved). It was demonstrated elegantly by Wolf and colleagues (Wolf et al., 1985) that purified PKC (consisting of a mixture of a, t , y) would interact with plasma membranes (inverted erythrocyte ghosts) in a Ca2+-dependent manner. Thus extraction of cells in the presence or absence of Ca 2+ leads to a differential distribution of PKC activity that reflects the Ca 2+-driven association of Ca2÷-dependent PKC activities with membranes. The quite distinct behavior of other PKC forms like e (Kiley et al., 1990) and c~(Olivier and Parker, 1991) highlights the Ca 2+-independence of these activities (Fig. 4). The possible physiological consequences of Ca 2+ -dependent and -independent forms of PKC are further discussed in Section 4.
3.3. DIACYLGLYCEROLAND PHORBOLESTER RESPONSIVENESS The activation of PKC by DAG (in the presence of phospholipids) is the common feature of this group of kinases. Structure/function analyses of DAG stereoisomers and analogs have highlighted the specificity for 1,2 sn DAG and the need for a free 3-OH (e.g. Bonser et al., 1988; Ganong et al., 1986; Rando and Young, 1984). Replacement of acyl with alkyl linkages has also been shown to abolish activation suggesting importance for the carboxyl groups (Bonser et al., 1988). However others have observed activation by ether and alkyl linked diradylglycerols (Ford et al., 1989; Heymans et al., 1987). This would appear to be an unresolved issue, however one of some importance given the potential source of DAGs (see below). To date no substantial differences in activation constants for DAGs have been noted for
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the different PKC isotypes; this is consistent with the high degree of conservation of the putative D A G binding site. The ability of phorbol esters to activate PKC through binding at the D A G site has aroused much interest. Structure/function analysis for this class of activators has largely been carried out on mixtures of PKC ~, fl and 7 enzymes. However more recently highly purified individual PKC enzymes have been assessed for their phorbol ester-dependence and furthermore for their response to various related structures. This has provided evidence for a distinct pattern of responsiveness to different phorbols that themselves have individual functional profiles in biological systems (Evans et al., 1991). One practical conclusion from these observations is that the nature of the biological response to these agents may be defined by the particular set of expressed PKC enzymes that can respond to the particular phorbol; this would provide some rationale for the diverse effects of these agents. That the phorbol esters can apparently distinguish particular PKC enzymes is also an encouragement to those seeking to obtain selective activation/inhibition of these enzymes. One further aspect of such studies is that they may reveal the existence of novel PKC activities as appears to be the case for resiniferatoxin (Evans et al., 1990).
substrate for recombinant PKC E* (Schaap and Parker, 1990), PKC r//L (Bacher et al., 1991; Osada et al., 1990) and PKC 6 (Olivier and Parker, 1991) (although it has been shown that a 'natural' PKC activity immunoreactive with a 6-selective antibody will phosphorylate histones very efficiently (Leibersperger et al., 1990); the basis for this discrepancy has yet to be resolved but may suggest the role of some stable association/modification in the regulation of PKC 6 activity/specificity). A more systematic approach has been made in the case of PKC ct, fll and 7 by addressing the role of N- and C-terminal basic residues in defining primary structural requirements for substrates. These studies indicate that while PKC ~ and fl show a very similar pattern of specificity, PKC 7 exhibits a strong preference for substrates with a C-terminal basic amino acid (Marais et al., 1990). The studies outlined above indicate that although there is as yet limited information on physiological targets, there are relative and absolute distinctions in substrate specificity between PKCs. It may be concluded that the expression of particular PKC enzymes will have a significant impact on the cellular consequences of triggering this signalling pathway.
4. F U N C T I O N A N D R E G U L A T I O N OF PKC 3.4. SUBSTRATESPECIFICITIES
IN VIVO
In contrast to the wealth of information accumulated on the activation of this signalling pathway and the structures of the components within it, there is a dearth of information on physiological substrates for PKC and in particular those whose phosphorylation has a defined effect, As a consequence there are few data on the specificity of different PKC isotypes for physiological substrates. Two that have been addressed are the E G F receptor which is phosphorylated on Thr654 and the fl protein phosphorylated on Ser32. In the case of the E G F receptor PKC • and fl have been shown to be significantly better catalysts than ~ in this phosphorylation (Ido et al., 1987); the relative activities being ~t > fl > 7, whilst for fl the rank order of effectiveness is fll > ~t > ~ (Sheu et al., 1990). These differences between isotypes although modest are likely to be significant in vivo where through the action of phosphatases steady state as opposed to initial rate considerations apply. Further distinctions in specificity have been documented through the use of nonphysiological and synthetic PKC substrates. The major (probably) nonphysiological substrate employed for PKC assays is the lysine-rich histone fraction (HI). Interestingly in addressing the function of members of the PKC family unearthed by homology screening with cDNA probes, it has been noted that histone is a poor
This section is subdivided for consideration of intracellular messenger sources (Section 4.1), the means available for assessing PKC activation and consequences to PKC itself (Section 4.2) and to the cell (Section 4.3).
*Although a poor substrate for PKC-E, histone is readily phosphorylated if PKC-E is proteolytically activated. Thus in this instance the specificity is not an intrinsic property of the catalytic domain but is in part imposed by the regulatory domain. Formal proof of this has been obtained through the construction ofa PKC ~/~,chimeric polypeptide (Pears et al., 1991).
4.1. SOURCES OF DIACYLGLYCEROL The trigger for the activation of the signal transduction pathway in which PKC lies, is the production of diacylglycerol. Historically this has been considered to arise from agonist induced activation of phospholipase Cs which act upon inositol lipids, in particular phosphatidylinositol 4,5-bisphosphate (Ptdlns4,5P2) (reviewed in Berridge, 1987). The delineation of this pathway and the control of the relevant phospholipases have recently been reviewed elsewhere (Meldrum et al., 1991). Suffice it to say that there is compelling circumstantial evidence for agonist-induced breakdown of Ptdlns4,5P2 with the consequent activation of PKC through the D A G produced. The precursor role of Ptdlns4,5P 2 is of particular significance in that the inositol 1,4,5 trisphosphate coincidently produced evokes a rise in cytosolic Ca 2÷ (Streb et al., 1983). This clearly will have an impact upon PKC ~, fl~/2 and 7 which display a Ca2+-dependence in their interaction with membranes (see Section 3.2); by increasing the membrane occupancy of these particular PKCs, it is anticipated that they will be more effectively activated by the limiting D A G produced. Evidence that this is the case, is suggested by the observations that elevated Ca 2+ appears to prime PKC responses and also to increase the apparent affinity for PDBu binding in vivo (for example see Akers and Routtenberg, 1987).
Protein kinase C Interestingly recent studies on the time course of D A G production in a number of distinct cell types has revealed a second prolonged phase of agonistinduced production. This has been readily observed through the use of postextraction conversion of D A G to riP-labelled phosphatidic acid (PA) using bacterial D A G kinase (Walsh and Bell, 1986). This provides an extremely effective procedure for analysis of D A G production without the need to label a particular (unknown) lipid pool in vivo. Analysis of the types of acylation present in this second phase DAG, has led to the conclusion that it is derived predominantly from phosphatidylcholine (PtdCho) (e.g. Pessin et al., 1990). While D A G can of course be produced by PLC hydrolysis of PtdCho, there is accumulating evidence that D A G is also produced via the combined action of a PLD (recently reviewed in Billah and Canthes, 1990) and phosphatidic acid phosphohydrolase (reviewed in Brindley, 1984). This latter enzyme is a microsomal enzyme (in its membrane bound state) suggesting that not only is Ptd-Cho-derived D A G temporally shifted relative to Ptdlns-derived D A G but also may be spatially distinct. As such it is not clear whether the 'translocation' assay successfully employed for plasma membrane D A G activation of PKC will necessarily yield information on PKC activation by microsomal D A G (the distinct properties ofmicrosomal membranes may alter the stability of the putative lipid/DAG/PKC complex). Thus although 'second phase translocation' has not been observed, the conclusion that PKC is not activated has yet to be corroborated. Indeed quite the opposite, since evidence has been presented to indicate that during the second phase response to TRH stimulation in GH4C 1 cells, PKC E becomes down-regulated (Akita et al., 1990; Kiley et al., in preparation). This would appear to be a typical down-regulation that is a consequence of chronic stimulation. Interestingly in this instance
85
it is a selective down-regulation since PKC ~ and/~ which are also expressed in these cells are not downregulated. This suggests that either PKC E is more susceptible to down-regulation or that in the absence of an elevation of cytoplasmic Ca 2+ PKC ~ and 13are not effectively activated by this second phase DAG. There is another de novo route for production of D A G for which there is evidence for agonist stimulation. The extent to which this represents a metabolic as opposed to an intracellular messenger response is not clear although it is pertinent to note that D A G is likely to be free to 'flip' within the membrane and as such may become available to cytoplasmic PKC during for example microsomal triglyceride synthesis/breakdown. An ability to monitor PKC activation in this compartment is evidently an important objective. The degree to which different PKCs respond selectively to the production of D A G in a contextual (Ca 2÷ or no Ca 2+) and spatial (plasma membrane, microsomal, etc.) manner remains to be determined. However it is now well established that Ptdlns4,5P2 is not the only (lipid) precursor for D A G production (as indicated in Fig. 6). As such there is no doubt that a number of regulatory mechanisms remain to be elucidated concerning the precise means by which agonists induce the PLC/PLD directed breakdown of the noninositol lipids. 4.2. ACTIVATION,TRANSLOCATIONAND DOWN-REGULATIONOF PKC The two most commonly used procedures for assessing the activation of PKC in vivo are (i) assessment of the phosphorylation of PKC specific substrates and (ii) measurement of the translocation of PKC. The first of these strategies has been best documented for the '80 k' protein originally identified
PtdCho
PtdIns 4,5 P2 PLC Ins 1,4,5 P3
A
ChoP ~
I
Cho
PA
c~ ÷
Glycerol
DAG
~
PKC
DAG
FIG. 6. Sources of DAG. Shown are possible sources of DAG including phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate 0atdIns4,5Pz) and phospholipase C and D-catalyzed hydrolysis of phosphatidylcholine (PtdCho). A de novo route is also indicated.
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S. STABELand P. J. PARKER
by Rozengurt and colleagues in Swiss 3T3 cells* (Rozengurt et al., 1983). This protein appears to be a selective PKC substrate and as a relatively abundant protein with a characteristic pL it has served as a useful marker (see Section 4.3 for further discussion). A major shortcoming of this general strategy of using PKC substrates is that in the absence of selective physiological substrates for the different PKCs it is not possible to distinguish whether one or more become activated at particular times. The translocation assay is based upon the phenomenon illustrated above in Fig. 4. In essence, the production of an active complex of PKC-lipid-DAG stabilizes the membrane associated form to extraction in the presence of Ca2+-chelators. Thus in 'PKCactivated' cells there is observed an increase in the membrane associated PKC relative to control cells, with a parallel decrease in cytosolic activity.t As mentioned above there are no specific substrates for different PKC isotypes that have been described that would permit a selective assessment of activation at a functional level, but selectivity can be introduced in this assay through the use of monospecific antisera; by immunoblot it is possible to monitor the behavior of individual PKC isotypes (e.g. Kiley et al., 1990). A consequence of chronic activation of PKC in vivo is down-regulation. This has been shown to be due to increased proteolysis (Ballester and Rosen, 1985; Woodgett and Hunter, 1987; Young et al., 1987) and for PKC ct appears to be due to cleavage in the V3 domain (Young et al., 1988; see Section 2). The mechanism involved in the increased proteolysis has not been elucidated, however the increased susceptibility of the active conformation of PKC to proteases in vitro (Kishimoto et al., 1989) suggests that in this passive manner increased proteolysis occurs in vivo. In contrast to this interpretation it has been reported that a mutant form of PKC ct which is inactive as a kinase does not down-regulate suggesting that substrate phosphorylation or some autophosphorylation event is involved (Ohno et al., 1990). However we have shown that in distinct cellular contexts both mutant (kinase negative) PKC ~ (Pears and Parker, 1991) and PKC y (Freisewinkel et al., 1991) will down-regulate. Thus it is clear that in these cases intramolecular autophosphorylation is not a prerequisite for down-regulation. It is certainly the case that different cellular contexts yield distinct patterns of induced down-regulation and it may be concluded that perhaps distinct pathways are involved for the different PKCs. The extent to which this 'pharmacological' induction of down-regulation reflects a universal physiological process is unclear. However it has been noted that in situations where there is chronic diacylglycerol *The bovine brain homologue of 80 k has been cloned and has been coined the MARCKS protein--myristylated, alanine rich, c-kinase substrate (Stumpo et al., 1989). tit should be noted that this is the simplest scenario. Due to down-regulation of the activated PKC the loss of cytosolic PKC may exceed the increase in membrane associated enzyme. Furthermore there are a number of reports describing acute losses of PKC activity (but not protein) suggesting a distinct plane of regulation (e.g. Cochet et al., 1986).
production, e.g. ras transformation and TRH stimulation, down-regulation has been observed (Akita et al., 1990; Wolfman and Macara, 1987). It may be concluded that the 'normal' production of endogenous DAG has a finite effect on PKC proteolysis. Beyond the acute membrane association of PKC there are a number of reports demonstrating that PKC also becomes associated with the Triton X-100 insoluble fraction of cells. This includes both association with the cytoskeleton (e.g. Kiley and Jaken, 1990; Mochly-Rosen et al., 1990; Zalewski et al., 1990) and the nucleus (e.g. Masmoudi et al., 1989). In the case of the cytoskeleton interaction we have noted that activated mutant forms of PKC ct are increased in their association (C. Pears and P. J. Parker, unpublished) suggesting that activation may be primarily responsible for sequestration in this compartment. Whether these associations reflect a necessary event that is responsible for some element of the physiological response is not yet known. 4.3. ANALYSISOF THE PKC SIGNALLING PATHWAY
There is little information as yet about the biochemical events mediating the proposed connection between PKC activation and cellular responses. One important goal therefore has been to identify and characterize physiological substrates for PKC. A detailed review of in vitro and potential in vivo substrates for PKC can be found in Woodgett et al. (1987). An acidic protein of 87 kDa (the MARCKS protein; see above) with multiple p l species within the range of 4.0-4.9 was shown to be a substrate for PKC in cell lysates from rat brain or 3T3 cells and became an early candidate for a specific PKC substrate (Isacke et al., 1986; Rodriguez-Pena and Rozengurt, 1986; Wu et al., 1982). The protein showed a widespread occurrence in different species and tissues with the highest levels in brain, spleen and lung and lowest levels in heart and skeletal muscle (Albert et al., 1986). Its distribution follows the distribution of PKC (Kuo et al., 1980; Minakuchi et al., 1981) and therefore made this protein a likely mediator of at least certain PKC functions. Phosphorylation of the 80 kDa protein was also seen in intact fibroblasts (Rozengurt et al., 1983); its rapid increase in phosphorylation was believed to reflect activation of PKC. Evidence for this was (1) that addition of biologically active phorbol esters to quiescent cells induces phosphorylation within 15 sec, (2) stimulation of lipid breakdown by PDGF or exogenous PLC or addition of the synthetic diacylglycerol OAG also led to rapid 80 k phosphorylation in quiescent cells. These phosphorylations can be prevented by down-regulation of PKC by prolonged treatment of cells with biologically active phorbol esters (Rodriguez-Pena and Rozengurt, 1986). The 80 k protein was found in all rat tissues examined and was most abundant in brain (Blackshear et al., 1986). Phosphorylation of 80 k could not be detected after addition of insulin or dibutyryl-cAMP. Comparison of the peptide maps of 80 k labelled in vivo after TPA treatment and of 80 k phosphorylated in vitro by PKC showed nearly identical patterns of phosphopeptides suggesting that 80 k is indeed a PKC substrate in vivo (Blackshear et al., 1986) and underlining the potential usefulness of 80 k
Protein kinase C phosphorylation as an apparently specific and sensitive marker of PKC activation in a wide variety of cell types (Isacke et al., 1986). Purification of this protein from bovine brain and amino acid composition analysis showed that it contained 28.6 mol% alanine and from the axial and frictional ratios it was concluded that the 87 k protein is an extremely elongated monomer and that phosphorylation of the purified protein by PKC in vitro occurred exclusively on serines (Albert et al., 1987; Patel and Kligman, 1987). A related 80 k protein was purified from rat brain which had 17.8 mol% alanine and may be related to the 87 k from bovine brain (Morris and Rozengurt, 1988). While phosphorylation of an 80 k protein has been observed within seconds of PDBu addition to quiescent Swiss 3T3 cells, phosphorylation of other proteins including one of 87 k has been reported after minutes of treatment (Rozengurt et al., 1983). Addition of PDBu or bombesin to quiescent Swiss 3T3 cells induces phosphorylation of 80 k and 87 k within 5 rain (Morris and Rozengurt, 1988). Sequence analysis of peptides of 80 k purified from rat brain indicated that there may exist a family of these PKC substrates since the two sequences are not identical (Erusalimsky et al., 1989). The 80 k protein is myristoylated cotranslationally (James and Olson, 1989). A cDNA clone for the 80-87 k bovine protein has been isolated (Stumpo et al., 1989) which codes for an open reading frame of 335 amino acids with a predicted molecular weight of 32 kDa. Despite its predicted molecular weight the protein migrates with an apparent molecular weight of 80-87 k following expression from the cDNA in mammalian cells lacking the protein. Upon addition of TPA to the cells this protein was phosphorylated (Stumpo et al., 1989). The unusual behavior upon gel electrophoresis is probably due to the acidic p I and general hydrophilicity. Striking features of this protein are its high content of alanine residues (28.4 mol%) and the relative abundance of glycine, proline and glutamate. Apart from the initiator methionine it contains no other methionine residues and it carries an N-terminal myristoylation consensus sequence. The phosphorylation sites for PKC on the 80 k protein have been determined (Graft et al., 1989); two of them are found in an unusual highly basic stretch of 25 amino acids K K K K K R F S F K K S F K L S G F S F K K N K K which carries a repeat of the sequence F S F K K . Data from the purified protein indicate that all 4 serines in this basic domain are phosphorylated by PKC in vitro and that the protein does not contain major phosphorylation sites outside this domain after phosphorylation in vitro or in vivo. The basic stretch which contains the PKC phosphorylation sites is perfectly conserved in the M A R C K S protein from beef, human and chicken (Stumpo et al., 1989). The sequence of the 80 k protein has revealed no similarity to known protein sequences. Two other major endogenous PKC substrates have been examined in detail. One, a 40-47 k protein in platelets (Nishizuka, 1984), has been purified (Imaoka et al., 1983) and was tentatively identified as Ins(145)P 3 5'-phosphomonoesterase (Connolly et al., 1986). Immunological screening of a eDNA expression library from HL-60 cells yielded a cDNA which codes for a cytosolic 40k protein with a
87
potential CaZ+-binding 'EF-hand' structure at the C-terminus, a potential PKC phosphorylation sequence and with no homology to any known protein (Tyers et al., 1988). Peptide sequences from the 47 k substrate in platelets are found in this cDNA. 1047 is enriched in HL-60 cells upon differentiation with retinoic acid and it may have a common function in platelets and leukocytes since it is found in hemopoietic cell lines of myeloid and lymphoid origin but not in cell lines from liver, fibroblasts, skin, lens or nerve tissue. The gene appears to be well conserved in vertebrates but is not detected in insects or yeast suggesting an important role for p47 in highly developed hemopoietic systems. Interestingly, p47 has several potential PKC phosphorylation sites one of which ( Q K F A R K STRRSIRL) closely resembles the (pseudo)substrate sequence of PKC ( R F A R K G S L R Q ) . The sequence of lO47 does not yield any further direct clues as to its possible functions and it appears that the Ins(145)P3 5'-phosphomonoesterase activity can be partially separated from the platelet p47 (Tyers et al., 1988). In the absence of any known function and enzymatic characteristics the name PLECKSTRIN has been proposed for p47 (platelet and leukocyte C kinase substrate with the most probable phosphorylation site K F A R K S T R R S I R ) (Tyers et al., 1988). A second brain protein most often referred to as B-50, fl or GAP-43 has been purified and has been hypothesized to modulate the activity of phosphatidylinositol-4-phosphate kinase (Benowitz et al., 1987; Chan et al., 1986; Van Dongen et al., 1985). While there is debate about the precise function of the fl protein, the phosphorylation of this synaptosomal protein is circumstantially associated with long term potentiation (Routtenberg, 1986). The structure of the protein has been elucidated by cDNA cloning (Rosenthal et al., 1987a). As indicated above, in vitro there is some relative specificity for phosphorylation of fl by PKC ct, fl, V but it is not known whether such specificity operates in vivo. With the availability of eDNA clones coding for the individual enzymes another approach has been used to investigate the role of PKC in cellular regulation. By overexpressing individual PKC enzymes through transfection of their cDNAs into mammalian cells their contribution to defined cellular events can be examined. When PKC fl~ is overexpressed 10- to 50-fold as judged by activity assays in rat or mouse fibroblasts the addition of phorbol ester results in exaggerated responses compared to control cells (altered morphology and increased growth rate). Some of the cell lines but not all show formation of small colonies in soft agar (Housey et al., 1988; Krauss et al., 1989). These effects are also visible without the addition of activators of PKC probably due to the production of intracellular D A G through the presence of growth factors in the culture medium. Introduction and overexpression of the 7-type of PKC in NIH 3T3 mouse fibroblasts had similar consequences with alterations in saturation density and reduced dependence on serum for growth (Persons et al., 1988). Selection of cell lines which express about 3-fold higher levels of this enzyme did not lead to morphological alterations under normal culture conditions.
88
S. STABELand P. J. PARKER
The mitogenic responses to TPA and cardiolipin, claimed to specifically activate PKC ~ in vitro (Huang et al., 1988) were enhanced as was anchorage-independent growth, but only in the presence of activators (Cuadrado et al., 1990). In no case, however, has expression of wild-type PKC sequences and activation by activators led to a fully transformed phenotype emphasizing the nononcogenic action of unmutated PKC. It is not known whether mutated, e.g. constitutively activated PKC types could show a fully transforming activity, although isolation of a transforming mutant of PKC from a murine fibrosarcoma has been reported (Megidish and Mazurek, 1989). Introduction of normal PKC ~ into Swiss 3T3 cells and up to 10-fold overexpression did not lead to a transformed phenotype of the cells, not even in the presence of TPA (Eldar et al., 1990), but as with PKC 13overexpressing cells exhibited an enhanced growth rate and reduced serum requirement. Overproducing cells had less EGF receptors on their surface due to decreased EGF receptor mRNA levels; there was no change in the affinity for EGF. This study therefore concluded that PKC ~ is involved in cellular mechanisms regulating the expression of EGF receptor molecules in Swiss 3T3 cells. A loss of EGF-binding sites on the cell surface had been described earlier as one of the properties of certain virally or chemically transformed fibroblasts (Moses et al., 1981; Todaro et al., 1976). Whether these early observations are related to the observed reduction here in at least three independent cell lines overexpressing PKC ~ is not known. Involvement of individual PKC enzymes in celltype-specific reactions can also be excluded by this approach, e.g. through the introduction of PKC flj into IL-3-dependent haematopoietic cells. Although selected cell lines have increased PKC fl~ levels the cells were not more sensitive to IL-3 than control cells nor did they grow in a factor-independent fashion in soft agar (Kraft et al., 1990). Interestingly, however, cells overproducing PKC/?~ showed a 10-fold increase in the formation of transformed colonies when cotransfected with the c - H - r a s oncogene (Hsiao et al., 1989), suggesting a synergistic action between ras oncogenes and PKC. Synergism between ras and PKC has been demonstrated by a number of reports (Fleischman et al., 1986; Hsiao et al., 1984; Lacal et al., 1987; Wolfman and Macara, 1987) the mechanism, however, by which ras transformation and PKC activation could be linked is unclear. It has been speculated that this synergistic effect could be due to the increased levels of DAG reported for ras-transformed cells (Fleischman et al., 1986; Preiss et al., 1986) which may be at least partially responsible for this effect. This does, however, not discriminate between the possibilities that ras acts downstream of PKC (Yu et al., 1988) or upstream of PKC (Lacal et al., 1987). In contrast to this speculation which assumes the synergism between ras and PKC to occur at the polypeptide level, more recently it has been reported that ras transformation can alter the relative expression PKC types present in a cell (Borner et al., 1990). In rat fibroblasts and liver epithelial cells which express the types ~ and c transformation by the ras oncogene or inducible
expression of ras from an introduced construct leads to increased expression of PKC c~ and decreased expression of PKC E at the protein and mRNA level, thereby offering a novel interpretation of ras/PKC cooperation namely by alterations in the set of PKC enzymes within the cell. It will be interesting to determine whether this reflects differential roles of and E in cell growth with high levels of ~ and low levels of E being required for growth. Such a situation could be similar to the one described for the cellular rrg gene product, low expression of which has been shown to be crucial for ras transformation (Contente et al., 1990). A quite suggestive function for PKC has recently been described in T-lymphocytes (reviewed by Berry and Nishizuka, 1990). Evidence was presented that activation of the T-cell receptor by phytohaemagglutinin or a CD3-specific monoclonal antibody leads to activation of cellular p21ras measured as a roughly 10-fold increase of ras in the GTP-bound, i.e. activated, state (Downward et al., 1990). This activated state is the result of decreased hydrolysis of the GTP bound to the ras protein and not of altered nucleotide exchange rates. The evidence is compelling that the GTP hydrolysis which is normally effected by the GTPase activating protein GAP is decreased due to inhibition of the GAP protein. All available evidence suggests that PKC either directly or indirectly is responsible for this inhibition. In this context it is interesting to note that activation of the T-cell receptor has also recently been claimed to lead to phosphorylation and activation of another cytoplasmic serine-/threonine-specific kinasc c-Raf through an apparently PKC-dependent pathway (Siegel et al., 1990). The physiological function of the Raf kinase is not known although it seems to be involved in growth control and intracellular signal transduction pathways which finally lead to gene activation (Jamal and Ziff, 1990; Kolch et al., 1991: Li et al., 1991). Activation of PKC can, itself have effects on transcription factor transactivation function. Evidence has been presented that in resting epithelial cells and fibroblasts activation of PKC results in site-specific dephosphorylation of the c-Jun protein which coincides with increased AP-I binding activity and transactivation by c-Jun (Boyle et aL, 1991). The data suggest that induction of TPA-induciblc promoter element (TRE) function by PKC activators (Angel et al., 1987) is mediated by enhanced binding and transcriptional activity of a dephosphorylated AP-1 complex. The region identified, adjacen~ lo the DNA-binding domain on the c-Jun protein, coincides with a region previously identified to be essential for activation of c-Jun in vitro and in vit, o and had been termed activation domain A2 (Baichwal and 7jian, 1990). The transcriptional activator NF-kB presents another intriguing example where a clearer picture of the role of PKC in the induction of gene expression may be emerging. NF-kB was detected as a prc,~ein that could complex to a 10 base pair site in the k immunoglobulin light chain enhancer called kB (Sen and Baltimore, 1986). Because it was constitutively present only in those B cells of the appropriate stage for light chain expression and was crucial for k
Protein kinase C enhancer function, NF-kB first appeared to be a tissue-restricted transcription factor (Atchison and Perry, 1987). It was, however, soon found to be present in a covert cytoplasmic form in a number of other cells where phorbol esters can induce the factor to move to the nucleus and to exhibit specific DNA binding (Baeuerle et al., 1988) NF-kB is now considered a rather ubiquitous intracellular messenger that mediates inducible and tissue-specific gene expression in many systems (reviewed in Lenardo and Baltimore, 1989). A protein of about 35 kDa which appears as 70 kDa on gel filtration and hence may form a dimer, is able to specifically inhibit the DNA binding activity of NF-kB and has therefore been named inhibitor-kB (I-kB) (Baeuerle and Baltimore, 1988). The purified inactive complex NF-kB/I-kB can be induced to show DNA binding in vitro by phosphorylation with PKC in vitro which phosphorylates sites on the inhibitor protein rather than on NF-kB itself (Ghosh and Baltimore, 1990). The fully active DNA-binding complex of NF-kB appears to be a heterotetramer consisting of two molecules of p50 and two molecules of p65. Cloning of the p50 and the p65 subunits of this complex has revealed that there exists a family of NF-kB-like proteins, the rel family of polypeptides, including KBF1, a factor that binds to a class I major histocompatibility complex enhancer, the v-re! oncogene of the avian retrovirus Rev-T and the Drosophila morphogen dorsal (Ghosh et al., 1990; Kieran et al., 1990; Meyer et al., 1991; Nolan et al., 1991). Whereas both the p50 and the p65 subunits appear to participate in the DNA binding of the active NF-kB complex, only the p65 subunit mediates inhibition by the inhibitor protein I-kB, probably by direct interaction (Nolan et al., 1991). Dissociation of the inactive cytoplasmic NF-kB/I-kB complex by direct phosphorylation of I-kB by activated PKC provides a simple, direct strategy by which PKC activation at the cell surface affects nuclear events. The activation of PKC at the cell surface and the subsequent regulation of many cytoplasmic functions, has also led to the idea that a number of such responses may be effected through kinase cascades. This is typified by the phosphorylation of the ribosomal subunit $6 which can be stimulated by TPA treatment of cells but is effected by a specific $6 kinase that is itself regulated by phosphorylation (albeit not a direct substrate for PKC) (Ballou et al., 1988). It appears then that at some level PKC feeds into a pathway involving an $6 kinase-kinase and $6 kinase. A similar situation exists for the activation of the so-called mitogen-activated protein kinase (MAP kinase) (Ray and Sturgill, 1987) which can be stimulated following TPA (or mitogen) treatment of cells and is itself activated by a MAP kinase-kinase which is functionally TPA inducible (Adams and Parker, 1991). These kinase cascades presumably serve to permit (i) amplification (ii) phosphorylation events distal to PKC activation and (iii) integration of the 'PKC pathway' with other signalling systems. So it appears that the pleiotropic effects that are ascribed to PKC activation may be due to the fact that this enzyme family may impinge on or directly control a number of important intracellular control circuits which are only now beginning to emerge.
89 NOTE ADDED IN PROOF
Resequencing of the PKC-L eDNA confirmed that PKC-L also has two complete zinc-fingers and is indeed the human homolog of PKC-~/ (Bacher et al., correction submitted). gratefully acknowledge Marek Liyanage (Max-Delbriick-Labor) for help with the sequence alignments. We should also like to thank Amanda Wilkinson for preparation of the manuscript.
Acknowledgements--We
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JPT
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95
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