Cellular Signalling Vol. 4, No. 6, pp. 595-609, 1992.

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MINI REVIEW ROLE OF COFACTORS IN PROTEIN KINASE C ACTIVATION JULIANNE J. SANDO,*Jf MURIEL C. MAURER,~ ELIZABETHJ. BOLEN* and CHARLESM. GRISHAM~ Cancer Center and Departments of *Pharmacology and ,Chemistry, University of Virginia, Charlottesville, VA 22908, U.S.A. (Received 12 August 1992; and accepted 18 August 1992) Key words: Protein kinase C, phospholipids, membranes, calcium, signal transduction.

1. INTRODUCTION

phosphatidylinositol (PI) hydrolysis which results in production of DAG and inositol phosphates, the latter leading to increased intracellular calcium (reviewed in [5]).

PROTEIN KINASEC (PKC) was first identified by Nishizuka and colleagues as a precursor for a cofactor-independent protein kinase, protein kinase M (PKM), which was generated by proteolysis [1, 2]. Shortly thereafter, intact PKC was found also to exhibit kinase activity when it was associated with membranes [3], thus providing a reversible mechanism for activation that was independent of proteolysis. Purification of the activating components from membrane revealed that acidic phospholipids, especially phosphatidylserine (PS), could support PKC activity at high (millimolar) calcium concentrations [3]. PS was not as effective as intact membranes and further fractionation of the membranes identified diacylglycerol (DAG) as an additional cofactor that could increase maximal activity and lower the concentrations of calcium required to physiologically relevant (micromolar) levels [4]. This discovery linked PKC to hormone-stimulated

2. STRUCTURAL FEATURES OF PROTEIN KINASE C

t Author to whom correspondenceshould be addressed. Abbreviations: eDNA-----complementarydeoxyribonucleic acid; DAG-----diacylglycerol;EGTA----ethyleneglycol-bis(flaminoethyl ether)-N,N,N',N'-tetraacetic acid; EPR-electron paramagnetic resonance; LUVs---largeunilamellar phospholipid vesicles; MLVs---multilamellar phospholipid vesicles; NMR--nuclear magnetic resonance; PA--phosphatidic acid; PC--phosphatidylcholine; PE--phosphatidylethanolamine; PI--phosphatidylinositol; PKC--protein kinase C; PKM--protein kinase M; PS---phosphatidylserine. 595

Early enzyme purification [6, 7] as well as cloning [8-11] revealed that PKC is actually a family of isozymes. Sequence analysis demonstrates four regions of homology among the isozymes, constant domains C1-C4, separated by variable regions, domains V1-V5. The structure and function of each of these regions have been reviewed recently by Stabel and Parker [12]. Calpain [13] and trypsin [14J-mediated proteolysis occurs in the V3 region generating the active catalytic C-terminal fragment (PKM) containing C3 and C4 domains, and an N-terminal regulatory fragment containing C1 and C2 domains which retains membrane binding functions [15-17]. The catalytic domain is homologous to the catalytic subunit of other kinases including the recently crystallized cAMP-dependent kinase [18]. The C3 domain contains a consensus ATP binding sequence [12] and, based on homology with other kinases, the C4 domain is hypothesized to be involved in substrate recognition. The originally identified isozymes ct, fit, fltl, and ~ contain all of these domains, but the more recently

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discovered 6, e, ~, and r/isozymes lack the C2 domain and have been found to be CaE÷-independent (reviewed in [12]). House and Kemp first recognized that the N-terminal part of the C1 domain contains a basic-rich sequence similar to the phosphorylation sequence for many substrates except that an alanine replaces a phosphorylatable serine or threonine [19]. Based on the similar sequence in the inhibitory protein of cAMP-dependent kinase [20], they proposed that this region serves as an autoinhibitory or pseudosubstrate domain and showed that synthetic peptides with this sequence inhibited PKC, whereas peptides in which the alanine is replaced with a serine serve as substrates [19]. This model explains activation by proteolysis which eliminates this autoinhibitory domain. It also predicts that cofactors or substrates may activate the enzyme by inducing or stabilizing an 'open' enzyme conformation in which the inhibitory domain has been removed from the active site. Markowske and Rosen provided additional evidence for this model by showing that an antibody directed at the pseudosubstrate sequence activates the enzyme [21], and Pears et al. showed that mutations in this region also generated a cofactor-independent kinase [22]. Orr et al. have provided more direct support for this model by demonstrating that association of PKC with PS-containing membranes causes a conformational change that exposes the pseudosubstrate region to cleavage by an added endoprotease [23]. While the pseudosubstrate model is widely accepted and supported, it does not predict how cofactors relieve the autoinhibition or explain the co factor-independent activity observed with some substrates (see below). Further characterization of the cofactors, coupled with the sequence analysis of the enzymes, has led to development of several models for PKC activation. These models continue to undergo revision as new information is gathered. The following sections will review the cofactor requirements and the current evidence in support of various activation models. The significance of the activation mechanisms for

cellular signal transduction will be discussed at the end.

3. CHARACTERIZATION OF PROTEIN KINASE C COFACTORS D A G p h o r b o l ester requirements

Castagna et al. first observed that the DAG requirement for activation of PKC could be replaced by phorbol ester tumour promoters [24]. This provided the first suggestion that PKC was the phorbol ester receptor. The effects of phorbol esters on apparent translocation of PKC from cytosol to membrane [25, 26], the similar cofactor requirements for phorbol ester binding and PKC activation [27-29], and the demonstration that DAGs compete for phorbol ester binding [30] further supported this idea. Co-purification of the two activities [31-33] and expression of both activities from cDNAs [10, 34] confirmed the association. Phorbol ester binding was speicfic [27-29] and stoichiometric [31], arguing for direct binding to a specific site on the enzyme. Stereospecific PKC activation [35, 36] and saturable, stoichiometric binding [37] by DAGs have also been shown. Other tumour-promoting compounds of diverse structure have also been found to activate PKC. These include teleocidins [38], aplysiatoxins [38], bryostatins [39], lyngbyatoxins [40, 41], and mezereins [42]. Computer modelling based on common structural motifs of the activators has been carried out by several groups [43--46] in an attempt to characterize the structural features required in the activator and the corresponding structural features of the PKC binding site. Evidence from many laboratories supports the importance of the free hydroxyl and both carbonyl groups of DAG, and these have been matched by modelling to structures in the tumour promoters. Rando and Kishi have recently analysed DAG/tumour promoter structures based on function of a series of analogues of the conformationally rigid debromoaplysiatoxins [47]. Using this pharmacophore approach, they argue strongly for involvement of different functional groups

Activation of protein kinase C on some of the tumour promoters than has been predicted with computer modelling. In addition to specific functional groups, all activators also contain a hydrophobic domain. In the case of DAGs, this is provided primarily by a long acyl chain in the 1-position and an equal or shorter one in the 2-position. Active DAGs could be achieved with trans- as well as cisunsaturated [36], saturated [36], and short chain [48] acyl groups, leading to the hypothesis that hydrophobicity sufficient for membrane partitioning is the relevant feature. Potency of phorbol esters is prominently affected by the nature of the 12- and 13-acyl chains as well. Recent deletion analysis of cloned PKC isozymes indicates that the phorbol ester binding site is localized to the cysteine-rich sequences of the regulatory C 1 domain [49, 50]. These sequences have recently been found to contain tightly bound zinc ions which are required for phorbol ester binding [51-53]. There are two such sequences in all but the isozyme which fails to bind phorbol esters [54]. Burns and Bell have reported that both of these domains can bind phorbol esters [50], raising questions about the stoichiometry of binding. However, other deletion studies suggest that the second cysteine-rich sequence is more important for conferring phorbol ester binding [55, 56]. The cysteine-rich sequence in ( more closely resembles the first such sequence in the other isozymes (see [12]). Phospholipid requirements

Early kinase [3] and phorbol ester binding [27-29] studies indicated that PKC requires acidic phospholipids. PS was preferred, but some activity could be obtained with phosphatidic acid (PA) and phosphatidylinositol (PI). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were ineffective. Extensive structural analysis by Lee and Bell indicated that phosphate, carbonyl, and amino groups of the PS are required for PKC activity [57]. Stereospecificity for L-serine and precise distance between the functional groups suggested that PKC might specifically bind to

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PS head groups. Phospholipid requirements for phorbol ester binding were similar, except that the amino group was not required and phosphatidyl-D-serine was effective. Many reports have shown a requirement for multiple PS molecules to activate PKC [ 3 7 , 57-60]. Unsuccessful attempts to demonstrate activation with monomeric short-chain PS [61] argued that the enzyme needed PS at a membrane surface or in a hydrophobic domain. Evidence that large unilamellar phospholipid vesicles (LUVs) were better than multilamellar vesicles (MLVs) was consistent with a surface requirement [36]. Mixtures of phospholipids were also examined and addition of PE to PS was found to increase activity, whereas addition of PC with PS decreased activity [62, 63]. A specific site on PKC for interaction with phospholipids has not yet been identified. While the regulatory fragment can provide this function, possible lipid interactions with the catalytic region have also been suggested [64]. Metal requirements

As mentioned above, all isozymes have the cysteine-rich C1 domain and are thus expected to contain tightly bound zinc ions. The calcium requirement for PKC activation is specific to isozymes ~t, fl~, ft., and ~. Effects of other metals have been examined with mixed results. Speizer et al. suggested that conflicting results may be due to indirect stimulation of PKC at low metal concentrations via liberation of Ca 2÷ from EGTA buffers, followed by direct inhibition of PKC at higher concentrations [65]. The mechanism for PKC inhibition by metals has not been determined; however, we have recently obtained evidence that Gd 3+ can inhibit the enzyme by formation of GdATP which competes at the active site [66]. It is possible that the inhibition of PKC activity at high Ca 2+ concentrations is due to a similar mechanism. Direct Ca 2+ binding to PKC has only been examined in one study. Using equilibrium dialysis, Bazzi and Nelsestuen demonstrated that a mixture of isozymes binds at least 8 tool 45Ca2+/mol/enzyme in a PS-dependent manner

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[67]. These sites are hypothesized to reside in the C2-domain which is absent in the Ca2+-independent isozymes. We have used Gd 3+ as a paramagnetic probe for Ca 2+ sites in NMR studies and also observed a class of multiple metal sites (>/8) on PKC0t and PKCfl [68]. However, these were detected in the presence of short-chain PC micelles which do not bind metal, as determined by lack of Ca 2÷ effect on the critical micelle concentration of the PC [69] and absence of detectable Gd3+-PC binding in NMR experiments [68]. These observations argue that the metal sites are intrinsic to the enzyme, although whether the PC is needed to expose the sites is unknown. In addition, we observed a single, high-affinity Gd 3+ site unique to PKC0t [68]. Interaction of the Gd 3÷ with both nucleotide and peptide substrates suggests that this site is located at or near the active site [68]. Although no classical E-F hand type Ca 2+ binding sites have been identified in the PKC sequences, the V3 region of PKC~t was noted to most closely resemble such a site [8]. The observation that the V3 region of PKCfl can undergo autophosphorylation by an intramolecular mechanism [70] suggests that the V3 region has access to the catalytic site. The hypothesis that the V3 domain of PKC0t provides the unique Ca 2+ site remains to be evaluated.

lated better with activity [73]. Membrane binding in the absence of activity was also observed when other acidic phospholipids were substituted for PS in both vesicle- [60] and Triton-mixed micelle [57, 74, 75] systems or when DAG was omitted in Triton-mixed micelles [37]. Some studies have even demonstrated irreversible inactivation of PKC upon its association with membranes containing acidic lipids [64, 76, 77]. This inactivation was timedependent and did not occur in the presence of Mg 2÷ [64] or MgATP [76, 77]. Huang and Huang observed that separated catalytic and regulatory fragments could each interact with PS in the absence of divalent cations, whereas inclusion of divalent cations allowed only the regulatory fragment to interact with the lipid, thus preventing the inactivation [64]. Phorbol ester binding activity was retained in the inactivated PKC [64]. The mechanism for this lipidinduced inactivation of the catalytic activity has not been elucidated, although protection against the inactivation may be due to occupation of a Mg 2÷ or MgATP site on the enzyme. Hannun and Bell have provided kinetic evidence that free Mg 2+ and MgATP can interact with the catalytic fragment as well as the intact enzyme independent of cofactors

[78]. 4. MODELS FOR PKC ACTIVATION AT THE MEMBRANE The majority of data from in vitro systems (e.g. [3]) as well as intact cells (e.g. [25, 26]) suggests that PKC becomes active when it associates with membrane. However, several studies have shown that membrane binding is not sufficient for activity. Wolf et aL observed binding of PKC to red cell ghosts at concentrations of Ca 2+ insufficient for kinase activity [71]. Others have similarly identified more than one form of membrane-bound enzyme on the basis of chelator sensitivity and other properties using defined lipid vesicles [60] or cellular membranes [72]. Bazzi and Nelsestuen noted that appearance of the more slowly generated, chelator-insensitive membrane binding corre-

Interdependence between the PKC cofactors and elimination of cofactor requirements in some circumstances have suggested that there may be several ways to activate PKC. Alternatively, all activating conditions may share common features and support PKC activation by a unified mechanism. Available evidence supports a model in which the pseudosubstrate sequence is removed from the PKC active site, but the possibility of several active PKC conformations or distinct activation states exists. Evidence in support of a number of models will be reviewed. Role of Ca 2÷ The calcium bridge model. The PKC requirements for Ca 2+ and acidic phospholipids

Activation of protein kinase C suggested that Ca2+-PS interactions might be important for the activation. Bell's group used a Triton-mixed micelle system to determine a stoichiometry for PKC activation of four PS molecules, 1 Ca 2+ and 1 DAG [37, 58, 59]. This led them to propose a Ca 2+ bridge activation model in which the acidic lipid head groups coordinate the Ca 2+ which then interacts with PKC. This mechanism--predicting a requirement for a charged membrane surface and lacking a requirement for intimate PKC-lipid interaction--has been widely embraced for a number of years. This model is supported by the demonstration of multiple PS-dependent binding sites for 45Ca2+ on mixed isozymes of PKC [67]. Some other data cannot be explained by a Ca2+-PS bridge model for membrane binding. First, the more recently identified isozymes 6, e, ~, and ~/do not require Ca 2+ for activation and yet do require phospholipids/membrane binding [79-83]. Second, we observed activation of mixed isozymes [69] or separated ~t and fl PKC [61] with PC micelles replacing the acidic PS vesicles. Furthermore, this system eliminates a Ca 2+ requirement for PKCfl but not PKC0t [61]. The multiple Gd 3+ sites we identified on PKC~ and /3 may be consistent with a metal bridge mechanism; however, acidic lipids were not required to observe these sites [68]. It is possible that a Ca 2+ bridge structure forms and is required for activation of PKC0t, /3, and when PS is present, but the metal binding observed in the PC system is not necessary for activity of PKCfl. Ca 2+ activation of PKC~. The role of the active site Ca ,+ in PKCet is unknown. This isozyme requires 10-fold less Ca 2+ than does PKCfl in vitro, suggesting that occupation of this site may eliminate some of the dependence on the other Ca 2÷ sites. It may be noteworthy in this regard that PKCct is less susceptible to lipid-stimulated inactivation than is PKCfl or PKCy [64, 77]. Occupation of this site also seems to be essential, since conditions that eliminate the Ca 2+ requirement for PKCfl and 7 (short-chain PC micelles [61, 66], fatty acid long-chain PC vesicles [84]) do not eliminate the

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Ca 2÷ requirement for PKC~t. Depending on the location of the site, this Ca 2+ might stabilize an active conformation of the enzyme or even participate in catalysis. Ongoing N M R and EPR analyses should help to answer these questions.

Role of phospholipid headgroup Cooperative interactions with PS. Cooperative activation of PKC by PS has been noted by a number of investigators [37, 59, 60, 63, 78, 85, 86]. Unusually high Hill coefficients of 4-11 were observed in the PS-Triton micelle system [37, 59, 63, 78], whereas more typical values of 2-3 are observed in vesicle systems [60, 85, 86]. Sandermann and Duncan suggest that the unusually high Hill coefficient for PS-dependence in Triton micelles may be due to a PS trapping artifact, such that much of the PS is not available [87]. The cooperativity in both systems has suggested that PKC interacts sequentially with multiple PS molecules. Bazzi and Nelsestuen showed self-quenching of fluorescently labelled PA or phosphatidylglycerol upon binding of PKC to mixed-lipid vesicles, thus supporting a model in which PKC sequesters acidic lipids [88]. Since binding was also highly cooperative with respect to Ca 2+ [89], they suggested that Ca 2+ may participate in the phospholipid clustering. Although the stereoselectivity for PS head groups [57] suggests the existence of specific PS binding sites on PKC, Newton and colleagues [63, 74, 75] and Lee and Bell [90] have shown that a portion of the PS requirement for activity can be replaced with other phospholipids, arguing for two types of interaction with PS. Orr and Newton show that PKC can bind to PA-Triton mixed micelles in a manner that is affected by ionic strength and surface charge, but not DAG, and that such binding does not result in activity [74, 75]. Binding to PS-Triton micelles is relatively insensitive to ionic strength and surface charge but dependent on DAG and Ca 2+. Activity of the PA-bound enzyme is still cooperatively dependent on PS [74]. These results are consistent with specific binding of

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several PS molecules plus less specific interactions dependent on surface charge. Cooperativity with respect to PS is also observed under more physiological conditions of P E P S mixtures [63, 91]. Bazzi and Nelsestuen showed that replacement of PC with PE in mixed PS vesicles significantly lowered the Ca 2÷ requirement for binding and activity and that, at very high Ca 2÷, activity could be observed with PC-PE mixtures in the absence of PS [91]. Although activation by PE-PC vesicles [91] or PC micelles [61, 69] is unlikely to occur physiologically, it does raise questions about the PS requirement. Final assessment of possible PKC-PS binding awaits additional physical studies. Membrane binding via basic amino acids. Recent evidence from McLaughlin's group shows that peptides corresponding to the PKC pseudosubstrate region can bind to phospholipid vesicles via cooperative binding of the basic amino acids to individual PS molecules [92-94]. This mechanism also features a charge interaction at the membrane surface, but no Ca 2+ is involved. It is proposed to provide a net binding energy of 6 kcal/mol enzyme toward stabilizing an 'open' form of the enzyme with the pseudosubstrate region removed from the active site. This model is hypothesized to explain the high cooperativity of PKC activation as a function of PS based on 'reduction of dimensionality' that would occur as PKC binds successive PS head groups at the surface of the membrane. This model remains to be tested with intact PKC, but the PS-specific exposure of the pseudosubstrate to an exogenous protease [23] is consistent with such a possibility. Role of phospholipid physical state Evidence from micelle systems. The above activation models do not depend on the physical state of the membrane, although binding via charge interactions could well be affected by changes in the physical state of the membrane that would affect head group spacing or charge density. The finding that

short-chain PCs can replace the requirement for acidic head groups led us to hypothesize that a charge interaction was not essential for PKC activation and that some other sort of PKC-membrane interaction was occurring [69]. The fact that short-chain PC micelles support activity while long-chain PC vesicles do not suggested that physical features of the lipid must be important. The dynamic state of micelle vs vesicle lipids and the increased head group spacing in micelles would be expected to facilitate interactions between PKC and the hydrophobic region of the membrane, leading us to favour an insertion model for PKC-membrane interaction. Evidence from vesicle systems. It is clear from the short-chain PC studies that PS head groups are not necessary for activation of PKC in micelles; nor are PS head groups sufficient in vesicle systems. We have recently observed that PC-PS-DAG (70:25:5) vesicles cannot support PKC activity if all of the lipid acyl chains are saturated [95]. The requirement for acyl chain unsaturation--in either the PS or the background PC lipid--argues that the physical state of the membrane is critical for PKC activation. This conclusion is also supported by work of Epand et al. showing that branched acyl chains increase PKC activity [96]. It is not clear at this point what physical properties of the membrane are important for PKC activation. The dynamic properties of micelles and the fluidizing properties of unsaturated vesicle lipids prompted us to consider that PKC might require a fluid membrane, e.g. for an insertion event [95]. However, this hypothesis was not supported by experiments in which cholesterol was added to decrease the fluidity. Activity was increased rather than decreased. Other possibilities under consideration are phase-separation and head group spacing. Addition of unsaturated acyl chains or cholesterol to the membrane would each have the effect of increasing head group spacing. Inclusion of branched chain lipids [96] would have a similar effect. In addition, these conditions destabilize the bilayer and lead to an increase in hydrocarbon volume. Mitchell et aL have shown that acyl chain unsaturation and

Activation of protein kinase C cholesterol also affect activation of the intrinsic membrane protein rhodopsin from the meta I to the meta II state by affecting bulk phospholipid acyl chain packing [97]. That PKC would require physical features of the membrane similar to those required for intrinsic membrane proteins furthers the argument that insertion of PKC into the membrane may be required for the conformational change that relieves the autoinhibition and exposes the active site of the enzyme. It is possible that PE contributes to PKC activation in part by this mechanism since it is noted for its ability to induce hexagonal phase structures which have increased acyl chain volume [98]. Evidence for membrane insertion. An insertion mechanism is most directly supported by the monolayer insertion studies of Bazzi and Nelsestuen [99] and Souvignet et al. [100]. Fluorescent energy transfer between PKC and a probe in the hydrophobic region of vesicle lipids [101] supports an insertion mechanism in bilayer vesicles as well. Models based on physical properties of the membrane do not explain the requirement for PS head groups in vesicle systems. One possibility is that interactions (of PKC, substrate, or Ca 2÷) with PS provide a means of overcoming a barrier to insertion. Such a barrier is expected to be minimal in a micelle system. Role of diacylglycerol

The direct phorbol ester binding studies and DAG competition argue strongly for a specific binding site in the C1 domain of PKC, as discussed above. How this binding contributes to activation is not fully apparent. It is possible that bound DAG sterically hinders association of the pseudosubstrate domain with the catalytic portion of the molecule and that a permissive membrane is required primarily to permit access of PKC to the hydrophobic environment where DAG is found. A more commonly offered speculation is that DAG strengthens the PKC-membrane binding by 'anchoring' the enzyme in the membrane. This explanation would seem to be more consistent with the

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observation that activation can be achieved without DAG given sufficient Ca 2+, PS, and/or unsaturation in the phospholipid acyl chains. However, it seems likely that at high concentrations, DAGs can also contribute to physical properties of the membrane required for PKC activity. Fully saturated PC-PS vesicle systems could be rendered effective for PKC activation but only at very high (25 mol %) concentrations of DAG [96]. The DAG, in this case, may be contributing to physical properties of the membrane instead of or in addition to direct binding to the enzyme. While such DAG concentrations will not occur physiologically, localized concentrations of DAG could contribute to activation by effects on membrane properties as well as via direct PKC binding. Das and Rand [102] and Epand [103] have shown that DAG destabilizes the lamellar structure of membranes. Concentrations of 5-10mo1% caused lamellar-to-hexagonal phase transition in PE, whereas 30 mol % was required for the same transition in PC [102]. The source of the DAG has also been questioned. Although PI hydrolysis was originally identified, PC hydrolysis has also been linked to PKC activation (for review, see [104]). Based on analysis of acyl chain composition, Leach et al. argued that only PI-derived DAG led to activation of PKC in cells [105]. It may be noteworthy that the PKC isozymes examined in that study were Ca2+-dependent. The possibility that Ca2+-independent hydrolysis of PCs gives rise to activation of Ca2+-independent PKC isozymes remains to be tested. In support of isozyme-specificity at the DAG binding site, Ryves et al. have recently reported isozyme specificity in the order of potency for activation by phorbol ester [106]. 5. OTHER MODELS FOR PKC ACTIVATION Role of fatty acids

Many studies have shown that cis-unsaturated fatty acids can also activate PKC [36, 84, 107-115] but the mechanism for this activation has been controversial. Activation can occur in

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the absence of phospholipids [107-113] leading to the hypothesis that soluble PKC might be activated by soluble fatty acid generated in response to distinct signal transduction pathways. This soluble PKC would be expected to have access to different substrates. The strongest arguments for PKC activation by monomeric fatty acids are provided by Murakami et aL [109] who show activation at fatty acid concentrations significantly below the critical micelle concentration of the pure lipid, and by Khan et al. [113] who show that partitioning of fatty acid into Triton-mixed micelles decreases the effectiveness of fatty acid as a PKC activator. Other studies have shown that fatty acids synergize not only with DAGs [84, 107, l ll, ll4, ll5] but also with PS vesicles [114, ll5]. Chen et aL have even shown that addition of cis-fatty acids rendered long-chain unsaturated PC effective as an activator of PKC in the absence of PS [84]. Unlike DAG, fatty acids were not found to compete for phorbol ester binding to PKC [112]. Although Ca 2+ was not required for PKC activation by fatty acids in some studies [109], Ca2+-dependence is reported in others [107, l l0, l l2]. The difference in Ca2+-dependence is likely to be due to differences in isozyme composition. Sekiguchi et al. have shown marked differences between isozymes ~, fl, and 7 in responsiveness to fatty acids and Ca 2+, with PKC~ exhibiting the most dramatic activation by both [ll0]. Murakami and colleagues show that synergistic activation of PKCct by fatty acid and DAG dramatically decreases the Ca 2+ requirement [115] and that fatty acid-PC vesicle systems eliminate the Ca -'+ requirement for PKCfl and ? but not for PKCct [84]. While it is clear that cis-fatty acids can activate PKC in vitro, the role that this activation mechanism plays in the cell remains to be established. It has been hypothesized that delayed accumulation of fatty acids may account for activation of PKC at later times after stimulation of cells [110]. Role of other proteins The substrate aggregation model. In 1987, Ferrari et aL [116] and Bazzi and Nelsestuen

[117] provided evidence that the PKC requirement for cofactors was substrate-dependent. In fact, it had been noted in the first PKC paper from the Nishizuka lab [2] that PKC phosphorylates protamine in a cofactor-independent manner. Bazzi and Nelsestuen showed that protamine sulphate, not protamine was the effective substrate [118] and they presented a correlation between the cofactor requirements for substrate aggregation and cofactor requirements for PKC-mediated phosphorylation of the substrate [117]. Evidence was presented [85] that this model could explain the PS concentration-dependence that formed the basis for the Ca 2+ bridge model. PKM activity was also stimulated by phospholipids, probably via effects on the substrates [119]. Many in vivo PKC substrates are localized to the membrane but the validity of the substrate aggregation model in intact cells remains to be established. It does not explain the observed autophosphorylation of PKC unless the PKC itself is aggregated in this situation. Most reports argue for an intramolecular autophosphorylation mechanism that is independent of enzyme aggregation [120, 121]; however, some data are consistent with a requirement for PKC aggregation (see below). The mechanism for PKC activation by cofactor-independent substrates is not established. Membrane binding via P K C receptor proteins.

Two 1986 reports suggested that PKC binds membranes via interaction with proteins present in the membrane [72, 122]. Several candidate proteins have been identified via binding of PKC to cellular proteins separated on a polyacrylamide gel [122, 123]. This binding is dependent on the usual PKC cofactors, including DAG and PS. Mochly-Rosen and coworkers identified one binding protein as annexin I [123] and showed that a peptide corresponding to the region of homology between annexin I [124] and a protein inhibitor of PKC [125] could inhibit PKC-protein binding [126]. They have recently provided evidence that this interaction is mediated by the PKC C2 domain [127]. Fragments of the synaptic vesicle protein p65 which contain

Activation of protein kinase C regions homologous to the PKC C2 domain bound to the same PKC binding proteins, and binding of p65 and PKC were mutually exclusive [127]. Although it is clear that PKC can interact with membrane lipids in the absence of additional proteins, it is suggested [126, 127] that these PKC binding proteins may help to target the enzyme to specific sites within the cell such as focal adhesions [128] or the nuclear envelope [129]. James and Olson have recently provided evidence that activation of PKC~ exposes a nuclear localization site that may recognize PKC binding proteins in the nuclear membrane [130]. Deletion analysis suggests that this site includes portions of the unique PKCa V3 region. Further genetic and structural analysis should help to localize specific sites on PKC involved in protein-protein interactions. Protein inhibitors o f PKC. Many studies have demonstrated the existence of endogenous inhibitors of PKC (e.g. [126, 131-134]) and a report of a PKC activator also exists [135]. A complete review of this area is beyond the scope of this Mini Review, but several already sequenced inhibitors are noteworthy. Pearson et al. have cloned a 13,700 M r inhibitor termed PKCI-1 [131] which binds zinc via a novel sequence motif [132]. Aitken and co-workers have purified and cloned a set of PKC inhibitors that has homology to the annexin family of proteins and a brain protein known as 14-3-3 [125, 133]. It is possible that these inhibitors interact with the C2 domain of PKC as discussed above. Only C2-containing isozymes of PKC would be expected to be regulated by inhibitors employing this sort of interaction. Schlaepfer et al. have recently reported that annexin V is also a potent inhibitor of PKC and have speculated that a pseudosubstrate-like sequence unique to the exposed surface of this annexin may mediate the inhibition [134]. The mechanism of action and relative importance of the various inhibitors is yet to be determined. Role o f P K C phosphorylation

PKC has long been known to undergo autophosphorylation [70, 1 2 1 , 136-140] and

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resulting effects on activity have been noted. The autophosphorylation was reported to exhibit a lower Km for ATP than heterologous phosphorylation and to increase enzyme activity by decreasing the Ka for Ca 2÷, increasing affinity of phorbol ester binding [138], and decreasing the Km for histone [140]. Wolf et al. observed that MgATP reversed membrane binding of PKC [141], suggesting decreased affinity of the phosphorylated enzyme for membrane. Based on studies with kinase-negative PKC in which the lysine at the ATP site was replaced with arginine, Ohno et al. suggested that autophosphorylation was required for proteolytic down-regulation [142]; however, studies in which kinase activity was eliminated by conversion of the lysine to methionine [143, 144] or by use of PKC inhibitors [145] found that unphosphorylated PKC was equally susceptible to down-regulation. Several studies have shown an increase in PKC phosphorylation upon its activation in cells (e.g. [146-148]). Borner et al. provided evidence for at least two phosphorylation states leading to increased activity [146]. The first phosphorylation event could not be reproduced with purified PKC in vitro, arguing for involvement of an additional kinase [146]. Casein kinase I [149] has been found to phosphorylate PKC in synergy with the autophosphorylation, and casein kinase II was also reported to phosphorylate PKC [150], suggesting candidate kinases for the in vivo result. Differences between autophosphorylation and histone phosphorylation have raised some questions about the mechanisms involved. The autophosphorylation required more PS than did histone phosphorylation [63] and autophosphorylation was not supported by fatty acids whereas histone phosphorylation was [84, 112]. Autophosphorylation per unit PKC did not change as PKC was diluted with respect to total assay solution [138] or PS-Triton mixed miceile concentration [121], suggesting that it is an intramolecular event. This conclusion was consistent with gel filtration data arguing that active PKC is a monomer [37]. Flint et al. have reported that the autophosphorylation occurs

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on several sites scattered throughout the PKC molecule with only one site phosphorylated in a given molecule and suggest that PKC can exist in multiple conformations [70]. These sites did not exhibit the basic-rich consensus sequence typical of other PKC substrates. Data consistent with dimerization also exist. Mochly-Rosen and Koshland observed elution of autophosphorylated enzyme from a sizing column at a higher molecular weight [140]. We found that autophosphorylation [151], like histone phosphorylation [68], decreased as concentration of short-chain PC was increased above the critical micelle concentration. Such an effect would be expected if dimerization occurs at a micelle surface or lipid aggregate, and enzyme is diluted out with respect to lipid. The synergism between autophosphorylation and casein kinase I-mediated phosphorylation is also dificult to understand in the context of low stoichiometry autophosphorylation on scattered sites in the PKC molecule. Given the potential significance of PKC phosphorylation in cells, further analysis of the mechanism and consequences is warranted. Role of proteolysis Although PKC can be activated by proteolysis in vitro, the possible role of proteolytic activation in the cell has been controversial. In support of a physiological role for proteolytic activation, Horecker and co-workers have shown that neutrophil activation is associated with loss of PS/Ca2÷-dependent kinase activity, coincident with appearance of cofactor-independent activity [152-155], and that both effects are blocked by an antibody to calpain [156]. Calpain I has been shown to cleave PKC at specific sites in the V3 region and is stimulated by PS and by micromolar Ca 2÷ [157]. Although down-regulation of PKC subsequent to its activation is of wide-spread occurrence, many investigators have been unable to detect the catalytic fragment using specific antibodies (e.g. [150, 158]), suggesting that any generation of PKM is a very transient step on a pathway to complete proteolysis. Pelech et al. [150] have

suggested that phosphorylation of PKC may generate a cofactor-independent form of the enzyme without proteolysis and suggested that this effect might explain results such as those in neutrophils. Differences in proteolytic sensitivity of the isozymes have been observed with PKC~t exhibiting greatest resistance [155, 157, 159]. PKCe was down-regulated in response to thyrotropin-releasing hormone [159] or ionomycin but not phorbol esters [160]. While all of these studies suggest that PKC proteolysis occurs physiologically, whether it serves to generate PKM or to terminate responses may be isozyme- and/or cell type-specific. 6. SUMMARY AND CELLULAR SIGNIFICANCE It must be emphasized that the above mechanisms are not mutually exclusive. In addition, some features may be more important than others in certain circumstances. A cartoon incorporating various models for PKC-membrane binding and activation is shown in Fig. 1. Here it is suggested that PKC can insert into the hydrophobic domain of the membrane to stabilize a conformation in which the pseudosubstrate region is removed from the active site. Physical properties of the membrane such as head group spacing or lipid acyl chain interactions are proposed to affect the ability of PKC to undergo the insertion event. This part of the model suggests that PKC activity may be sensitive to alterations in physical properties of the membrane affected by membrane active drugs or even changes in dietary lipids. Examples of such agents or manipulations affecting PKC activity already exist. Phenothiazines [161], palmitoyl carnitine [162, 163] and local anaesthetics such as dibucaine [164] inhibit PKC activity, and organic solvent anaesthetics such as chloroform [165] activate the enzyme. Incorporation of cis-unsaturated fatty acids from cell culture medium into cellular phospholipids has been shown to prolong PKC activation in lymphocytes [166] and to enhance translocation of PKC to membrane following prolactin treatment of mammary epi-

Activation of protein kinase C

605

@

@

@ C

FIG. 1. Models for activation of PKC.

thelial cells [167]. Whether the individual PKC isozymes will require different physical properties of the membrane is not yet known. Such differences could favour localization of specific isozymes to certain membranes and affect access to different substrates. Also shown is the direct binding of the basic amino acid-rich pseudosubstrate sequence to PS head groups which is hypothesized to help stabilize the 'open' or active conformation. This mechanism would be common to all PKC isozymes but direct binding of this region of the intact enzyme to PS has not yet been demonstrated. It is possible that this PS-pseudosubstrate binding accounts for the apparent co-operativity observed as a result of a reduction in dimensionality as more PS is bound. The initial membrane binding may involve binding of the C2 domain via a PKC-Ca2÷-PS bridge as depicted. This mechanism could explain the large number of Ca :÷ ions as well as

PS molecules required. However, it would not be available with the Ca2+-independent PKC isozymes that lack a C2 domain, nor would it be required in PC or fatty acid micelle systems which minimize any barriers to PKC-membrane insertion. The hypothesized sequestering of PS around the enzyme could occur via the Ca2+-dependent C2 binding and/ or via the Ca2÷-independent pseudosubstrate binding. D A G or phorbol esters are proposed to stabilize the membrane-bound form of PKC and in this summary model would bind only after the enzyme has associated with membrane. The interdependence between D A G requirements and PS-Ca 2÷ requirements may suggest that either cofactor set can provide the same stabilization of membrane-anchored PKC but via different mechanisms. DAGs as well as other membrane-associated molecules or perhaps a PS-Ca 2÷ interaction, could also contribute to formation of the appropriate

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physical state of the membrane for P K C insertion as discussed above. The active site Ca 2+ of PKC~t is hypothesized in the illustration to be localized to the V3 hinge region, which more closely resembles a portion of an E-F hand site than does the comparable region of PKCfl. Whether occupation of this site minimizes the requirement for other cofactors is not well established. However, it seems likely that this unique site will permit activation of PKC~t under conditions that are distinct from those that activate PKCfl or other isozymes. N o t illustrated in the figure are models in which P K C is activated by proteolysis, by fatty acids in solution, by phosphorylation, or by interaction with other proteins. Cellular evidence in support of these mechanisms has been mentioned. The relative importance of the various mechanisms is unknown but it seems that P K C is poised to respond in several ways to a number of perturbations in its environment. The nature of the perturbation may determine the subcellular site of activation, the isozymes which can respond, and the access to substrates which mediate downstream events. Acknowledgements--We are grateful to CINDY KLEVICKiS for assembling Fig. 1 and we thank DRS ALEXANDRANEWTONand DARIAMOCHLY-ROSENfor sharing preprints of their manuscripts in press. This work was supported by NIH grant GM-31184.

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