Summary Phospholipase C is a family of cellular proteins believed to play a significant role in the intracellular signaling mechanisms utilized by diverse hormones. One class of hormones, polypeptide growth factors, elicits its influence on cellular function through stimulation of cell surface receptor tyrosine kinase activity. Certain growth factors appear to stimulate cellular phospholipase C activity by selective, receptor-mediated tyrosine phosphorylation of the phospholipase C-y, isozyme. While the role of phospholipase C activity in growth factor regulation of cell proliferation remains to be clarified, the selective growth factor-stimulated tyrosine phosphorylation and activation of phospholipase C - y l is an interesting example of enzyme-substrate interaction at the crossroads of two important intracellular signaling pathways.

Growth Factors in Cell Proliferation Cell proliferation plays a fundamental role in the development and maintenance of organisms. In the embryo, there is rapid, programmed cell division, progressive, selective induction and differentiation of tissues, and organogenesis. In the adult, there is continued rapid renewal of many epithelial tissues and hematopoietie lineages. A variety of biological factors are known to influence cell proliferation. including the polypeptide growth factors. A large numbcr of growth factors purified from biological sources are characterized by their ability to influence the proliferation of specific cell lineages"). The basis for differential cell responsiveness to growth factors is complex. and results from the selective expression of receptor proteins on the cell surface, the tonic influence of other growth regulatory factors in the biological milieu, and the presence of intrinsic signaling or processing mechanisms. Disruption of these regulatory processes leads to absent (aplastic) , inappropriate (metaplastic) , or persistent (neoplastic) cell proliferation. Since the biochemical pathways required for growth homeostasis also underlie expression of the pathological states, an understanding of thc molecular basis for growth factor effects helps to define the mechanisms of aberrant cell growth.

EGF and its Receptor Epidermal growth factor (EGF) is one of the most thoroughly investigated peptide growth factors and in many ways has been a model system for revealing the biological and biochemical mechanisms of growth control. EGF was originally purified thirty years ago, the assay being based on its capacity to induce precocious eye-opening and inciser-eruption in newborn mice(2). Since then, EGF has been demonstrated to regulate growth and differentiation, especially of epithelial cell types in the intact animal. EGF is present in detectable quantities in many biological fluids, and in the normal animal it may function in an autocrine or paracrine fashion, produced and acting within a local cellular or tissue en~ironment(~). However, the specific physiological roles of endogenous EGF in the intact animal remain uncertain. The capacity of EGF to influence cell growth or metabolism depends primarily on the expression of the EGF receptor on the surface of target cells. Like other members of the growth factor receptor family (insulin, insulin-like growth factor-I, colony-stimulating factor1, platelet-derived growth factor, and fibroblast growth factor reccptors), the EGF receptor contains an extracellular, a single transmembrane, and an intracellular domain(". The extracellular domain of the EGF receptor selectively binds with high affinity EGF and several other polypeptides homologous to EGF. One hydrophobic mem brane-spanning domain anchors the rcceptor on the cell surface. The intracellular domain is especially interesting, since it contains an intrinsic enzymatic activity, i.e., tyrosine kinase, that i? activated by EGF binding. Receptors in Signaling Pathways Cells may express several classes of receptors, which respond to distinct agcmists and couple to one or more intraccllular signaling pathways. Signaling pathways are the changes in cellular metabolism, often involving small molecules, that are induced by hormones, and that result in changes in cellular function or state. Physiological, biochemical, and genetic analyses have been employed to examine structural and functional relationships among the receptor, effector, and regulatory proteins that participate in the growth factor signaling pathways. The EGF receptor transmits within the cell a biochemical signal after EGF binds to the receptor extracellular domain("'). Transmission of this signal at the cell's plasma membrane is a complex process in which there are receptor-receptor interactions. stimulation of receptor tyrosine kinase activity, and phosphorylation of multiple tyrosine residues near the intracellular (-carboxyl terminus) tail of the receptor. Activation of the receptor tyrosine kinase after EGF binding (Fig. 1) allows the receptor to contact and covalently modify cellular proteins (by phosphorylation of tyrosine residues), and through these interactions to provoke additional cellular responses. These signaling

It has been a challenging task to link any of the known effects of EGF. related or unrelated to cell proliferation, to the receptor-mediated tyrosine phosphorylation of specific cell proteins. Several protein substrates for the EGF receptor, and other growth factor receptors, have been identified during the past d e ~ a d e ( ~ -Of ~ )this . group of substrates. a small number have identifiable enzyme activities that have been speculated to play a role in cell growth control. This short list includes: MAP, a mitogen-activated protein serine/threonine k i n a ~ e ( ~ , ~ ) , which stimulates the GTPase activity of as, a cellular homolog of an onco ene; type T phosphatidylinositol (PtdIns) kinasc(”>2 ) , which phosphorylates the 3’ position of the membrane lipid PtdIns; and phospholipase C-yl (PLC-yl)(’‘), which hydrolyzes Ptdlns and it5 phosphorylated derivatives Ptdlns 4-P and Ptdlns 4.5-P2. This review will focus on the EGF receptor interaction with PLC-yI.

events are initiated within seconds or minutes after growth factor binding. Furthermore, as the receptor initiates the cell response to the growth factor, the cell also begins a process of down-regulation of the activated receptor and its signal.

5

r+--

TIE’:”@

p

PLC in the Phosphoinositide Signaling Pathway An early event in the intracellular signaling cascades elicited by many hormones, including EGF, is a stimulation of the metabolism of the plasma membrane phospholipid PtdIns 4.5-P2(14).Tn the phosphoinositide signaling pathway (Fig. 2), hydrolysis of PtdIns 4,S-P2 by PLC generates two molecules that have significant roles in hormonal regulation of intracellular processes. Ins 1,4,S-P3, a water-soluble product, mediates, by an incom letel defined mechanism, an oscillatory pattern r&ase and storage within the hormoneof CE+!

Y-P

‘Y

n

3IC ADP

Y

Fig. 1. Tyrosine phosphorylation of cell proteins by a hormonestimulated receptor tyrosine kiiiase is depicted. After binding of hormone (H) to the extracellular (EC) domain o f a reccptor in the cell plasma membrane (PM), the intracellular (IC) kiriase domain transfers phosphate (P) from adenosinc triphosphate (ATP) to tyrosine residues (-Y) of itceli and other protein substrates (S1 . S2).

...................................

...................

...

(%) .........

.............

.........

....

1

Ins 1,4,5P

....

f

\

.

... ....

Y-P .............

Ins 1,4,5-P

1 2+

+ Ca

Pig. 2. The role of PLC in generating an intraccllular signal is depicted. PLC can be activated (PLC*) by hormone-stimulated receptors (Rl*-G, R2*). PLC* hydrolyzes PIP2 (PtdIns 4,5-P2) to generate Ins 1.4,5-P3 and DAG. Ins L,4,S-P3releases Ca” from intracellular stores while DAG stimulatcs protein kinase C (PKC*) activity. Two distinct receptor classes are depicted, R1-G (receptors containing seven membrane domains and linked to G-proteins) and R2 (growth factor receptors), and as discussed in thc text these classes appear to couple with distinct PLC isozymes.

stimulated Ca2+ is well-characterized as a regulator of many enzyme activities, including protein kinases, proteases, lipases, and calmodulin-dependent enzymes. Understanding the role of Ins 1,4,S-P3 in CaZ+ oscillations will be instructive regarding both the roles of PLC in Ins 1,4,5-P3formation and the action of Ca2+ in cell function. 1,Zdiacylglycerol (l,a-DAG), a lipid-soluble product of PtdIns 4,5-P2 hydrolyqis, may stimulate the activity of protein kinase C(l6).This Ca2+ and phospholipid-dependent serine/threonine kinase is the cellular target for tumor-promoting phorbol esters, which like 1,2-DAG activate the enzyme. Among the substrates for activated protein kinase C are the EGF receptor(‘), and PLC isozymesLL7).

-

PLC lsozymes Structure and Distribution cDNA sequence and immunoreactivity analyses suggest the existence of at least nine distinct PLC isozymes ( a , PI, A, ~ 1 ,y2, 4, 82, 83). Certain homology relationships amongst members of the PLC family, revealed by their nucleotidc sequences, and the chronological order of their identification is the basis for the Less is understood of the respective enzymatic characteristics of the isozymes, their differential expression in cells, and how those features function in signaling pathways. In the /3, y, and 6 species (Fig. 3) there are two regions (A and B) with highly conserved amino acid sequences believed necessary for catalytic activity(18). The y1 and y2 isozymes are distinguished from the rest by the presence of sequences similar to regions of the src

a,

B

A

A

SH2

SH2 S H ~

B

Yl 47iY-P

A

? 7 1 h 7fSY-P

S H ~SH2

SH3

1254Y-P

B

Y2 759Y

Fig. 3. Represcntative members of the PLC family are depicted. ‘ A and ‘€3’ are regions required for catalytic activity in the /3, y, and 6 isozymes. PLC-yl, and y2 contain regions (SH2: SH3) similar to the amino terminal portion of src. The positions of tyrosine residues of PLC-y1phosphorylated by the EGF receptor (Y-P) are indicated. A tyrosine residue (Y759) of PLC-y2, homologous to PLC-y, Y783 is also shown, although no cvidence for phosphorylation at this sitc has been published. as yet.

kinase (a non-receptor tyrosine kinase) and src-likc proteins(1x,2”’Z1) . Th e presence of src-homology (SH) regions within the PLC yl and y2 species and several other proteins believed to be involved in cellular signaling (e.g.- GAP) has been interpreted as meaning that SH regions function in protein-protein interactions during signaling eventdZ2).This possibility is supported by the recent observations that the crk p r ~ t c i n ( ~ ~ ) . which contains SH2 and SH3 regions, associates with tyrosine kinases and proteins containing phosphotyros1ne(23.2’) . Mutational analyses of PLC-y, and y2 indicate that deletion of the portion of the protein containing the SH regions does not destroy catalytic activity, supporting a regulatory role (rather than enzymatic) for these regions in the function of y species(19, 6.27) In order to identify the role of specific PLC isozymes in cellular responses to hormones, it is necessary to examine the distribution of these isozymes in cells. Immunohistochemical and in situ mRNA hybridization analyses of PLC a, PI, yl, y2, and d1 isozymes in animal tissues reveals a differcntial distribution of thesc In the brain, certain isozymes are iso~ymes‘”-~~). expressed in patterns that reflect the functional stratification of the organ and arallel the distribution of certain receptor classes(P’). Most information regarding growth factor receptor-PLC interactions derives from observations of growth factor-responsive cultured cell lines, rather than intact animal studies. As with the tissue studies, information regarding PLC isozyme expression in cultured cells is incomplete. One interesting finding, however, is that PLC-y, is expressed in most, if not all, cultured cell lines (S. G . Rhee. personal communication) while PLC-y2 is expressed selectively in hematopoietic cells(’”). Whether the ubiquitous expression of PLC-:/ specics reflects a fundamental role for them in cell proliferation (since cultured cells are selected for a capacity to proliferate) is not clear. Additional work is needed to clarify the mechanisms that regulate expression of PLC isozymes with respect to cell origins, cell functions and hormonal responsiveness. Receptor-PLC Coupling Because of the number of PLC isozymes, several of which may be expressed in a single cell, it is not unrealistic to assume that distinct classes of hormone receptors present on the cell surface interact only with specific PLC subtypes. Accurate assignment of receptor:PLC isozyme relationships in signaling pathways has been difficult because of a lack of clear, distinguishing isozyme characteristics - enzymatic, physical, or biological properties - such as selective localization in membrane or cytosol compartments, substrate (phosphoinositide) selectivity, sensitivity to cations or inhibitors. Studies of the interaction of manv hormone receptors with cellular PLC activi ties(31) indicate a significant role for a guanine nucleotidebinding protein (G-protein). In general, those receptor proteins believed to stimulate PLC activity through a

SH regions, and 1254 is near the carboxyl terminus of G-protein-requiring step are unlike the growth factor the protein. receptors, in that: (i) they lack a tyrosine kinase Since the PLC-y, and yz isozymes are structurally and domain; (ii) they most often have seven membrane perhaps functionally similar, those sites relevant to spanning regions rather than one; and (iii) they interaction of y species with tyrosine kinases may be probably influence signaling pathways through allosconserved. In fact, PLC-yl, tyrosine residue 783 is the teric interactions with regulatory proteins. A novel PLC only phosphorylation site for which there is a conserved isozyme, purified from turkey erythrocytes. capable of tyrosine residue (759) in PLC-1'2 (Fig. 3). Thus this coupling with the P2-purinergic receptor, and moduresidue ~ ) . alone may be significant for receptor-PLCy lated by a G-protein, was recently d e ~ c r i b e d ( ~ ~ , ~ interaction. Cloning and sequencing the cDNA for this PLC If growth factor-stimulated PtdIns 4 5 P 2 metabolism isozyme will clarify the identity of the class of derives, in part or in whole, from receptor-mediated G-protein-linked PLC isozymes. It is possible that one tyrosine phosphorylation of PLC-yl, then a functional or more G-proteins (or other regulatory proteins) characteristic of PLC-yl, related to its enzymatic modulate receptor interactions with each PLC isozyme. activity, should be altered coincidentally with phosIdentification of the G-protein(s) relevant to hormonal regulation of PLC activity(35)will certainly advance the phorylation . Our laboratory recently defined reaction conditions that, although non-physiological, allow understanding of selective receptor coupling to PLC isozymes. detection of a marked influence o f t rosine phosphorylation on PLC-yl catalytic activity(45. EGF treatment of In contrast with many other hormones, little evidence exists that a G-protein-requiring step participates in cells induced a 3- to 6-fold increase in PLC activity growth factor regulation of PLC activity. (On the other present in PLC-y, immunoprecipitates. Furthermore, hand, no evidence conclusively rules out such a role.) in vitro EGF receptor tyrosine phosphorylation of PLCBecause the growth factor receptor's intracellular signal yl immunoprecipitated from untreated cells activated includes covalent modification of proteins, it has been the enzyme. Analysis of PLC-yl reaction kinetics possible to document receptor interactions with other indicates an increase in apparent V,,, and decrease in proteins by examining protein phosphotyrosine conapparent K for PtdIns 4,5-P2 after tyrosine phostent. Experimentally, this has facilitated the identifiphorylation@").It is possible that physiological presencation of a selective interaction between certain growth tation of the substrate to the activated enzyme may factor receptors (EGF, platelet-derived growth factor require a PtdIns 4,5-P2 hinding protein, such as (PDGF), and fibroblast growth factor (FGF)) and the profilin("), and/or a specific plasma membrane lipid PLC-y, isozyme. Although we will focus on the EGF environment. receptor interaction with PLC-yl, many similar obserThe role of tyrosine (and serine) phosphatases in vations have been made with PDGF and, to a lesser growth factor signaling pathways was until recently an extent, FGF receptors. underappreciated area of investigation compared to interest in kinased"). The kinetics of PLC-yI tyrosine phosphorvlation and dephosphorylation in vivo at 4°C PLC-y, Tyrosine Phosphorylation and 37°C739>49.50) are consistent with the hypothesis that PLC-yl is readily accessible to a cellular protein Cells expressing high levels of functional receptors for tyrosine phosphatase (PTPase). In vitro dephosphorylEGF respond to stimulation by the growth factor with a ation of PLC-y, was accomplished at a slow rate with rapid increase in hydrolysis of PtdIns 4,S-P2, formation purified T cell PTPase, but not at all by CD45 of Ins 1,4,5-P3and 1,2-DAG, mobilization of Ca2+ and P T P ~ s ~ ( ~ 'Removal ). of tyrosine phosphate from activation of protein kinase C('). This response, and activated PLC-y, by the T cell PTPase resulted in a many others, is abolished in cells that express a decrease in enzyme activity("). lnterestingly , using the mutated, kinase-deficient form of the growth factor EGF receptor as a substrate, these two PTPases show receptor(3637).Since PLC is a key enzyme regulating the opposite selectivity(4s). The PLC-yl and EGF phosphoinositide metabolism, it is a likely receptor receptor PTPases in growth factor-sensitive cells have kinase substrate in this signaling pathway. not been identified; however, the differential sensitivity In fact, PLC-y, is rapidly and selectively phosphorylof PLC-yl in in vitro PTPase assays and the loss of ated on both tyrosine and serine residues after activity following dephosphorylation suggests that a stimulation of cells with EGF(38-43').In intact cells, highly selective PLC-yl PTPase may play a significant 50-70% of the total PLC-yl pool can be phosphor 1 ated within a few minutes of hormone treatment('Yp I). role in this pathway. The apparent K, for the in vitro phos horylation of PCLy, Serine Phosphorylation Receptor~). PLC-yl by the EGF receptor is S I ~ M ~ mediated phosphorylation occurs at tyrosine residues In contrast to thc low level of phosphotyrosine present 472, 771, 783, and 1254(",") (Fig. 3). It appears that on PLC-yl in an unstimulated cell, the basal level of 771, 783, and 1254 are major sites, whereas 472 is a phos horylation of serine residues is relatively minor site(44).The tyrosine residue 472 is adjacent to highg9,4"). EGF stimulation of cells results in a the 'A' region within PLC-yl, 771 and 783 are within the significant increase in phosphorylation of PLC-yl serine putative regulatory domain, adjacent to and between Serine phosphorylation of PLC-yl is

7

P-

rapid and coincident with tyrosine phosphorylation. Since PLC-y, serine (as well as tyrosine) phosphorylation is rapid in cells treated with growth factors at a , th e growth factorlow temperature (4 0C)(39,40.49) stimulated serine kinase is easily accessible for and probably a direct or indirect substrate for the receptor kinase. The identity of the growth factor-stimulated PLC-11, serine kinase is uncertain, although there are several candidates. The CAMP-dependent protein kinase phosphorylates PLC-y, serine residues both in vitro and in viva("), protein kinase C phosphorylates serine residues in vitro (S.G. Rhee, personal communication) but not significantly in vivo (refs 40,51, Wahl et aE. unpublished data), and the rufkinase is also capable of phosphorylation of serine residues in vitrd5')). PLC-yl is an in vitro substrate of the serine phosphatase 2A(4'), whose action is without detectable effect on PLC activity. The regulation of dephosphorycation of growth factor-sensitive and CAMP kinase serine phosphorylation sites in vivo remains to be characterized. Selective PLC-y, Interaction with Receptor Kinases A fundamental concern in the study of growth factor regulation of cell proliferation is the definition of molecular characteristics required for kinase-substrate interaction. PLC-y, is an ideal protein for examination of this issue, since phosphorylation on tyrosine residues is efficient in vivo and in vitro,phosphorylation can be correlated with a change in enzymc activity, and phosphorylation demonstrates selectivity in terms of both kinase and substrate molecules. In intact cells, PLC-yl is phosphorylated on tyrosine residues after stimulation of the receptors for EGF, PDGF, and FGF(39-41.54,55). In contrast, PLC-yl, is not phosphorylated in cells stimulated by insulin, insulinlike growth factor-I, or colony stimulating factorThe structural features that distinguish the receptor kinases capable of phosphorylating PLC-y, (EGF, PDGF, FGF) from those incapable (insulin, IGF-1, CSF-1) have not been characterized. One structural motif that distinguishes certain members of the growth factor receptor family is a short span of amino acids that divides the intracellular kinase domain into two pard4). The presence (absence) of the 'kinase insert' domain does not correlate with the phosphorylation of PLC-y. One significant issue that has not been addressed is which, if any, of the large number of non-receptor (srclike) tyrosine kinases phosphorylate PLC-yl and modulate its activity. Circumstantial evidence suggests a role for tyrosine phosphorylation in the stimulation of PLC activity after activation of the T cell (lymphocyte) receptor complex("). One or more of the src-like tyrosine kinases may function in the stimulated T lymphocyte to activate a yisozyme. Since intact cells express several PLC isozymes, receptor-mediated tyrosine phosphorylation of PLC-yl I(39,m75h.s7).

(and possibly PLC-y2) is selective, in that it occurs in the absence of tyrosine phosphorylation of other PLC is~zymes(~'~").The selcctivity of receptor tyrosine kinase interaction with PLC-7, is supported by the observations that: (1) agonists such as ATP, bradykinin, bombesin, and NaF stimulate cellular PLC activity without effectin PLC-yl tyrosine (or serine) phos%'); and (ii) PDGF treatment of cells ph~rylation(~~.""~ overexpressing PLC-yl led to enhanced cellular PLC activity, but no change in cell responsiveness to G-protein-linked a g ~ n i s t s ( ~Comparison ~). of the efficiency of phosphorylation of purified PLC isozymes (PI, yl,01)by purified EGF receptor demonstrated that the selectivity for PLC-y, was reproducible in virro("3). This result indicates that no accessory proteins are required for selective receptor-PLC- y, interaction. Furthermore, a protein corresponding to the EGF receptor cytoplasmic domain selectively phosphorylates PLC-y, in Therefore, the receptor-PLC- y, interaction is independent of receptor transmembrane and extracellular domains. As previously discussed, PLC-yl and yz contain a putative regulatory domain with src-like sequences. This domain may distinguish the y isozymes from other PLC isozymes with regard to phosphorylation by receptor kinases. In fact, the regulatory domain of PLC-.)Iwas , itself phosphorylated on tyrosine residues when overexpressed in cells(''). Additional mutational analyses of PLC-yl is required to identify sequences required for selection by kinases. Activation of the receptor kinase after growth factor binding enhances interaction of receptor and PLC(40,41,5(7). F~rmation of a receptor-PLC-yl complex Yl may require prior receptor autophosphorylation on tyrosine residues("). Some evidence supports the hypothesis of a growth factor 'signaling complex'(53). including activated receptor plus one or more of the identified receptor substrates (i.e., PLC-yl, raf, GAP, type I PtdIns kinase). Most conclusions regarding the receptor-PLC-yl interaction have been based upon observation of co-precipitation of proteins in immune complexes. More complete understanding of its significance awaits purification of intact receptor-PLC--yl complexes, and characterization of the stoichiometry, enzyme activities, and associated proteins, if any. PLC-y, Activation/ Deactivation Cycle Hormone-stimulated modulation of PLC-yl activity through tyrosine phosphorylation and dephosphorylation is a dynamic process. To define the multiple possible sites for cell regulation of this signaling pathway, it is helpful to conceptualize the activation process in a biochemical cycle (Fig. 4). In an unstimulated cell, PLC-y, is predominantly in a soluble, nonmembrane-associated state(61), with little or no phosphorylation on tyrosine residues, and possibly in a complex with other proteins(3974o0). Growth factor binding to a cell surface receptor results in activation of receptor tyrosine kinase activity. Receptor activation rapidly induces redistribution of PLC-)I, to a mem-

inositide metabolism involves direct. selective communication of a receptor species having an intrinsic enzyme activity (i.e.. tyrosine kinase) with an effector species having an intrinsic regulatory domain (i.e., SH regions), rather than through an intermediate regulatory species (i.e.. G-proteins). Furthermore, the EGF receptor interaction with PLC-y,, in thc generation of a cellular response to EGF, occurs in the context of multiple other regulatory influences on receptor and PLC-yl activities. It will be interesting to identify the specific importance that activation of PLC-yl plays in cellular growth regulation by EGF, and to clarify the additional biochemical mechanisms that modulate this crossroads of intracellular signaling.

Cytosol

Membrane

a-I

I

DAG Fig. 4. Activation of PLC-1.1 by tyrosine phosphorylation a s il biochemical cycle. The EGF rc c c ptor is activated (*) after binding EGF, t h e n interacts with PLC-y1. Tyrosine phosphorylation (P) activates (*) PLC-yl,,;which may associate with othe r membrane proteins (X). Activatcd PLC-y, hydrolyzes PtdTns 1.5-P2to p r o d u c e Ins 1.4,S-P3a n d DAG. Dephosphorylation by PTPases deactivates the enzyme.

Acknowledgements The authors thank Ms. Susan Heaver for preparation of the manuscript and Ms. Suzanne Carpenter for preparation of the figures. The authors receive support from National Institutes of Health grants GM07347 (M.W.) and CA43720 and CA24071 (G.C.). References

1 SPORN, M. U. AUD ROBERTS, A. €3.. eds (1990). Peptide growth factors and their receptors. Hand. Exper. P/zcrrmacol. Vol. 95, Springer-Verlag, brane-associated state(61), and PLC-yl selectively asHeidelberg. sociates with the autophosphorylated receptor("). The 2 COHEN, s. (1962). laolalion of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening iii thc newborn animal. receptor-associated form of PLC-yl may represent a J. Biol. Chem. 237, 1555-1562. catalytically activated state, although this is not clear at 3 CARPEWER. G. AND WAHL.M. I. (1990). The epidermal growth factor family ol mitogcns. In 'Peptide tirowth Factors and their Keceptors.' In Handbook of present. PLC-yl phosphorylation on tyrosine residues is Expe+wztaI Pharnmcology (ed. A. U . Sporn, M. B. Roberts). Vol. 9.5, rapid and does result in a demonstrable activated 69-171, Springer-Vcrlag, Heidelberg. state(4'). After PLC-yl tyrosine phosphorylation, PLC4 C4RPENTER. G. (1987). Receptors for epidermal growth factor and other polypcptide mitogens. ilrmu. Rep. BiucAem. 56, 881-914. y1 may dissociate from the receptor and remain 5 ULLKICII. A . ANT) SCHLESSLNGEK. J . (1990). Signal transduction hy receptors associated with the membrane through interaction with with tyrosine kinase activity. Cell 61. 203-212. either membrane phospholipid components or a 6 HUNTER, T. A N D C O O P ~JK .A , . (1985). Protein-tyrosine kinases. Annu. Rev. Biochem. 54, 8'3-930. binding protein(s) ('X' in Fig. 4) that regulates its 7 ROsSOMANDO, A . J . . PAYNE.M. D., WEBLR,M J. AND STIJRGILL, T. W. interaction with phosphoinositide molecules. Hydroly(1989). Evidence that pp4Z2.a major tyrosine kinast. larget ptotcin, is a mitogenactivated serine/threoiiine protein kinase. Proc. Natl Acad. Sci. tiSA 86, sis of PtdIns 4,S-P2 generates second messenger 6940-6943. molecules. Tyrosine phosphorylated PLC-y, may dis8 ANDERSON. N . G.. MALLLK, J. L., TONKS. N.K. A N D SIURGILL, T. W. (1990). sociate from the membrane, since this species is also Requirement for integration of signals from two distinct phosphorylation for activation of MAP kinase. Nature 343, 651-653. . prescnt in a soluble fraction of a cell ~ y s a t e ( ~ * . ~ l )pathways 9 MoLLw, c. J . , BOTTARO, D . P.. FLtnlNG, T. P., MARSHALL. M. S.. GIBBS,J. Finally, PLC-yl is dephosphorylated by protein tyrosine B. .4ND AAROKSOU. S. A. (1989). PDGF induction of tyrosine phosphorylation of GTPase activating protein. ,Vurtun~342. 711-713. phosphatases, although the cellular site of this process 10 ELLIS, C.. MORAK, M . , MCCORMICK. 1'. AND PAWSON. T. (1990). is not known. Dephos horylation may require a specific Pliosphorylation of GAP and GAP-associated proteins hy transforming and tyrosine phosphataseg'). mitogenic tyrosine kinases. i h j n r r m 343. 377-381. 11 WHITMAN, M. AND CANTLEY. L. (1988). Phosphoinositidc mctaholism and This representation of selective growth factor moduthe control of cell prolileration. Biochirn. Uiuphys A r m 948. 327-344. lation of PLC-7, activity in a biochemical cycle must be 12 BJoRwi. J . , CHAN.T.-0.. ANTCL'ZK, M., KLNG, H.-J. AND FUJITA, 1). J. (1?90). Activated type I phosphatidylinositol kinase is asrociated with the modified in the future by inclusion of a variety of other epidermal growth factor (EGF) receptor following EG F stimulation. Proc. Nor/ factors, e.g., the PLC-7, serine kinase(s), other PLCArad. sci. US.4 87, 3816-3820. y,-associated proteins, tyrosine and serine phospha13 WAHL. M. I . , NISHIBE,S. AND CARPENTER, G. (1989). Growth factor signaling pathways: phosphoinositide metabolism and phosphorylation ol tases. Equally relevant but undefined issues are the phorpholipase C . Cmcer Cells 1, 101-107. reaction rates and affinity constants describing steps 14 ABDDL-LA~F,A. A. (1986). Calcium-mohilizing receptors. within the cycle, and coordination with other cellular polpphosphninositides and the generation of second messengers. Pliarniacol. Rev. 38, 227-272. processes, such as receptor activation, deactivation, 15 BFRRIDGE.M. J. (199Oj. Calcium oscillations. .I. Biol. Chcm. 265, down-regulation, and interaction with proteins of 9583-9586. 16 NISHIZVKA, Y. (1984). The role of protein kinase C in cell surface signal distinct signaling pathways. tranaduction and tumor promotion. Nature 308. 693-698. 17 RYU.S . H.. KIM, U-H., WAHL.M. I., BRowx, A. B.. CAKPLKTER. G.: I~~~A K.-P. N Gn :m RHEF.S. G. (199U). Phosphorylation of phospholipase C-B by protein kinase C. J. Biol. Chem. 265. in prcss. Conclusion 18 RHta, S. G.. PAVN-GHILL. S . , SLNG-HO. R. AND LEE, s. Y . (1989). Studies In summary, the growth factor stimulation of phosphoof inoailol phospholipid-specific phospholipase C . Sr.imce 244. 546-550.

19 KRIZ.R.. LIN,L.-L., SULTZMAN. L., ELLIS,C.. HLLOIN, C.-H., PAWSON,T. AND KxoPr. J. (1990). Phospholipase C isozynies: Structural and functioiial similarities. I n Prom-Oncogenes iri c'ell Developmenr (Ciba Foundatiou symposium 150). 112-127. 20 STAHL. M. L., FERENZ, C. R.. KOLLEHFR, R . 1.. KRIZ.R. W . A ~ KNOPF, O J. L. (1988). Sequence similai-ity of phospholipace C with thc non-catalytic region of 5rc. Nature 332, 269-272. 21 OIITA.S . , Mrsur, A , . NAZAWA, Y . AND K.AGAWA, Y. (1988). Complete cDNA encoding a putative phospliolipase C from Iransiormcd human lymphocytes. FEES Lem, 242, 31-35. 22 PAWSON, T. (1988). Nori-catalytic domains of cytoplasmic protein-tyrosine kinases: Regulatory elements in signal transduction. Uncogew 3, 491-495. 23 M A Y ~ RB. S ..I., Z~AMAGUCHI, M. A Y U HANARJSA. H. (1988). A nuvel viral oncogene with structural similarity to phospholipasc C. 24 MATSCDA. M.. MAYES, B. .I..FUKUI,Y. AKU H.ANAFT of tranqforming protein. ~ 4 7 8 " r - to ~ ~ a~ .iiruad rang containing protcins. Science 248, 153771539, 25 M A I S U D .M., ~ . MARSHALL, C. P. AXLI HAN.AFVSA, H. (1990). Purification and characlerization of P47gog~crk expressed in insect cclls. J. B i d . Chem. 265, 12 000- 12004. 26 EMOKI, Y.. HOMMA. Y., SORIMACIII. H.. KAWASAKI. H., NAKANISHI. O., S U Z I ~K., A N D TAKEN-~WA, T. (1989). A second type of rat phosphoinositidespecific pliospholipase C containing a si-c-rclated sequence not essentkdl for phosphoiiiositide-hydrolyzingactivity. J , Biol. Chpm. 264. 21 885-21 890. 27 UKISTOL. A , , H&LL. S. M.. KRIZ,R . W..STAHI.,M. L., FAY.Y. S . , RYERS. M. G.. EDDY,R. I>.,SIIOWS; T. B. AND KNUYF, J. L. (1988). Phospholipase C148: Chromosomal localion and delction mapping of functional domains. Cold Spring Horb S y q ~Q ~ ~ u i iUiot. t. 53. 915-920. 28 SLTH, P . G., RYC,S. H., CHOI.W. C.. LEE,K. Y . AND RHFE,S. G. (1988). Monoclonal antibodics to three phospholipase C iwzymes from bovine brain. J. Biol. ChPm. 263. 14397- 145114. 29 Ross, C. A , , MACCUMBER, M. W., GLATT, C. E. AKD SNYDER. S. H. (19x9). Brain phospholipase C isozynics: Diflerential mRNA localizations hy in situ hybridization. Pior. Norl Acad. Sci. U S A 86. 2923-2927. 30 HOMMA. Y.. TAKENAWA, 'Y.. EMORI, Y . , SORIMACHI, H. AND SUZLiKI. K. (1989). Tissue- and cell type-specific expression of mRNAs for four types of inositol phospholipid-specific phospholipase C. Riochem. B i o j ~ h ? ~ REX. . Conimun. 164, 406-412. 31 GERTLN. C. R.. C H O I , h'. C . . SUH, P. G . h N D RHEF, s. G . (1988). Phospholipase C I and TI brain isozyincs: Imrriiinohistochcmical localization i n iicuroiial systems in rat brain. Pror. Nati Acid Scr. U S A 85, 32118-3212. 32 F A I NJ. , N . (1990). Regulation of phosphoinositide-bpecific phospholipase c'. Biochern. Biophys. Act0 1053, 81-88. 33 MOKKIY, A. .I.. WALDO. G . L.. DOWNF.S, C. P.AND HARDFN, T. K. ( M U ) . A rcceptor and ti-protci 11-regulated polyphosplioino,itide-specificphospholipasc C from turkey erythrocytes. I . Purification and properties. J. Bid. Chon. 265, 13501-13507. 34 MORRIS; A. J.: Wsr.no. G . L.. DOWNES, C:. P. A N D HARDEN, T. K. (1990). A receptor and G-protein-regulated polyphosplioinnsitirle-specificyhocpholipase C from turkey erythrocytcs. 11. P2,,-purinergicreccptor and C;-protein-mediated regulation of the purified enzyme rcconstituled with turkcy erythrocyte ghosts. .I. Rial. C'i~enz.265, 13508- 13 514. 35 TAYLOR, S. J., SMITH,J. A . AND EXTOV, J. H. (199W). Purification from bovine liver membrancs of a guanine iiucleotide-tlepeiident activation of phospholipase C. Immunologic identification as a novel G-protein LY subunil. J. Brol. C'hem. 265. 17150-17156. 36 CHEK,W. S.. LAZAR. C . S . . POENIF., M.. TSILN,R. Y., GILL,G , N. AND ROSENPELU. M. G. (1987). Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. Namre 328. 820.823. 37 MOOI.EUA.AR. W.H.. BIERVAN, A. .I..TILLY. B. C.. VERLAAN. I., DFF17E: L. H. K.. HOYEGGER. A. M., ULLRICH. A. AND SCHLESSIVGER, J . (1988). A poinl mulation at thc ATl-binding sitc of the LGF receptor abolishes signal transduction. E M B O .I. 8, 707-710. 38 WAHI. M. I., DANIEI, 1'. 0. AND CARPLNIER. G. (1988). Antiphosphotyrosine recovery of phospholipase C activity after EGF trcatnienl of A-431 cells. Science 241, 968-970. 39 W.4HL. M. I., NISHIBE,S., SUH, P.-G.. RHEII,s. G. A N D CARPENIBK. G. (1989). Epicleiinal growth factor stimulates tyrosine phosphorylation of phospholipase C-gamma iiidcpentlcntly of intcrnalimlion and cxtracellular Ca'+. Prooc. Xarl. Acad. Sci. USA 86, i568-1572. 40 MEISENHLLDER. J., SUH,P. G., RHF.P..S. G . AND HUKILK,T. (1989). Phospholipase C - y is a substrate for the PDGF and EGF rcccptor proteintyrosine kinascs in v i m and in i , i l r o . Ccdl 57. 1109-1122. 41 MARGOLIS. B., RHFE,S . G . , FELDER. S.. MERVIC.M.. LYAIL, R . , LEVITZKI, A , , Z~LBERSTFIN, A. h h SCHISSSINCER. ~ J . (1989). EGI; induces A., ULLRICH. tyrosine phosphorylalion of phospholipase C-11: A potentid mechaniwi Cur EGF receptor signaling. Crlf 57; 1101-1107. 42 WAHL. M. I . , NISHIBE.S., KIM. J. W.. KIM, H.. RHEE. S. G . AXD

CARPFNTER. G. (1990). Identification of thc major EGF sensitive tyro4ne phosphorylation sites of phospholipase C-yin intact HSC-1 cells. J. B i d . Chcnz. 265. 394-3948, 43 NISHIBE.S.. WAIII.,M. 1.. RHLE,S . G. AND CAKYONTER. G. (19x9). Tyrosine phosphorylation of phospholipasc C-I1 in vitro by the epidermal growth factor receptor. B i d . Chem. 264. 10335-10338, 44 KIM, J. W.. SIM,S. S.. KIM,U . H.. NISHIRE, S . , WAHI..M. I., CARPENTER, G. A A O RHEE,S. G. (lY90). Tyrosine residues in bovine phospholipase C-1, phosphorylated by thc EGF receptor in i,itr.o. J . Bid. Chom. 265, 3940-3943. 45 NISHIBE. s., WAHL, M. I., HLRNnYDEZ-SoTOh~.4rER,S. M. T.i TONKS.N., GR. .(1990). Incrcase of the catalytic activity nf RHFE,S . G. AND C A R P ~ K T E PLC-y, by tyrosinc phosphorylation. Science. in pt-es\. 46 NISHIBE. S., WAIIL.M. I . . R H ~ E . G. AND CARPENTFR, 6. (1990). from epidermal growth tactorProperties of activaled phospholipase trcatcd A431 cells. Submitted. 47 ~ ~ ~ L D S C H M I D I - C LP.EJR. ,AMACHESKY. ~ ~ E ~ . ~ , L. M.. BAIDASSAKI!. J . J. AND POILARD. T. D . (1990). The actin-hinding protcin profilin binds to PIP2 and inhibits its hydroly-sis by phospholipase C'. Science 247. 1575-1578. 48 HUNTER, 7. (1989). Protein-tyrosinc phosphntases: Thc other side of the coin. Cdf 58. 1013-1016. 49 WAHL.M. I . . OLASHAW, N. E.. NISHIBE,S . , RHEE,S. G., PIEDGER. W. J. AYD C A R P E N ~ G. L R (19x9). , Platelet-derived growth factor induces rapid and sustaincd tyrobine phosphorylation of phospholipase C-gamma in quicscent IJALB/c 373 cclls. Molec. Cell. Biol. 9 , 2934-2943. 50 KUMJIAN.D. A.. WAHL.M. 1.- RIIOB,S . G. A N D DANIEL.7. 0. (1989). PDGF binding proniu1es physical aisociation of PDGk receptor wilh phospholipase C. Proc. h i d h a d . Sci. U S A 86, 8232-839. 51 KIM, I!.-H.. KIM, J . W. AND KHEE. S. G. (1989). Phosphorylation of phospholipasc C-y by CAMP-dependent protcin kinase. J. B i d . Chrm. 264. 20 167-20 1711. 52 DECKFR. s. J.. ELLIS,C., PAWSON. f . AND V t L L . 'I.(1990). Effects of substitution of lhreonine 6.54 of the epidermal growth factor receptor on epidermal growth lactor-mediated activation of pho\pholipase C. .I. Biol. Cheni. 265, 7009-7015 53 MORRISON. D . K., KAPLAN, D . K., RHEE.S. G. AND WILLIAMS. L. T. (1990). Platelet-derived growth factor (PDGF)-dependciit association of phospholipise C-2,. with the PDGF receptor signaling complex. ; I f o l ~ .Cell. B i d . 10, 2359-2366. 54 RUTA,M., BURGESS. W., GnoL. U., EPSTEIN. J., NEIGOK, N., KAPLOW. .I., CKUULEY, G., DIOYNI.,C . , JAYE.M . A N D SLHLESSINGER, J . (1989). Rcceptor for acidic fibroblast growth factor is rclated Lo the tyrosjnc kinnac encoded by the his-like gene (flg). Proc. Nutl Acad. Sci. US4 86, 8722-8726. 55 BLIKG~SS. W. H.: D L O ~ NC. E . A,, K ~ P L W V J.,. MUDD, K., FRIESF.~, R.. J. om JAY^. ,M. (1990). Characterization and ZJI-B~RSTLIN, A , : SCHI.F.SSINGEK, c D N 4 cloning of phospholipase C-y. a niaior substrate for heparin-binding growth factor 1 (acidic fibrolila\l growth factoi-)-activated tyrosine kinase. ~ ~ i t rCPK . . B i d . in. 000-ow. 56 DOWNING, J. R.. MARGOLIS. U. L., ZILBERSTEIN. A , . ASHMIU,K. A., ULLKICH, A , , SHFRR,C. J. AKD SCHIF.SSINGLR. J. (1989). Phoapholipasc C-y. a suhctrate tor PDGF receplor kinase, is not phosphorylatcd on tyrosine during the mitogenic response to CSF-1. LIMBO .I. 8 > 3345-3350. 57 NISHIBE. s., WI\IIL. M. I.. WFDGAERTNER, P. B., KIM. J. J., KHEE.s. G. AND CARPENI~ G.K (1989). , Selectivity-of phospholipase C phosphorylation by the EGF receptor, thc insulin receptor and their cytoplasmic domain. I'voc. Naiari. Acad. Sci. USA 87, 424-428. 58 MUSTEIJN, T., C U G ~ ~ E S1 ,HK. A IM., ISAKOV, N. ALLMAN, A. (1990). T cell antigcn receptor-mediatcd activation of phospholipase C requires tyrosine phosphorylation. Science 247, 1584-1 587. 59 ~ ~ A R G O I - I S U.. , ZILBERSTEIK. A., FRAFKS. C., F ~ L O E RS.. . KREWLK, S.. ULLKICH. A.. RHEE,S. G.. SKORFCKI. K. AND SCHI.F.SSINCEK. J. (1990). Effect of phospholipasc C-1. overexpression on PDGF-induccd second messcngerc and mitogenesis. Science 248, 607-610. 60 MARGOLIS. €5.. UELLOT, F., H O N L W ~A K.. M.. LLLRICH, A , . SCHI.FSSIYG~K, J. AND ZILEERSTFTN, A . (1990). Tyrosine kinase activity is essential for the association of phospholipasc C - y with the epidermal growth factor rcceplor. M&c. Cell. Bid. l a , 435-441. 61 TODDERUD, G . , WAHL. M. 1.. RHEE. S . c?.AND CAKPENTFR. G. (1990). Stimulation of phospholipase c-11, membrane association by EtiF. Srieizcr 249. 296- 299.

Matthew Wahl" and Graham Carpenter are at the Department of Biochemistry, Vaiiderbilt Univcrsity School of Medicine, Nashville, TN 37232-0146, USA. *To whom correspondence should he scnt.