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Regulation of phospholipase C isozymes
Progress in Growth Factor Research, Vol. 4, pp. 97-106. Printed in Great Britain. All rights reserved.
REGULATION
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OF PHOSPHOLIPASE ISOZYMES
0955-2235/92 Pergamon
$15.00 Press Ltd
C
Gwenith Jones and Graham Carpenter Department of Biochemistry Vanderbilt University School of Medicine Nashville, TN 37232-0146. U.S.A.
Phosphatidylinositol bisphosphate hydrolysis is an immediate response to many hormones, including growth factors. The hydrolysis ofphosphatidylinositol bisphosphate is catalyzed byphosphatidylinositol-spect3cphospholipase C. A number ofphospholipase C isozymes have been identified. Dtflerent isozymes are activated by dtyerent receptor classes. This review will summarize the dt#erent isozymes of phospholipase C, and the current knowledge of the mechanisms by which phospholipase C activity is modulated by growth factors.
Keywords: Phospholipase
C, tyrosine kinase receptors, G proteins, phosphorylation.
regulation.
INTRODUCTION Growth factors mediate their effect on cells through receptor-coupled signaling pathways. In response to growth factor-receptor interaction, there are metabolic changes resulting in the generation of second messenger molecules. It is these second messengers that modulate the activity of effector proteins that regulate proliferation. Among the best studied second messengers are inositol 1,4,5 trisphosphate (Ins 1,4,5 PJ and diacylglycerol. Ins 1,4,5 P, arises from the action of phospholipase C (PLC) on phosphatidylinositol-4,Sbisphosphate (PtdIns-4,5-P,) [ 1,2]. In contrast, diacylglycerol can be generated from more than one source and by multiple mechanisms. The most common mechanisms for the generation of diacylglycerol are from either phosphatidylinositol or from phosphatidylcholine by the action of a PLC or from the dephosphorylation of phosphatidic acid that is generated by phospholipase D action on phosphotidylcholine [3,4]. The direct effects of these second messengers are to stimulate Ca2+ release (Ins 1,4,5 P,) [2,5] and activate protein kinase C (diacylglycerol) [4]. Acknow~edgemenrs-The authors acknowledge support from NIH research grants CA 43720, CA 2407 1 and Training Grant CA09582. Abbreviufions-EGF~pidermal growth factor; PDGF-platelet-derived growth factor; PLC-phospholipase C; PtdIns-4, 5-P,-phosphatidyhnositol bisphosphate; SH-src homology. 97
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Stimulation of PtdIns-4,5-P, hydrolysis occurs when cells are challenged with certain agonists that bind to either tyrosine kinase receptors or to G protein-coupled receptors [7-l I]. Current research objectives in understanding receptor coupled activation of phosphatidylinositol hydrolysis are to determine the number of PLC isozymes, whether different receptor classes activate the same or different PLC isozymes, how PLC activity is regulated for each isozyme, and whether the activity of each isozyme is regulated by the same or different mechanisms. PHOSPHOLIPASE
C ISOZYMES:
STRUCTURAL
CHARACTERISTICS
Experimental evidence, such as protein purification [ 12,131, molecular cloning [ 1416] and immunological studies [12,13,17,18], have identified four distinct classes of phosphatidylinositol-specific PLC(p, y,S,&). All four classes of PLC isozymes consist of a single polypeptide chain with molecular weights of: 150-l 54 kDa for PLC-p, 1455148 kDa for PLC-y, 85-88 kDa for PLC-Gand 86 kDa for PLC-E. The sequence of PLC cx, which was identified by molecular cloning, was recently determined not to be a PLC isozyme [ 191. Isoforms have been isolated for PLC-p (PI, fl) [20], PLC- y ( yl , y2) [2023] and PLC-6(61, 62, 63) [20]. At this time little is known about the kinetic properties of PLC isozymes as compared to the data available for phospholipase Al. It is known that all four isozymes of PLC have similar enzymatic properties in that phosphatidylinositol, phosphatidylinositol-4-phosphate or PtdIns- 4,5-P2 can be used as substrates and Ca” is required for activity, though each class differs in the optimal pH and Ca’+ concentration required for maximal in vitro activity [ 12.131. Structural similarities between PLC-y, -Sand -6have been observed (Fig. 1). These isozymes have two regions of sequence homology, denoted X and Y, that are 60% and 40% identical, respectively [19]. Because these regions are conserved among the isozymes, it has been suggested that these regions are domains that provide catalytic function. Deletion of either the X or Y region of PLC- yl or -y2 expressed in COS cells [20] or E. coli [21], respectively, results in the loss of catalytic activity. It is not known if the X and Y regions exist in PLC-E, because it has not been cloned as of this date. PLC-p
--I
x
H
Y
I
PLC-6 Y1254
PLC-Y, PLC-r2 FIGURE 1. Structural characteristics of the PLC isozymes. Comparison of PLC isozyme sequences have revealed distinct regions of homology. AU isozymes, -a -yand -6, possess two regions of homology, X and Y. In the case of PLC-yl and -y2, the X and Y region are split by SHZ/SH3 domains. PLC-yl is tyrosine phosphorylated. Tyrosine phosphorylation sites Y771, Y783 and Y1254 are indicated.
PLC- yhas an additional region of sequence homology between the X and Y regions (Fig. 1) to other proteins. This region is related to sequences found in the src family of tyrosine kinases, which are termed src homology region 2 (SH2) and src homology
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region 3 (SH3) [ 14,221. Deletion of the SH2 and SH3 domains of PLC-~1 reduces, but does not eliminate, enzymatic activity; therefore, the SH domains may have a regulatory role [20,21]. In addition to non-receptor tyrosine kinases, such as src, SH2 and SH3 domains have been found in several other proteins, some of which are involved in signal transduction [24-311. These proteins include GTPase activating protein or GAP, [27], crk [28] and the 85 kDa subunit of phosphatidylinositol-3phosphate kinase [29-3 11. CELLULAR
AND SUBCELLULAR
LOCALIZATION
Because there are many isoforms of PLC, it would be interesting to know if there is tissue or cell specificity in the localization of each isoform. PLC-yl and PLC-81 have a wide tissue distribution [32,33], whereas PLC-I/;! is predominately expressed in lung, spleen and thymus [33]. PLC-pl is almost exclusively localized to the forebrain, as only low quantities of PLC-PI are detected in other tissues [32,33-361. Even within tissues, such as the central nervous system, there also can be differential expression of the PLC isozymes. PLC-yl and PLC- pl have been localized to neuronal cells, whereas PLC-Sl is localized to glial cells [37]. In cultured cells, PLC-yl is the most common isoform detected and only low level expression of PLC-61 and PLC-al has been detected [32]. Since in most tissues and in some cultured cells, multiple PLC isozymes are expressed simultaneously, the possibility exists that the different isozymes of PLC may be regulated by different receptor classes. The subcellular localization of the isozymes is also important. PLC-PI is primarily membrane-associated, whereas PLC-yl -61 and -&are primarily cytosolic [17.37]. The mechanism by which PLC-PI is membrane-associated is unclear from the available evidence. The cytosolic localization of other PLC isozymes presents a logistic problem since PtdIns-4,5- P, is located in the membrane. Translocation of the PLC-yl from cytosolic to membrane compartments has been observed in response to EGF and PDGF [38,39]. However, the mechanism by which this translocation takes place is unknown. PLC-yl does contain sequence similarities to a putative translocation domain, CalB [40]. This domain was first identified in protein kinase C as a Ca“ binding domain [41]. Work on cytosolic PLAz by Clark et al., suggests that this domain may be involved in Ca”-dependent translocation of the enzyme to the membrane [40]. Whether the ‘CalB’ sequence plays this role in PLC-yl translocation is unknown; however. PLC-y interaction with the membrane is not sensitive to EGTA [42] unlike PLA, [40]. Using immunohistochemical techniques, McBride et al. [43] localized a small pool ot PLC- yl to the cytoskeleton. An interaction of PLC- yl with the cytoskeleton may occur through the SH3 domain present in PLC-yl. SH3 sequences have been found in cytoskeleton proteins or proteins that interact with the cytoskeleton, leading to the hypothesis that SH3 domains may play a role in the anchoring of proteins involved in signal transduction to the cytoskeleton to form a ‘signaling complex’ [24,26]. REGULATION
BY TYROSINE
PHOSPHORYLATION
Certain ligands, such as EGF and PDGF, that activate tyrosine kinase receptors stimulate PtdIns-4,5-P, hydrolysis [44449]. The isoforms involved in tyrosine kinase-
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mediated PtdIns-4,5-P, hydrolysis are PLC- yl and - y2. While PLC- yl can be phosphorylated by both receptor and non-receptor tyrosine kinases [4454], not all tyrosine kinases phosphorylate PLC- yl. Tyrosine phosphorylation of PLC- yl is seen in response to EGF, FGF and PDGF, but not CSF or insulin [46,50,55]. No phosphorylation of PLC- yl, by src, a non-receptor tyrosine kinase, has been observed [46]. PLC- yl can also be phosphorylated on tyrosine in response to NGF in PC 12 cells [52,53] or CD3 in T cells [51,54]. Since the tyrosine kinase, trkB, has been shown to be a component of the high affinity NGF receptor [53], it is likely that trkB is responsible for the phosphorylation of PLC-yl in response to NGF. It is not clear how CD3 stimulates tyrosine phosphorylation of PLC- yl, since the T cell-antigen receptor does not possess an inherent protein kinase activity [56]. Association of the receptor with another protein possessing tyrosine kinase activity such as ~~56”” [56] or fyn [57] may be responsible for tyrosine phosphorylation of PLC- yl . Since some tyrosine kinases, but not others, can phosphorylate PLC- yl, it is tempting to postulate that different growth factors may activate specific signaling pathways. Less is known about the phosphorylation of PLC-~2, though crosslinking of membrane IgG in B cells leads to tyrosine phosphorylation of PLC-~2 [58], also transfection and expression of PLC-~2 in fibroblasts permit tyrosine phosphorylation of -~2 in response to PDGF [59]. In the presence of the appropriate ligand, phosphorylation of PLC-yl on tyrosine occurs rapidly, < 1 min [4547,49,55,60]. For tyrosine phosphorylation of PLC-yl to occur, PLC-yl must first associate with the receptor. Experiments have demonstrated the association of PLC-yl with the carboxy-terminus of the EGF and presumably the PDGF and FGF receptors [60-651. Since no association of PLC-yl with kinasenegative EGF receptors was detected, it was concluded that the receptor has to be tyrosine phosphorylated for PLC-yl association to occur [61,63]. In vitro experiments revealed reduced association of phosphorylated as compared to dephosphorylated PLC-yl with activated EGF receptors. These data suggest that after PLC-yl is phosphorylated, the enzyme rapidly dissociates from the receptor [61]. Association of PLC-yl with activated receptors is proposed to be mediated by SH2 domains. SH2 domains are regions capable of binding to sequences containing phosphotyrosine [24 27, 61, 621 and the preferential interaction of SH2 domains with tyrosine phosphorylated sequences would explain why PLC-yl specifically interacts with activated growth factor receptors. Recently Rotin et al. demonstrated that PLC-yl SH2 domains can prevent the dephosphorylation of both the PDGF and EGF receptors [62]. The EGF receptor residue that was specifically protected by PLC- yl SH2 domains was Y992. From this data it was concluded that Y992 was part of the high affinity binding site for PLC-yl. However, Sorkin et al. demonstrated that with an EGF receptor mutant in which Y992 was mutated to F992 tyrosine phosphorylation of PLC-yl occurred to the same extent as with the wild-type receptor [63]. Concomitant with an increase in tyrosine phosphorylation is an increase in PLC-yl activity [45,5 1,64,66-701. PLC- yl enzymatic activity is apparently controlled through a mechanism of tyrosine phosphorylation and dephosphorylation. Nishibe et al. demonstrated that when PLC-yl is phosphorylated by EGF receptor in vitro, which phosphorylates only tyrosine residues, an increase in PLC-yl activity occurred [66]. Incubation of the tyrosine phosphorylated PLC-yl with a tyrosine phosphatase resulted in a decrease in activity. The PLC-yl sites that are tyrosine phosphorylated in vivo and in vitro have been determined (Fig. 1). In vivo the major phosphorylation sites are Y771 and Y783,
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located between the SH2 and SH3 domains, and Yl254, located near the carboxyterminus [48]. Y783 is conserved in PLC-72, but it has not been determined if PLC-y2 is phosphorylated on this residue [20]. An additional phosphorylation site at Y472 was observed in vitro [7 11. To identify which sites are important for PLC- yl function, Kim et al. mutated individual tyrosine phosphorylation sites to phenylalanine and then overexpressed the mutant proteins in NIH 3T3 cells [70]. By measuring the capacity of these PLC-71 mutations to stimulate Ins-l, 4,5-P, production in response to PDGF, it was determined that Y783 and Y 1254 were important for PLC-yl function, while Y771 was dispensible.
J
OL
PIP*, (mole fraction) FIGURE 2. Kinetics of control and EFG-activated PLC+. PLC-yl was imuumoprecipitated from untreated and EGF- treated cells and then assayedfor activity in the presence of increasing mole fraction of Ptdlns+-P,. Open symbols: - EFG and closed symbols: + EGF.
Recently, Wahl et al. have used kinetic analysis to show that tyrosine phosphorylation acts as an allosteric modifier to activate PLC-yl [67]. Under control conditions, non-tyrosine phosphorylated PLC-yl displays strong negative cooperativity toward PtdIns-4,5-P,, whereas the EGF-activated enzyme does not (Fig. 2). At a low PtdIns-4,5-P, mole fraction, a 7-fold difference in the relative velocity between control and EGF-activated PLC-yl is observed. However, at a high PtdIns4, 5-P, mole fraction, approaching that of pure liposomes, the relative velocity of the control enzyme is similar to the EGF-activated enzyme. These data suggest that at a low PtdIns-4, 5-P, mole fraction (representing a PtdIns-4, 5-P, concentration closer to its physiological concentration) the control enzyme is comparatively inactive. Tyrosine phosphorylation increases PLC-yl activity by overcoming the cooperative effect. Goldschmidt-Clermont et al., using a different assay system, only observed a difference in activity between control and tyrosine phosphorylated PLC-yl in the presence of profilin [68,69]. Profilin binds PtdIns-4,5-P, and in the presence of profilin the effective substrate concentration (that available for hydrolysis by PLC-yl) is lowered. Regulation of PLC-yl activity by allosterism implies that PLC-yl is a multi-domain molecule with at least two binding sites for PtdIns-4,5-P, (Fig. 3). This model is reminiscent of the dual phospholipid hypothesis proposed by Dennis and colleagues for cobra venom PLA, [72]. In the dual phospholipid model, PLA, initially interacts with a micelle surface by binding to one phospholipid molecule. Once associated with the micellar surface, the enzyme can then bind another phospholipid molecule, which is
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fast
PIP2 I
site
2 PLCYI (active)
FIGURE 3. Putative model of EGF-activation of PLC-71. PLC-71 may have two sites that bind PtdIns-4,5-P,, site 1 and site 2. The bhuiing of PtdIns-rlJ-P, to site 1 is a rate-limiting step. Under basal conditions, binding of PtdIns-4,5-P, to site 1 of PLC-yl occurs slowly. Once PtdIns-4,5-P, binds to site 1, binding PtdIns-4,5-P, to site 2 occurs rapidly. Tyrosine phosphorylation of PLC-yl results in a kinetic change such that the slow step (binding of PtdIns-4,5-P, to site 1) is no longer rate-limiting.
hydrolyzed. The situation with PLC-yl is more complex than PLA, since there are two forms of the enzyme, phosphorylated and non-phosphorylated. The kinetic data (Fig. 2) suggest that the non-phosphorylated enzyme may have two binding sites for PtdIns4,5-P2. In this model (Fig. 3), binding of PtdIns-4,5-P, to the first site of the nonphosphorylated enzyme occurs slowly, resulting in the cooperative effect observed in Fig. 2. In contrast, binding of PtdIns-4,5-Pz to the tyrosine phosphorylated enzyme occurs more readily, overcoming the cooperative effect (Fig. 2). Kinetically, it is not possible to distinguish whether there are one or two binding sites for PtdIns-4,5-P, with the tyrosine phosphorylated enzyme. It is possible that the conserved regions X and Y are both PtdIns-4,5-Pz binding sites. REGULATION
BY SERINE PHOSPHORYLATION
Serine phosphorylation may also play a role in the regulation of PLC activity. Both PLC- yl and PLC-p are serine phosphorylated and can be phosphorylated OR serine residues in vitro by protein kinase C [73,74]. In vivo, phorbol ester activation of protein kinase C only phosphorylates PLC-P; no phosphorylation of PLC- yl occurs [73]. PLCyl can also be phosphorylated by CAMP-dependent kinase in vitro [75]. The role of
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serine phosphorylation for either PLC-yl or PLC-pis not known. In vitro treatment of PLC-yl with a serine phosphatase, protein phosphatase 2A, did not affect the activity of either the basal or EGF-activated enzyme [76]. REGULATION
BY G PROTEINS
Phosphatidylinositol hydrolysis is also regulated by G protein-coupled receptors by pertussis toxin-sensitive and -insensitive mechanisms [7-10,77-901. A 42 kDa pertussis toxin-insensitive G protein 01 subunit has been isolated that can stimulate PLC hydrolysis of PtdIns-4,5-P? in vitro [84,85,89,90]. This unique a subunit is designated G,. Incubation of G, with various PLC isozymes revealed that G, stimulates PLC-/?I activity but has no effect on PLC-yl or PLC-8activity [84,85,90]. How G, modulates PLC-/?I activity is not known. It is possible that association of PLC-j31 with G,*GTP results in an activated conformation of PLC-pl akin to Gm@GTP activation of adenylcyclase. Results of protein isolation, immunological cross-reactivity, and Northern analysis of G, proteins indicate there is a family of G, proteins that may regulate PLC isozymes [7-10,84,85,87-911. These G, proteins differ in their activation by AlFl,- and the Ca” requirement for PtdIns-4,5-P? hydrolysis [84, 85, 89,901. Regulation of phosphatidylinositol hydrolysis by G protein-coupled receptors can also occur in a pertussis toxin-sensitive manner [7-10.8688,92]. Yang et ul. identified in hepatocytes a G protein that complexes with PLC-yl in a EGF- dependent manner [92]. This G protein can be ADP-ribosylated by pertussis toxin and reacts with Gu, antibody. It was suggested that association of PLC-yl with this Ga,-like protein aids in the attachment of PLC-yl to the plasma membrane. However, it is not known if the association of PLC-yl with a Gcw,-like protein modulates PLC-yl activity. PLC-6may also be regulated by a pertussis toxin-sensitive G protein. In the Chinese Hamster lung fibroblast cell line CCL39, pertussis toxin inhibited thrombin-induced phosphatidylinositol hydrolysis [77]. A mutant of CCL39 defective in thrombin, serotonin and AlFl,provoked phosphatidylinositol hydrolysis, was found to lack PLC-0‘ [93].whereas PLC-6 was present in the wild-type cell line (CCL39) [77]. The activity of PLC-E is increased in the presence of GTP$, which suggests PLC-r may also be regulated by a G-protein [17]. Whether this G protein is pertussis toxin-sensitive or -insensitive is not known. CONCLUSION At this time seven isoforms of PLC, that vary in tissue and cell distribution, have been identified. It is likely that through the use of molecular biological techniques. more isoforms of PLC will beldentified. Figure 4 presents a model of how two of the most studied isozymes of PLC, PLC-yl and -/Il, may be regulated. PLC-~1 activity is modulated by tyrosine phosphorylation, whereas PLC-PI activity is modulated by a unique class of G proteins, G,. The evidence that, at least in hepatocytes, PLC-yl associates with a G protein subunit demonstrates that each isozyme may be regulated by more than one mechanism. Judging from the evidence already collected, regulation of PLC by growth factors may be as complex and varied as the number of PLC isozymes and the receptors that activate them.
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tyrosine kinase receptors
non-tyrosine kinase receptors
FIGURE 4. Schematic of PLC activation by tyrosine kinase and non-tyrosine kinase receptors. By diverse mechanisms, receptors can activate PLC to produce PtdIns-4,5-P, and diacylglycerol. Putative mechanisms of activation for PLC-pand - yl are diagrammatically represented. In this schematic PLC-/?l activity is modulated by receptor activabion of a G protein, whereas PLC-yl activity is modulated by tyrosine phosphorylatlon of the enzyme by a tyrosine kinaae receptor.
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69. Goldschmidt-Clermont PJ, Kim JW. Machesky LM, Rhee SG, Pollard TD. Science 1991; 252: 123 I 1233. 70. Kim HK, Kim JW, Zilberstein A, Margolis B. Kim JG. Schlessinger J, Rhee SG. CeN 1991; 65: 435441. 71. Kim JW, Sim SS, Kim U-H, Nishibe S, Wahl MI, Carpenter G, Rhee SG. J Biol Chem. 1990; 265: 394g-3943. 72. Henderickson HS, Dennis EA. J Biol Chem. 1984; 259: 57345739. 73. Ryu SH, Kim U-H, Wahl MI, Brown AB, Carpenter G, Huang K- P, Rhee SG. J Biol Chem. 1990; 265: 17941-17945. 74. Blank JL, Foster KA, Hawthorne JN. J Neurochem. 1991; 57: 15-21. 75. Kim U-H, Kim JW, Rhee SG. J Biol Chem. 1989; 264: 20167-20170. 76. Carpenter G, Htnandez-Sotomayor SMT. Nishibe S, Todderud G, Mumby M. Wahl M. Interactions among cell signalling systems.In: Widdow K, ed. CIBA Foundation Symposium, Vol. 164. New York: Wiley; 1992: 222-239. 77. Rath HM, Doyle GAR, Silbert DF. J Biol Chem. 1989; 264: 13387-13390. 78. Boyer JL. Downes CP, Harden TK. J Biol Chem. 1989; 264: 884890. 79. Glossman H, Baukal A. Catt RJ. J Biol Chem. 1974; 249: 664666. 80. Goodhardt M, Ferry N, Geymet P, Hanoune J. J Biol Chem. 1982; 257: 11577-I 1583. 8 I. Boyer JL, Garcia A, Posadas C, Garcia-Saine JA. J Biol Chem. 1984; 259: 80768079. 82. Evans T, Martin MW, Hughes AR, Harden TK. Molec Pharmac. 1985; 7: 32-37. 83. Evans T. Hepler JR, Masters SB, Brown JH, Harden TK. Biochem J. 1985: 232: 751-757. 84. Taylor SJ. Chae HZ, Rhee SG, Exton JH. Nature 1991; 350: 516518. 85. Smrcka AV, Hepler JR, Brown KO, Sternweis PC. Science 1991; 25 I : 804807. 86. Ueda H, Yoshihara Y, Misawa H, Fukushima N, Katada T, Ui M, Takugi H, Satoh M. J Biol Chem. 1989; 264: 3732- 3741. 87. Perney TM, Miller RJ. J Biol Chem. 1989; 264: 73 17-7327. 88. Cowen DS, Baker B, Dubyak GR. JBiolChem. 1990; 265: 16181-16189. 89. Waldo GL. Boyer JL, Morris AJ, Harden TK. J Biol Chem. 1991; 266: 14217-14225. 90. Blank JL, Ross AH, Exton JH. JBiol Chem. 1991; 266: 1820618216. 91. Wilkie TM, Schere PA, Strathmunn MP, Slepak VZ. Simon Ml. Proc Nufl.4cad Sci U&4. 1991; 88: 10049-10053. 92. Yang L, Batty G. Rhee SG, Manning D. Hansen CA, Williamson JR. JBiol Chem. 1991; 266: 2245122458. 93. Rath HM. Fee JA, Rhee SG. Silbert DF. J Biol Chem. 1990; 265: 3080-3087.
REGULATION
1992 cl1993
OF PHOSPHOLIPASE ISOZYMES
0955-2235/92 Pergamon
$15.00 Press Ltd
C
Gwenith Jones and Graham Carpenter Department of Biochemistry Vanderbilt University School of Medicine Nashville, TN 37232-0146. U.S.A.
Phosphatidylinositol bisphosphate hydrolysis is an immediate response to many hormones, including growth factors. The hydrolysis ofphosphatidylinositol bisphosphate is catalyzed byphosphatidylinositol-spect3cphospholipase C. A number ofphospholipase C isozymes have been identified. Dtflerent isozymes are activated by dtyerent receptor classes. This review will summarize the dt#erent isozymes of phospholipase C, and the current knowledge of the mechanisms by which phospholipase C activity is modulated by growth factors.
Keywords: Phospholipase
C, tyrosine kinase receptors, G proteins, phosphorylation.
regulation.
INTRODUCTION Growth factors mediate their effect on cells through receptor-coupled signaling pathways. In response to growth factor-receptor interaction, there are metabolic changes resulting in the generation of second messenger molecules. It is these second messengers that modulate the activity of effector proteins that regulate proliferation. Among the best studied second messengers are inositol 1,4,5 trisphosphate (Ins 1,4,5 PJ and diacylglycerol. Ins 1,4,5 P, arises from the action of phospholipase C (PLC) on phosphatidylinositol-4,Sbisphosphate (PtdIns-4,5-P,) [ 1,2]. In contrast, diacylglycerol can be generated from more than one source and by multiple mechanisms. The most common mechanisms for the generation of diacylglycerol are from either phosphatidylinositol or from phosphatidylcholine by the action of a PLC or from the dephosphorylation of phosphatidic acid that is generated by phospholipase D action on phosphotidylcholine [3,4]. The direct effects of these second messengers are to stimulate Ca2+ release (Ins 1,4,5 P,) [2,5] and activate protein kinase C (diacylglycerol) [4]. Acknow~edgemenrs-The authors acknowledge support from NIH research grants CA 43720, CA 2407 1 and Training Grant CA09582. Abbreviufions-EGF~pidermal growth factor; PDGF-platelet-derived growth factor; PLC-phospholipase C; PtdIns-4, 5-P,-phosphatidyhnositol bisphosphate; SH-src homology. 97
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Stimulation of PtdIns-4,5-P, hydrolysis occurs when cells are challenged with certain agonists that bind to either tyrosine kinase receptors or to G protein-coupled receptors [7-l I]. Current research objectives in understanding receptor coupled activation of phosphatidylinositol hydrolysis are to determine the number of PLC isozymes, whether different receptor classes activate the same or different PLC isozymes, how PLC activity is regulated for each isozyme, and whether the activity of each isozyme is regulated by the same or different mechanisms. PHOSPHOLIPASE
C ISOZYMES:
STRUCTURAL
CHARACTERISTICS
Experimental evidence, such as protein purification [ 12,131, molecular cloning [ 1416] and immunological studies [12,13,17,18], have identified four distinct classes of phosphatidylinositol-specific PLC(p, y,S,&). All four classes of PLC isozymes consist of a single polypeptide chain with molecular weights of: 150-l 54 kDa for PLC-p, 1455148 kDa for PLC-y, 85-88 kDa for PLC-Gand 86 kDa for PLC-E. The sequence of PLC cx, which was identified by molecular cloning, was recently determined not to be a PLC isozyme [ 191. Isoforms have been isolated for PLC-p (PI, fl) [20], PLC- y ( yl , y2) [2023] and PLC-6(61, 62, 63) [20]. At this time little is known about the kinetic properties of PLC isozymes as compared to the data available for phospholipase Al. It is known that all four isozymes of PLC have similar enzymatic properties in that phosphatidylinositol, phosphatidylinositol-4-phosphate or PtdIns- 4,5-P2 can be used as substrates and Ca” is required for activity, though each class differs in the optimal pH and Ca’+ concentration required for maximal in vitro activity [ 12.131. Structural similarities between PLC-y, -Sand -6have been observed (Fig. 1). These isozymes have two regions of sequence homology, denoted X and Y, that are 60% and 40% identical, respectively [19]. Because these regions are conserved among the isozymes, it has been suggested that these regions are domains that provide catalytic function. Deletion of either the X or Y region of PLC- yl or -y2 expressed in COS cells [20] or E. coli [21], respectively, results in the loss of catalytic activity. It is not known if the X and Y regions exist in PLC-E, because it has not been cloned as of this date. PLC-p
--I
x
H
Y
I
PLC-6 Y1254
PLC-Y, PLC-r2 FIGURE 1. Structural characteristics of the PLC isozymes. Comparison of PLC isozyme sequences have revealed distinct regions of homology. AU isozymes, -a -yand -6, possess two regions of homology, X and Y. In the case of PLC-yl and -y2, the X and Y region are split by SHZ/SH3 domains. PLC-yl is tyrosine phosphorylated. Tyrosine phosphorylation sites Y771, Y783 and Y1254 are indicated.
PLC- yhas an additional region of sequence homology between the X and Y regions (Fig. 1) to other proteins. This region is related to sequences found in the src family of tyrosine kinases, which are termed src homology region 2 (SH2) and src homology
Regulation
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region 3 (SH3) [ 14,221. Deletion of the SH2 and SH3 domains of PLC-~1 reduces, but does not eliminate, enzymatic activity; therefore, the SH domains may have a regulatory role [20,21]. In addition to non-receptor tyrosine kinases, such as src, SH2 and SH3 domains have been found in several other proteins, some of which are involved in signal transduction [24-311. These proteins include GTPase activating protein or GAP, [27], crk [28] and the 85 kDa subunit of phosphatidylinositol-3phosphate kinase [29-3 11. CELLULAR
AND SUBCELLULAR
LOCALIZATION
Because there are many isoforms of PLC, it would be interesting to know if there is tissue or cell specificity in the localization of each isoform. PLC-yl and PLC-81 have a wide tissue distribution [32,33], whereas PLC-I/;! is predominately expressed in lung, spleen and thymus [33]. PLC-pl is almost exclusively localized to the forebrain, as only low quantities of PLC-PI are detected in other tissues [32,33-361. Even within tissues, such as the central nervous system, there also can be differential expression of the PLC isozymes. PLC-yl and PLC- pl have been localized to neuronal cells, whereas PLC-Sl is localized to glial cells [37]. In cultured cells, PLC-yl is the most common isoform detected and only low level expression of PLC-61 and PLC-al has been detected [32]. Since in most tissues and in some cultured cells, multiple PLC isozymes are expressed simultaneously, the possibility exists that the different isozymes of PLC may be regulated by different receptor classes. The subcellular localization of the isozymes is also important. PLC-PI is primarily membrane-associated, whereas PLC-yl -61 and -&are primarily cytosolic [17.37]. The mechanism by which PLC-PI is membrane-associated is unclear from the available evidence. The cytosolic localization of other PLC isozymes presents a logistic problem since PtdIns-4,5- P, is located in the membrane. Translocation of the PLC-yl from cytosolic to membrane compartments has been observed in response to EGF and PDGF [38,39]. However, the mechanism by which this translocation takes place is unknown. PLC-yl does contain sequence similarities to a putative translocation domain, CalB [40]. This domain was first identified in protein kinase C as a Ca“ binding domain [41]. Work on cytosolic PLAz by Clark et al., suggests that this domain may be involved in Ca”-dependent translocation of the enzyme to the membrane [40]. Whether the ‘CalB’ sequence plays this role in PLC-yl translocation is unknown; however. PLC-y interaction with the membrane is not sensitive to EGTA [42] unlike PLA, [40]. Using immunohistochemical techniques, McBride et al. [43] localized a small pool ot PLC- yl to the cytoskeleton. An interaction of PLC- yl with the cytoskeleton may occur through the SH3 domain present in PLC-yl. SH3 sequences have been found in cytoskeleton proteins or proteins that interact with the cytoskeleton, leading to the hypothesis that SH3 domains may play a role in the anchoring of proteins involved in signal transduction to the cytoskeleton to form a ‘signaling complex’ [24,26]. REGULATION
BY TYROSINE
PHOSPHORYLATION
Certain ligands, such as EGF and PDGF, that activate tyrosine kinase receptors stimulate PtdIns-4,5-P, hydrolysis [44449]. The isoforms involved in tyrosine kinase-
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mediated PtdIns-4,5-P, hydrolysis are PLC- yl and - y2. While PLC- yl can be phosphorylated by both receptor and non-receptor tyrosine kinases [4454], not all tyrosine kinases phosphorylate PLC- yl. Tyrosine phosphorylation of PLC- yl is seen in response to EGF, FGF and PDGF, but not CSF or insulin [46,50,55]. No phosphorylation of PLC- yl, by src, a non-receptor tyrosine kinase, has been observed [46]. PLC- yl can also be phosphorylated on tyrosine in response to NGF in PC 12 cells [52,53] or CD3 in T cells [51,54]. Since the tyrosine kinase, trkB, has been shown to be a component of the high affinity NGF receptor [53], it is likely that trkB is responsible for the phosphorylation of PLC-yl in response to NGF. It is not clear how CD3 stimulates tyrosine phosphorylation of PLC- yl, since the T cell-antigen receptor does not possess an inherent protein kinase activity [56]. Association of the receptor with another protein possessing tyrosine kinase activity such as ~~56”” [56] or fyn [57] may be responsible for tyrosine phosphorylation of PLC- yl . Since some tyrosine kinases, but not others, can phosphorylate PLC- yl, it is tempting to postulate that different growth factors may activate specific signaling pathways. Less is known about the phosphorylation of PLC-~2, though crosslinking of membrane IgG in B cells leads to tyrosine phosphorylation of PLC-~2 [58], also transfection and expression of PLC-~2 in fibroblasts permit tyrosine phosphorylation of -~2 in response to PDGF [59]. In the presence of the appropriate ligand, phosphorylation of PLC-yl on tyrosine occurs rapidly, < 1 min [4547,49,55,60]. For tyrosine phosphorylation of PLC-yl to occur, PLC-yl must first associate with the receptor. Experiments have demonstrated the association of PLC-yl with the carboxy-terminus of the EGF and presumably the PDGF and FGF receptors [60-651. Since no association of PLC-yl with kinasenegative EGF receptors was detected, it was concluded that the receptor has to be tyrosine phosphorylated for PLC-yl association to occur [61,63]. In vitro experiments revealed reduced association of phosphorylated as compared to dephosphorylated PLC-yl with activated EGF receptors. These data suggest that after PLC-yl is phosphorylated, the enzyme rapidly dissociates from the receptor [61]. Association of PLC-yl with activated receptors is proposed to be mediated by SH2 domains. SH2 domains are regions capable of binding to sequences containing phosphotyrosine [24 27, 61, 621 and the preferential interaction of SH2 domains with tyrosine phosphorylated sequences would explain why PLC-yl specifically interacts with activated growth factor receptors. Recently Rotin et al. demonstrated that PLC-yl SH2 domains can prevent the dephosphorylation of both the PDGF and EGF receptors [62]. The EGF receptor residue that was specifically protected by PLC- yl SH2 domains was Y992. From this data it was concluded that Y992 was part of the high affinity binding site for PLC-yl. However, Sorkin et al. demonstrated that with an EGF receptor mutant in which Y992 was mutated to F992 tyrosine phosphorylation of PLC-yl occurred to the same extent as with the wild-type receptor [63]. Concomitant with an increase in tyrosine phosphorylation is an increase in PLC-yl activity [45,5 1,64,66-701. PLC- yl enzymatic activity is apparently controlled through a mechanism of tyrosine phosphorylation and dephosphorylation. Nishibe et al. demonstrated that when PLC-yl is phosphorylated by EGF receptor in vitro, which phosphorylates only tyrosine residues, an increase in PLC-yl activity occurred [66]. Incubation of the tyrosine phosphorylated PLC-yl with a tyrosine phosphatase resulted in a decrease in activity. The PLC-yl sites that are tyrosine phosphorylated in vivo and in vitro have been determined (Fig. 1). In vivo the major phosphorylation sites are Y771 and Y783,
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of Phospholipase
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located between the SH2 and SH3 domains, and Yl254, located near the carboxyterminus [48]. Y783 is conserved in PLC-72, but it has not been determined if PLC-y2 is phosphorylated on this residue [20]. An additional phosphorylation site at Y472 was observed in vitro [7 11. To identify which sites are important for PLC- yl function, Kim et al. mutated individual tyrosine phosphorylation sites to phenylalanine and then overexpressed the mutant proteins in NIH 3T3 cells [70]. By measuring the capacity of these PLC-71 mutations to stimulate Ins-l, 4,5-P, production in response to PDGF, it was determined that Y783 and Y 1254 were important for PLC-yl function, while Y771 was dispensible.
J
OL
PIP*, (mole fraction) FIGURE 2. Kinetics of control and EFG-activated PLC+. PLC-yl was imuumoprecipitated from untreated and EGF- treated cells and then assayedfor activity in the presence of increasing mole fraction of Ptdlns+-P,. Open symbols: - EFG and closed symbols: + EGF.
Recently, Wahl et al. have used kinetic analysis to show that tyrosine phosphorylation acts as an allosteric modifier to activate PLC-yl [67]. Under control conditions, non-tyrosine phosphorylated PLC-yl displays strong negative cooperativity toward PtdIns-4,5-P,, whereas the EGF-activated enzyme does not (Fig. 2). At a low PtdIns-4,5-P, mole fraction, a 7-fold difference in the relative velocity between control and EGF-activated PLC-yl is observed. However, at a high PtdIns4, 5-P, mole fraction, approaching that of pure liposomes, the relative velocity of the control enzyme is similar to the EGF-activated enzyme. These data suggest that at a low PtdIns-4, 5-P, mole fraction (representing a PtdIns-4, 5-P, concentration closer to its physiological concentration) the control enzyme is comparatively inactive. Tyrosine phosphorylation increases PLC-yl activity by overcoming the cooperative effect. Goldschmidt-Clermont et al., using a different assay system, only observed a difference in activity between control and tyrosine phosphorylated PLC-yl in the presence of profilin [68,69]. Profilin binds PtdIns-4,5-P, and in the presence of profilin the effective substrate concentration (that available for hydrolysis by PLC-yl) is lowered. Regulation of PLC-yl activity by allosterism implies that PLC-yl is a multi-domain molecule with at least two binding sites for PtdIns-4,5-P, (Fig. 3). This model is reminiscent of the dual phospholipid hypothesis proposed by Dennis and colleagues for cobra venom PLA, [72]. In the dual phospholipid model, PLA, initially interacts with a micelle surface by binding to one phospholipid molecule. Once associated with the micellar surface, the enzyme can then bind another phospholipid molecule, which is
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fast
PIP2 I
site
2 PLCYI (active)
FIGURE 3. Putative model of EGF-activation of PLC-71. PLC-71 may have two sites that bind PtdIns-4,5-P,, site 1 and site 2. The bhuiing of PtdIns-rlJ-P, to site 1 is a rate-limiting step. Under basal conditions, binding of PtdIns-4,5-P, to site 1 of PLC-yl occurs slowly. Once PtdIns-4,5-P, binds to site 1, binding PtdIns-4,5-P, to site 2 occurs rapidly. Tyrosine phosphorylation of PLC-yl results in a kinetic change such that the slow step (binding of PtdIns-4,5-P, to site 1) is no longer rate-limiting.
hydrolyzed. The situation with PLC-yl is more complex than PLA, since there are two forms of the enzyme, phosphorylated and non-phosphorylated. The kinetic data (Fig. 2) suggest that the non-phosphorylated enzyme may have two binding sites for PtdIns4,5-P2. In this model (Fig. 3), binding of PtdIns-4,5-P, to the first site of the nonphosphorylated enzyme occurs slowly, resulting in the cooperative effect observed in Fig. 2. In contrast, binding of PtdIns-4,5-Pz to the tyrosine phosphorylated enzyme occurs more readily, overcoming the cooperative effect (Fig. 2). Kinetically, it is not possible to distinguish whether there are one or two binding sites for PtdIns-4,5-P, with the tyrosine phosphorylated enzyme. It is possible that the conserved regions X and Y are both PtdIns-4,5-Pz binding sites. REGULATION
BY SERINE PHOSPHORYLATION
Serine phosphorylation may also play a role in the regulation of PLC activity. Both PLC- yl and PLC-p are serine phosphorylated and can be phosphorylated OR serine residues in vitro by protein kinase C [73,74]. In vivo, phorbol ester activation of protein kinase C only phosphorylates PLC-P; no phosphorylation of PLC- yl occurs [73]. PLCyl can also be phosphorylated by CAMP-dependent kinase in vitro [75]. The role of
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serine phosphorylation for either PLC-yl or PLC-pis not known. In vitro treatment of PLC-yl with a serine phosphatase, protein phosphatase 2A, did not affect the activity of either the basal or EGF-activated enzyme [76]. REGULATION
BY G PROTEINS
Phosphatidylinositol hydrolysis is also regulated by G protein-coupled receptors by pertussis toxin-sensitive and -insensitive mechanisms [7-10,77-901. A 42 kDa pertussis toxin-insensitive G protein 01 subunit has been isolated that can stimulate PLC hydrolysis of PtdIns-4,5-P? in vitro [84,85,89,90]. This unique a subunit is designated G,. Incubation of G, with various PLC isozymes revealed that G, stimulates PLC-/?I activity but has no effect on PLC-yl or PLC-8activity [84,85,90]. How G, modulates PLC-/?I activity is not known. It is possible that association of PLC-j31 with G,*GTP results in an activated conformation of PLC-pl akin to Gm@GTP activation of adenylcyclase. Results of protein isolation, immunological cross-reactivity, and Northern analysis of G, proteins indicate there is a family of G, proteins that may regulate PLC isozymes [7-10,84,85,87-911. These G, proteins differ in their activation by AlFl,- and the Ca” requirement for PtdIns-4,5-P? hydrolysis [84, 85, 89,901. Regulation of phosphatidylinositol hydrolysis by G protein-coupled receptors can also occur in a pertussis toxin-sensitive manner [7-10.8688,92]. Yang et ul. identified in hepatocytes a G protein that complexes with PLC-yl in a EGF- dependent manner [92]. This G protein can be ADP-ribosylated by pertussis toxin and reacts with Gu, antibody. It was suggested that association of PLC-yl with this Ga,-like protein aids in the attachment of PLC-yl to the plasma membrane. However, it is not known if the association of PLC-yl with a Gcw,-like protein modulates PLC-yl activity. PLC-6may also be regulated by a pertussis toxin-sensitive G protein. In the Chinese Hamster lung fibroblast cell line CCL39, pertussis toxin inhibited thrombin-induced phosphatidylinositol hydrolysis [77]. A mutant of CCL39 defective in thrombin, serotonin and AlFl,provoked phosphatidylinositol hydrolysis, was found to lack PLC-0‘ [93].whereas PLC-6 was present in the wild-type cell line (CCL39) [77]. The activity of PLC-E is increased in the presence of GTP$, which suggests PLC-r may also be regulated by a G-protein [17]. Whether this G protein is pertussis toxin-sensitive or -insensitive is not known. CONCLUSION At this time seven isoforms of PLC, that vary in tissue and cell distribution, have been identified. It is likely that through the use of molecular biological techniques. more isoforms of PLC will beldentified. Figure 4 presents a model of how two of the most studied isozymes of PLC, PLC-yl and -/Il, may be regulated. PLC-~1 activity is modulated by tyrosine phosphorylation, whereas PLC-PI activity is modulated by a unique class of G proteins, G,. The evidence that, at least in hepatocytes, PLC-yl associates with a G protein subunit demonstrates that each isozyme may be regulated by more than one mechanism. Judging from the evidence already collected, regulation of PLC by growth factors may be as complex and varied as the number of PLC isozymes and the receptors that activate them.
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tyrosine kinase receptors
non-tyrosine kinase receptors
FIGURE 4. Schematic of PLC activation by tyrosine kinase and non-tyrosine kinase receptors. By diverse mechanisms, receptors can activate PLC to produce PtdIns-4,5-P, and diacylglycerol. Putative mechanisms of activation for PLC-pand - yl are diagrammatically represented. In this schematic PLC-/?l activity is modulated by receptor activabion of a G protein, whereas PLC-yl activity is modulated by tyrosine phosphorylatlon of the enzyme by a tyrosine kinaae receptor.
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