Mechanism of Phosphorylation-induced Activation of Phospholipase C-γ Isozymes

The lipase activity of most phospholipases C (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y linker. In contrast, PLC-γ isozymes (PLC-γ1 and -γ2) are structurally distinct from other PLCs because multiple domains are present in their X/Y linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-γ isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-γ isozymes by their X/Y linker. Specifically, we identify the C-terminal SH2 domain within the X/Y linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y linker mediates high affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs. The lipase activity of most phospholipases C (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y linker. In contrast, PLC-γ isozymes (PLC-γ1 and -γ2) are structurally distinct from other PLCs because multiple domains are present in their X/Y linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-γ isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-γ isozymes by their X/Y linker. Specifically, we identify the C-terminal SH2 domain within the X/Y linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y linker mediates high affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs. IntroductionPhospholipase C (PLC) 2The abbreviations used are: PLCphospholipase CPtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphateTIMtriose phosphate isomeraseFGFRfibroblast growth factor receptorSEC-MALSsize exclusion chromatography-multi angle light scatteringITCisothermal titration calorimetryRTKreceptor tyrosine kinasenSH2N-terminal SH2cSH2C-terminal SH2sPHsplit pleckstrin homology. isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (1Harden T.K. Sondek J. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 355-379Crossref PubMed Scopus (112) Google Scholar). Diacylglycerol and inositol 1,4,5-trisphosphate subsequently activate PKC isozymes and liberate intracellular calcium stores, respectively, thereby initiating and propagating numerous cellular events including fertilization, proliferation, differentiation, and chemotaxis (2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar).The mammalian PLC family contains 13 isozymes subdivided into six subtypes (β, δ, γ, ϵ, η, and ζ) based on structural homology (1Harden T.K. Sondek J. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 355-379Crossref PubMed Scopus (112) Google Scholar). The highest sequence similarity among PLCs lies within the catalytic triose phosphate isomerase barrel comprised of two halves (X and Y boxes) separated by a highly degenerate X/Y linker. The active site is essentially invariant in PLC enzymes and ligates a calcium ion cofactor necessary for deprotonation of the PtdIns(4,5)P2 substrate and stabilization of the transition state (3Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (513) Google Scholar).Using structural and biochemical information, we recently showed that the X/Y linker in PLC-β, -δ, and -ϵ isozymes mediates auto-inhibition of phospholipase activity (4Hicks S.N. Jezyk M.R. Gershburg S. Seifert J.P. Harden T.K. Sondek J. Mol. Cell. 2008; 31: 383-394Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The X/Y linker of these PLCs is highly negatively charged, mostly disordered, and prevents PtdIns(4,5)P2 access to the active site by a combination of steric exclusion and electrostatic repulsion of negatively charged membranes. Consequently, we proposed a general mechanism of interfacial activation of these isozymes in which any upstream input that preferentially orients the active site toward PtdIns(4,5)P2-containing membranes would remove the X/Y linker to allow access of substrate for hydrolysis (4Hicks S.N. Jezyk M.R. Gershburg S. Seifert J.P. Harden T.K. Sondek J. Mol. Cell. 2008; 31: 383-394Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).In contrast to all other PLCs, the PLC-γ isozymes (PLC-γ1 and -γ2) possess highly elaborated X/Y linkers containing multiple domains: a split PH domain, two SH2 domains, and an SH3 domain (see Fig. 1A). Nevertheless, some reports suggest that PLC-γ isozymes are also auto-inhibited via their X/Y linkers. For example, deletion (5Horstman D.A. Chattopadhyay A. Carpenter G. Arch. Biochem. Biophys. 1999; 361: 149-155Crossref PubMed Scopus (19) Google Scholar) or proteolysis (6Fernald A.W. Jones G.A. Carpenter G. Biochem. J. 1994; 302: 503-509Crossref PubMed Scopus (27) Google Scholar, 7Horstman D.A. DeStefano K. Carpenter G. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 7518-7521Crossref PubMed Scopus (34) Google Scholar, 8Jones G.A. Wu Y. Arch. Biochem. Biophys. 2000; 375: 229-239Crossref PubMed Scopus (7) Google Scholar) of the X/Y linker modestly (2–5-fold) enhanced PLC-γ1 activity. This potential auto-inhibition has been neither rigorously confirmed nor evaluated. Moreover, the X/Y linker of PLC-γ isozymes lacks the high density of negative charge critical for interfacial activation of other PLCs, and because these isozymes are directly activated by phosphorylation, their mode of activation must be mechanistically distinct.Although it has been recognized for 20 years that activation of PLC-γ isozymes downstream of numerous receptor tyrosine kinases (RTKs) and cytosolic tyrosine kinases (2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar) involves membrane recruitment through the binding of the N-terminal SH2 (nSH2) within the X/Y linker and subsequent tyrosine phosphorylation of PLC-γ isozymes (9Wahl M.I. Nishibe S. Kim J.W. Kim H. Rhee S.G. Carpenter G. J. Biol. Chem. 1990; 265: 3944-3948Abstract Full Text PDF PubMed Google Scholar, 10Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (444) Google Scholar, 11Serrano C.J. Graham L. DeBell K. Rawat R. Veri M.C. Bonvini E. Rellahan B.L. Reischl I.G. J. Immunol. 2005; 174: 6233-6237Crossref PubMed Scopus (37) Google Scholar), the molecular mechanism linking phosphorylation to activation remains poorly defined.In this report, we used highly purified proteins and robust in vitro assays to discover that PLC-γ isozymes have modified the common mechanism of interfacial activation present in most PLCs to couple phosphorylation of a highly elaborated X/Y linker with release of auto-inhibition and consequential activation. IntroductionPhospholipase C (PLC) 2The abbreviations used are: PLCphospholipase CPtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphateTIMtriose phosphate isomeraseFGFRfibroblast growth factor receptorSEC-MALSsize exclusion chromatography-multi angle light scatteringITCisothermal titration calorimetryRTKreceptor tyrosine kinasenSH2N-terminal SH2cSH2C-terminal SH2sPHsplit pleckstrin homology. isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (1Harden T.K. Sondek J. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 355-379Crossref PubMed Scopus (112) Google Scholar). Diacylglycerol and inositol 1,4,5-trisphosphate subsequently activate PKC isozymes and liberate intracellular calcium stores, respectively, thereby initiating and propagating numerous cellular events including fertilization, proliferation, differentiation, and chemotaxis (2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar).The mammalian PLC family contains 13 isozymes subdivided into six subtypes (β, δ, γ, ϵ, η, and ζ) based on structural homology (1Harden T.K. Sondek J. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 355-379Crossref PubMed Scopus (112) Google Scholar). The highest sequence similarity among PLCs lies within the catalytic triose phosphate isomerase barrel comprised of two halves (X and Y boxes) separated by a highly degenerate X/Y linker. The active site is essentially invariant in PLC enzymes and ligates a calcium ion cofactor necessary for deprotonation of the PtdIns(4,5)P2 substrate and stabilization of the transition state (3Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (513) Google Scholar).Using structural and biochemical information, we recently showed that the X/Y linker in PLC-β, -δ, and -ϵ isozymes mediates auto-inhibition of phospholipase activity (4Hicks S.N. Jezyk M.R. Gershburg S. Seifert J.P. Harden T.K. Sondek J. Mol. Cell. 2008; 31: 383-394Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The X/Y linker of these PLCs is highly negatively charged, mostly disordered, and prevents PtdIns(4,5)P2 access to the active site by a combination of steric exclusion and electrostatic repulsion of negatively charged membranes. Consequently, we proposed a general mechanism of interfacial activation of these isozymes in which any upstream input that preferentially orients the active site toward PtdIns(4,5)P2-containing membranes would remove the X/Y linker to allow access of substrate for hydrolysis (4Hicks S.N. Jezyk M.R. Gershburg S. Seifert J.P. Harden T.K. Sondek J. Mol. Cell. 2008; 31: 383-394Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).In contrast to all other PLCs, the PLC-γ isozymes (PLC-γ1 and -γ2) possess highly elaborated X/Y linkers containing multiple domains: a split PH domain, two SH2 domains, and an SH3 domain (see Fig. 1A). Nevertheless, some reports suggest that PLC-γ isozymes are also auto-inhibited via their X/Y linkers. For example, deletion (5Horstman D.A. Chattopadhyay A. Carpenter G. Arch. Biochem. Biophys. 1999; 361: 149-155Crossref PubMed Scopus (19) Google Scholar) or proteolysis (6Fernald A.W. Jones G.A. Carpenter G. Biochem. J. 1994; 302: 503-509Crossref PubMed Scopus (27) Google Scholar, 7Horstman D.A. DeStefano K. Carpenter G. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 7518-7521Crossref PubMed Scopus (34) Google Scholar, 8Jones G.A. Wu Y. Arch. Biochem. Biophys. 2000; 375: 229-239Crossref PubMed Scopus (7) Google Scholar) of the X/Y linker modestly (2–5-fold) enhanced PLC-γ1 activity. This potential auto-inhibition has been neither rigorously confirmed nor evaluated. Moreover, the X/Y linker of PLC-γ isozymes lacks the high density of negative charge critical for interfacial activation of other PLCs, and because these isozymes are directly activated by phosphorylation, their mode of activation must be mechanistically distinct.Although it has been recognized for 20 years that activation of PLC-γ isozymes downstream of numerous receptor tyrosine kinases (RTKs) and cytosolic tyrosine kinases (2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar) involves membrane recruitment through the binding of the N-terminal SH2 (nSH2) within the X/Y linker and subsequent tyrosine phosphorylation of PLC-γ isozymes (9Wahl M.I. Nishibe S. Kim J.W. Kim H. Rhee S.G. Carpenter G. J. Biol. Chem. 1990; 265: 3944-3948Abstract Full Text PDF PubMed Google Scholar, 10Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (444) Google Scholar, 11Serrano C.J. Graham L. DeBell K. Rawat R. Veri M.C. Bonvini E. Rellahan B.L. Reischl I.G. J. Immunol. 2005; 174: 6233-6237Crossref PubMed Scopus (37) Google Scholar), the molecular mechanism linking phosphorylation to activation remains poorly defined.In this report, we used highly purified proteins and robust in vitro assays to discover that PLC-γ isozymes have modified the common mechanism of interfacial activation present in most PLCs to couple phosphorylation of a highly elaborated X/Y linker with release of auto-inhibition and consequential activation.