Title: Regulation of the Lck SH2 Domain by Tyrosine Phosphorylation
Abstract: Src homology 2 (SH2) domains bind to phosphotyrosine (Tyr(P)) residues in specific sequence contexts in other proteins and thereby mediate tyrosine phosphorylationdependent protein-protein interactions. The SH2 domain of the Src family kinase Lck is phosphorylated at tyrosine 192 in T cells upon T cell antigen receptor triggering. We have studied the consequences of this phosphorylation on the properties of the SH2 domain and on the function of Lck in T cell activation. We report that phosphorylation at Tyr192 reduced the capacity of the isolated SH2 domain to bind a high affinity peptide ligand and Tyr(P)-containing cellular proteins. This effect was mimicked by mutation of Tyr192 to an acidic residue. In intact T cells, where Lck participates in T cell antigen receptor signal transduction in an SH2 domain-dependent manner, phosphorylation of Tyr192 correlated with reduced downstream signaling. Our results indicate that tyrosine phosphorylation of the SH2 domain of Lck terminates its high affinity binding to ligands, thereby negatively regulating its participation in T cell antigen receptor signaling. This represents a novel mechanism for the regulation of the function of SH2 domains. Src homology 2 (SH2) domains bind to phosphotyrosine (Tyr(P)) residues in specific sequence contexts in other proteins and thereby mediate tyrosine phosphorylationdependent protein-protein interactions. The SH2 domain of the Src family kinase Lck is phosphorylated at tyrosine 192 in T cells upon T cell antigen receptor triggering. We have studied the consequences of this phosphorylation on the properties of the SH2 domain and on the function of Lck in T cell activation. We report that phosphorylation at Tyr192 reduced the capacity of the isolated SH2 domain to bind a high affinity peptide ligand and Tyr(P)-containing cellular proteins. This effect was mimicked by mutation of Tyr192 to an acidic residue. In intact T cells, where Lck participates in T cell antigen receptor signal transduction in an SH2 domain-dependent manner, phosphorylation of Tyr192 correlated with reduced downstream signaling. Our results indicate that tyrosine phosphorylation of the SH2 domain of Lck terminates its high affinity binding to ligands, thereby negatively regulating its participation in T cell antigen receptor signaling. This represents a novel mechanism for the regulation of the function of SH2 domains. INTRODUCTIONSH2 1The abbreviations used are: SH2Src homology 2mAbmonoclonal antibodyTCRT cell antigen receptorGSTglutathione S-transferase. domains are independently folded hemispherical units of ~100 amino acid residues, which are found in many signaling proteins (1Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Google Scholar, 2Eck M.J. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Google Scholar, 3Moran M.F. Koch C.A. Anderson D. Ellis C. Engand L. Martin G.S. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8622-8626Google Scholar, 4Pawson T. Nature. 1995; 373: 573-580Google Scholar, 5Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Google Scholar). Their physiological function is to bind Tyr(P) residues in specific sequence contexts in other cellular proteins, thereby facilitating the formation of tyrosine phosphorylation-induced multimeric protein complexes (1Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Google Scholar, 3Moran M.F. Koch C.A. Anderson D. Ellis C. Engand L. Martin G.S. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8622-8626Google Scholar).The ligand-binding surface of the SH2 domain of the Lck nonreceptor protein tyrosine kinase contains two pockets, one for the Tyr(P) residue and another for the amino acid residue three positions C-terminal to it, the +3 amino acid (2Eck M.J. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Google Scholar, 5Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Google Scholar). Although the first pocket is well conserved among SH2 domains, the residues of the SH2 domain that form and surround the second pocket vary more. These differences determine the depth and properties of the pocket and thereby the preferred amino acid ligand (6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Google Scholar, 7Songyang Z. Shoelson S.E. McGlade J. Oliver P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Google Scholar). In the case of the Lck SH2 domain, the optimal ligand is a Tyr(P) followed by two acidic residues followed by an isoleucine at position +3, a specificity largely determined by amino acid residues in β strands D and E and in the EF loop adjacent to the second pocket (6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Google Scholar, 8von Bonin A. Wienands J. Manning U. Zuber J.F. Baumann G. J. Biol. Chem. 1994; 269: 33035-33041Google Scholar). Recently, it was shown that a single amino acid substitution in the EF loop in the SH2 domain of c-Src (a T215W mutation) changed the ligand selection of the SH2 domain to that of the Grb2 SH2 domain (9Marengere L.E.M. Songyang Z. Gish G.D. Schaller M.D. Parsons J.T. Stern M.J. Cantley L.C. Pawson T. Nature. 1994; 369: 502-505Google Scholar), which has a tryptophan residue at the corresponding location in its EF loop. Conversely, a switch from tryptophan to threonine in the SH2 domain of Grb2 changed its ligand preference to that of the c-Src SH2 domain (9Marengere L.E.M. Songyang Z. Gish G.D. Schaller M.D. Parsons J.T. Stern M.J. Cantley L.C. Pawson T. Nature. 1994; 369: 502-505Google Scholar).Here we describe a more physiological regulation of the function of an SH2 domain, namely the effect of phosphorylation of a highly conserved tyrosine residue, Tyr192, in the end of β strand E in the Lck SH2 domain. This phosphorylation event, which can be catalyzed by Syk and possibly Zap nonreceptor kinases, leads to a profound down-regulation of the ligand binding capacity of the SH2 domain. The consequences for the function of Lck as a signal transducer in T cell activation were studied. INTRODUCTIONSH2 1The abbreviations used are: SH2Src homology 2mAbmonoclonal antibodyTCRT cell antigen receptorGSTglutathione S-transferase. domains are independently folded hemispherical units of ~100 amino acid residues, which are found in many signaling proteins (1Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Google Scholar, 2Eck M.J. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Google Scholar, 3Moran M.F. Koch C.A. Anderson D. Ellis C. Engand L. Martin G.S. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8622-8626Google Scholar, 4Pawson T. Nature. 1995; 373: 573-580Google Scholar, 5Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Google Scholar). Their physiological function is to bind Tyr(P) residues in specific sequence contexts in other cellular proteins, thereby facilitating the formation of tyrosine phosphorylation-induced multimeric protein complexes (1Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Google Scholar, 3Moran M.F. Koch C.A. Anderson D. Ellis C. Engand L. Martin G.S. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8622-8626Google Scholar).The ligand-binding surface of the SH2 domain of the Lck nonreceptor protein tyrosine kinase contains two pockets, one for the Tyr(P) residue and another for the amino acid residue three positions C-terminal to it, the +3 amino acid (2Eck M.J. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Google Scholar, 5Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Google Scholar). Although the first pocket is well conserved among SH2 domains, the residues of the SH2 domain that form and surround the second pocket vary more. These differences determine the depth and properties of the pocket and thereby the preferred amino acid ligand (6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Google Scholar, 7Songyang Z. Shoelson S.E. McGlade J. Oliver P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Google Scholar). In the case of the Lck SH2 domain, the optimal ligand is a Tyr(P) followed by two acidic residues followed by an isoleucine at position +3, a specificity largely determined by amino acid residues in β strands D and E and in the EF loop adjacent to the second pocket (6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Google Scholar, 8von Bonin A. Wienands J. Manning U. Zuber J.F. Baumann G. J. Biol. Chem. 1994; 269: 33035-33041Google Scholar). Recently, it was shown that a single amino acid substitution in the EF loop in the SH2 domain of c-Src (a T215W mutation) changed the ligand selection of the SH2 domain to that of the Grb2 SH2 domain (9Marengere L.E.M. Songyang Z. Gish G.D. Schaller M.D. Parsons J.T. Stern M.J. Cantley L.C. Pawson T. Nature. 1994; 369: 502-505Google Scholar), which has a tryptophan residue at the corresponding location in its EF loop. Conversely, a switch from tryptophan to threonine in the SH2 domain of Grb2 changed its ligand preference to that of the c-Src SH2 domain (9Marengere L.E.M. Songyang Z. Gish G.D. Schaller M.D. Parsons J.T. Stern M.J. Cantley L.C. Pawson T. Nature. 1994; 369: 502-505Google Scholar).Here we describe a more physiological regulation of the function of an SH2 domain, namely the effect of phosphorylation of a highly conserved tyrosine residue, Tyr192, in the end of β strand E in the Lck SH2 domain. This phosphorylation event, which can be catalyzed by Syk and possibly Zap nonreceptor kinases, leads to a profound down-regulation of the ligand binding capacity of the SH2 domain. The consequences for the function of Lck as a signal transducer in T cell activation were studied.