The Activated Form of the Lck Tyrosine Protein Kinase in Cells Exposed to Hydrogen Peroxide Is Phosphorylated at Both Tyr-394 and Tyr-505
1997; Elsevier BV; Volume: 272; Issue: 41 Linguagem: Inglês
10.1074/jbc.272.41.25429
ISSN1083-351X
AutoresJames S. Hardwick, Bartholomew M. Sefton,
Tópico(s)Renin-Angiotensin System Studies
ResumoMembers of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H2O2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H2O2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members. Members of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H2O2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H2O2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members. p56lck, a member of the Src family of non-receptor tyrosine protein kinases (1Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 2Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (161) Google Scholar), is expressed predominantly in T cells. Lck function is critical both for T-cell development in the thymus (3Allen J.M. Forbush K.A. Perlmutter R.M. Mol. Cell. Biol. 1992; 12: 2758-2768Crossref PubMed Scopus (105) Google Scholar,4Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar) and activation of mature T cells in the periphery by antigen (5Karnitz L. Sutor S.L. Torigoe T. Reed J.C. Bell M.P. McKean D.J. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1992; 12: 4521-4530Crossref PubMed Scopus (180) Google Scholar, 6Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). Lck stably associates with the inner surface of the plasma membrane as a result of myristoylation of Gly-2 and palmitoylation of Ser-3 and Ser-5 (7Marchildon G.A. Casnellie J.E. Walsh K.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7679-7682Crossref PubMed Scopus (67) Google Scholar, 8Paige L.A. Nadler M.J. Harrison M.L. Cassady J.M. Geahlen R.L. J. Biol. Chem. 1993; 268: 8669-8674Abstract Full Text PDF PubMed Google Scholar, 9Kwong J. Lublin D. Biochem. Biophys. Res. Commun. 1995; 207: 868-876Crossref PubMed Scopus (46) Google Scholar, 10Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar). There it binds to the T-cell receptor-associated glycoproteins CD4 and CD8 as well as other proteins through its unique amino terminus (11Rudd C.E. Trevillyan J.M. Dasgupta J.D. Wong L.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (613) Google Scholar, 12Veillette A. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 44: 301-308Abstract Full Text PDF Scopus (1122) Google Scholar, 13Campbell M.-A. Sefton B.M. EMBO J. 1990; 9: 2125-2131Crossref PubMed Scopus (161) Google Scholar, 14Stefanova I. Horejsi V. Ansotegui I.J. Knapp W. Stockinger H. Science. 1991; 254: 1016-1019Crossref PubMed Scopus (762) Google Scholar, 15Shenoy-Scaria A.M. Timson Gauen L.K. Kwong J. Shaw A.S. Lublin L.M. Mol. Cell. Biol. 1993; 13: 6385-6392Crossref PubMed Scopus (225) Google Scholar). The activity of Lck is regulated by phosphorylation of two conserved tyrosine residues. Tyr-505 (equivalent to Tyr-527 in c-Src) is located near the carboxyl terminus of Lck and, when phosphorylated, associates intramolecularly with the SH2 domain in the amino-terminal half of the protein. This helps stabilize Lck in a conformation that, biologically, is relatively inactive (16Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 17Marth J.D. Cooper J.A. King C.S. Ziegler S.F. Tinker D.A. Overell R.W. Krebs E.G. Perlmutter R.M. Mol. Cell. Biol. 1988; 8: 540-550Crossref PubMed Scopus (182) Google Scholar, 18Sieh M. Bolen J. Weiss A. EMBO J. 1993; 12: 315-321Crossref PubMed Scopus (185) Google Scholar, 19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar). In the absence of phosphorylation at Tyr-505, intramolecular binding of the carboxyl terminus to the SH2 domain does not occur, and Lck exhibits increased activity in vivo. In contrast, phosphorylation of Tyr-394 (equivalent to Tyr-416 in c-Src) stimulates the catalytic activity of Lck (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 23Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1079) Google Scholar). Phosphorylation of Tyr-394 allows the formation of hydrogen bonds between the phosphate of Tyr(P) 1The abbreviations used are: Tyr(P), phosphotyrosine; PVDF, polyvinylidene difluoride. 1The abbreviations used are: Tyr(P), phosphotyrosine; PVDF, polyvinylidene difluoride.-394 and amino acid residues in the catalytic cleft. These interactions allow the enzyme to assume a conformation resembling that of activated cyclic AMP-dependent protein kinase A (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar).We have previously demonstrated that hydrogen peroxide, a potent activator of Lck, acts by inducing phosphorylation of Lck on Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 37Yurchak L.K. Hardwick J.S. Amrein K. Pierno K. Sefton B.M. J. Biol. Chem. 1996; 271: 12549-12554Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). It is likely that the effects of exposing cells to H2O2 are mediated by global inhibition of tyrosine phosphatases (27Hecht D. Zick Y. Biochem. Biophys. Res. Commun. 1992; 188: 773-779Crossref PubMed Scopus (247) Google Scholar, 28Sullivan S.G. Chiu D.T. Errasfa M. Wang J.M. Qi J.S. Stern A. Free Radical Biol. Med. 1994; 16: 399-403Crossref PubMed Scopus (104) Google Scholar, 29Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar, 30Bevan A.P. Drake P.G. Yale J.F. Shaver A. Posner B.I. Mol. Cell. Biochem. 1995; 153: 49-58Crossref PubMed Scopus (113) Google Scholar, 31Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. Chem. 1997; 272: 843-851Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). The increase in phosphorylation of Lck at Tyr-394 that we observe in the presence of H2O2 may therefore result largely from reduced dephosphorylation of this site. Our previous work did not address the question of whether or not activation of Lck by H2O2-induced phosphorylation of Tyr-394 required dephosphorylation of Tyr-505. Thus, the extent to which the H2O2-activated population of Lck molecules was phosphorylated on Tyr-505 was unclear. Here we show that the population of Lck that is phosphorylated on Tyr-394 in response to H2O2 exposure is also phosphorylated at Tyr-505. Therefore, dephosphorylation of Tyr-505 and untethering of the SH2 domain is not a prerequisite for either phosphorylation of Lck at Tyr-394 or activation of the kinase by Tyr-394 phosphorylation. These results indicate that phosphorylation of Tyr-394 positively regulates Lck activity and is dominant over any negative regulation induced by phosphorylation of Tyr-505.DISCUSSIONThe catalytic activity of Lck is greatly influenced by the phosphorylation state of Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 38Shibuya H. Kohu K. Yamada K. Barsoumian E. Perlmutter R. Taniguchi T. Mol. Cell. Biol. 1994; 14: 5812-5819Crossref PubMed Scopus (35) Google Scholar, 39Veillette A. Fournel M. Oncogene. 1990; 5: 1455-1462PubMed Google Scholar). Mutation of Tyr-394 to phenylalanine not only decreases Lck activity in unstimulated cells, but also prevents activation of Lck by oxidative stress. In addition, Lck that has been genetically activated by mutation of Tyr-505 to phenylalanine loses its transforming ability when Tyr-394 is also mutated to phenylalanine (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 40Caron L. Abraham N. Pawson T. Veillette A. Mol. Cell. Biol. 1992; 12: 2720-2729Crossref PubMed Scopus (80) Google Scholar). We previously showed that the extent of Tyr-505 phosphorylation in Lck from H2O2-stimulated cells was at least as great as that of Tyr-394. Thus, H2O2 activation appeared not to require Tyr-505 dephosphorylation. These observations suggested that Tyr-394 phosphorylation could override any negative regulation of Lck due to Tyr-505 phosphorylation. However, it was impossible to rule out the possibility that two differentially phosphorylated subpopulations of Lck existed in H2O2-stimulated cells, a relatively inactive population phosphorylated only on Tyr-505 and an activated population phosphorylated only on Tyr-394. Thus, it was formally possible that activation of Lck by Tyr-394 phosphorylation occurred only in the absence of phosphorylation of Tyr-505.Through the use of a phosphorylation state-specific antibody, we have now shown formally that Lck that is phosphorylated on Tyr-505 may be additionally phosphorylated on Tyr-394. If dephosphorylation of Tyr-505 were required for phosphorylation of Tyr-394, we would expect that Lck immunoprecipitated by α-Tyr(P)-416 sera would only be phosphorylated on Tyr-394. This was not seen. The observation that the Tyr(P)-505:Tyr(P)-394 ratio is approximately 1:1 suggests that the molecules phosphorylated on Tyr-394 are also phosphorylated on Tyr-505. Thus, the activating effects of Tyr-394 phosphorylation are dominant over the inhibitory effects of Tyr-505 phosphorylation. Apparently, even when Lck is in a “closed” conformation with Tyr(P)-505 bound to the SH2 domain and the SH3 domain bound to the polyproline type II helix in the linker region between the SH2 and catalytic domains (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar), Tyr-394 is still accessible as a substrate for phosphorylation. Comparison of the crystal structure of Lck phosphorylated at Tyr-394 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar) with that of Src and Hck lacking phosphorylation at this site (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar) suggests that phosphorylation of Tyr-394 allows the formation of hydrogen bonds between Tyr(P)-394 and Arg-387 and Arg-363. These interactions appear to induce the repositioning of Glu-288, Leu-385, and Arg-387 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar), and this in turn allows Lys-273, Glu-288, Asp-364, Asn-369, and Asp-382, residues critical to ATP binding and phosphate transfer, to assume positions characteristic of an active catalytic site (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar). Our data suggest that such repositioning of residues in the catalytic site of Lck can occur while the SH3 domain is still intramolecularly bound to the SH2-kinase linker region.Our results agree with data presented by others who showed that the Src tyrosine kinase retains activity when phosphorylated on Tyr-416 and Tyr-527 (41Boerner R.J. Kassel D.B. Barker S.C. Ellis B. DeLacy P. Knight W.B. Biochemistry. 1996; 35: 9519-9525Crossref PubMed Scopus (84) Google Scholar). 3J. D. Bjorge and D. J. Fujita, personal communication. Previous work in our laboratory as well as kinetic data by other groups suggest that the activating phosphorylation of Tyr-394 in Src family members is an intermolecular event rather than intramolecular reaction (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar,42Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (136) Google Scholar, 43Barker S.C. Kassel D.B. Weigl D. Huang X. Luther M.A. Knight W.B. Biochemistry. 1995; 34: 14843-14851Crossref PubMed Scopus (158) Google Scholar, 44Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar). 4K. Pierno and B. M. Sefton, unpublished results. Intermolecular phosphorylation of Tyr-394 may be carried out by Lck in vivo, but it is quite possible that other Src family members, or non-Src tyrosine kinases, may also act to phosphorylate Tyr-394 and activate Lck. Consistent with this second possibility is our finding that H2O2 stimulates the activity of an as yet unidentified tyrosine kinase in Lck-deficient JCaM1.6 cells that can phosphorylate Lck on Tyr-394.2Our results show that it is possible for the Src family member Lck to be phosphorylated at both the conserved tyrosine in the activation loop and the C-terminal tyrosine simultaneously. Because H2O2 activates Lck while inducing an increase of phosphorylation of both Tyr-394 and Tyr-505, these results suggest that Lck phosphorylated on Tyr-394 and Tyr-505 is catalytically activated. Thus, activation of Lck can occur in the absence of Tyr505 dephosphorylation or SH3 domain disassociation from the SH2-kinase linker. It is reasonable to predict that phosphorylation of the absolutely conserved tyrosine in the activation loop of other Src kinases will also activate them in the absence of dephosphorylation of the conserved carboxyl-terminal tyrosine. p56lck, a member of the Src family of non-receptor tyrosine protein kinases (1Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 2Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (161) Google Scholar), is expressed predominantly in T cells. Lck function is critical both for T-cell development in the thymus (3Allen J.M. Forbush K.A. Perlmutter R.M. Mol. Cell. Biol. 1992; 12: 2758-2768Crossref PubMed Scopus (105) Google Scholar,4Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar) and activation of mature T cells in the periphery by antigen (5Karnitz L. Sutor S.L. Torigoe T. Reed J.C. Bell M.P. McKean D.J. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1992; 12: 4521-4530Crossref PubMed Scopus (180) Google Scholar, 6Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). Lck stably associates with the inner surface of the plasma membrane as a result of myristoylation of Gly-2 and palmitoylation of Ser-3 and Ser-5 (7Marchildon G.A. Casnellie J.E. Walsh K.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7679-7682Crossref PubMed Scopus (67) Google Scholar, 8Paige L.A. Nadler M.J. Harrison M.L. Cassady J.M. Geahlen R.L. J. Biol. Chem. 1993; 268: 8669-8674Abstract Full Text PDF PubMed Google Scholar, 9Kwong J. Lublin D. Biochem. Biophys. Res. Commun. 1995; 207: 868-876Crossref PubMed Scopus (46) Google Scholar, 10Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar). There it binds to the T-cell receptor-associated glycoproteins CD4 and CD8 as well as other proteins through its unique amino terminus (11Rudd C.E. Trevillyan J.M. Dasgupta J.D. Wong L.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (613) Google Scholar, 12Veillette A. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 44: 301-308Abstract Full Text PDF Scopus (1122) Google Scholar, 13Campbell M.-A. Sefton B.M. EMBO J. 1990; 9: 2125-2131Crossref PubMed Scopus (161) Google Scholar, 14Stefanova I. Horejsi V. Ansotegui I.J. Knapp W. Stockinger H. Science. 1991; 254: 1016-1019Crossref PubMed Scopus (762) Google Scholar, 15Shenoy-Scaria A.M. Timson Gauen L.K. Kwong J. Shaw A.S. Lublin L.M. Mol. Cell. Biol. 1993; 13: 6385-6392Crossref PubMed Scopus (225) Google Scholar). The activity of Lck is regulated by phosphorylation of two conserved tyrosine residues. Tyr-505 (equivalent to Tyr-527 in c-Src) is located near the carboxyl terminus of Lck and, when phosphorylated, associates intramolecularly with the SH2 domain in the amino-terminal half of the protein. This helps stabilize Lck in a conformation that, biologically, is relatively inactive (16Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 17Marth J.D. Cooper J.A. King C.S. Ziegler S.F. Tinker D.A. Overell R.W. Krebs E.G. Perlmutter R.M. Mol. Cell. Biol. 1988; 8: 540-550Crossref PubMed Scopus (182) Google Scholar, 18Sieh M. Bolen J. Weiss A. EMBO J. 1993; 12: 315-321Crossref PubMed Scopus (185) Google Scholar, 19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar). In the absence of phosphorylation at Tyr-505, intramolecular binding of the carboxyl terminus to the SH2 domain does not occur, and Lck exhibits increased activity in vivo. In contrast, phosphorylation of Tyr-394 (equivalent to Tyr-416 in c-Src) stimulates the catalytic activity of Lck (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 23Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1079) Google Scholar). Phosphorylation of Tyr-394 allows the formation of hydrogen bonds between the phosphate of Tyr(P) 1The abbreviations used are: Tyr(P), phosphotyrosine; PVDF, polyvinylidene difluoride. 1The abbreviations used are: Tyr(P), phosphotyrosine; PVDF, polyvinylidene difluoride.-394 and amino acid residues in the catalytic cleft. These interactions allow the enzyme to assume a conformation resembling that of activated cyclic AMP-dependent protein kinase A (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar). We have previously demonstrated that hydrogen peroxide, a potent activator of Lck, acts by inducing phosphorylation of Lck on Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 37Yurchak L.K. Hardwick J.S. Amrein K. Pierno K. Sefton B.M. J. Biol. Chem. 1996; 271: 12549-12554Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). It is likely that the effects of exposing cells to H2O2 are mediated by global inhibition of tyrosine phosphatases (27Hecht D. Zick Y. Biochem. Biophys. Res. Commun. 1992; 188: 773-779Crossref PubMed Scopus (247) Google Scholar, 28Sullivan S.G. Chiu D.T. Errasfa M. Wang J.M. Qi J.S. Stern A. Free Radical Biol. Med. 1994; 16: 399-403Crossref PubMed Scopus (104) Google Scholar, 29Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar, 30Bevan A.P. Drake P.G. Yale J.F. Shaver A. Posner B.I. Mol. Cell. Biochem. 1995; 153: 49-58Crossref PubMed Scopus (113) Google Scholar, 31Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. Chem. 1997; 272: 843-851Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). The increase in phosphorylation of Lck at Tyr-394 that we observe in the presence of H2O2 may therefore result largely from reduced dephosphorylation of this site. Our previous work did not address the question of whether or not activation of Lck by H2O2-induced phosphorylation of Tyr-394 required dephosphorylation of Tyr-505. Thus, the extent to which the H2O2-activated population of Lck molecules was phosphorylated on Tyr-505 was unclear. Here we show that the population of Lck that is phosphorylated on Tyr-394 in response to H2O2 exposure is also phosphorylated at Tyr-505. Therefore, dephosphorylation of Tyr-505 and untethering of the SH2 domain is not a prerequisite for either phosphorylation of Lck at Tyr-394 or activation of the kinase by Tyr-394 phosphorylation. These results indicate that phosphorylation of Tyr-394 positively regulates Lck activity and is dominant over any negative regulation induced by phosphorylation of Tyr-505. DISCUSSIONThe catalytic activity of Lck is greatly influenced by the phosphorylation state of Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 38Shibuya H. Kohu K. Yamada K. Barsoumian E. Perlmutter R. Taniguchi T. Mol. Cell. Biol. 1994; 14: 5812-5819Crossref PubMed Scopus (35) Google Scholar, 39Veillette A. Fournel M. Oncogene. 1990; 5: 1455-1462PubMed Google Scholar). Mutation of Tyr-394 to phenylalanine not only decreases Lck activity in unstimulated cells, but also prevents activation of Lck by oxidative stress. In addition, Lck that has been genetically activated by mutation of Tyr-505 to phenylalanine loses its transforming ability when Tyr-394 is also mutated to phenylalanine (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 40Caron L. Abraham N. Pawson T. Veillette A. Mol. Cell. Biol. 1992; 12: 2720-2729Crossref PubMed Scopus (80) Google Scholar). We previously showed that the extent of Tyr-505 phosphorylation in Lck from H2O2-stimulated cells was at least as great as that of Tyr-394. Thus, H2O2 activation appeared not to require Tyr-505 dephosphorylation. These observations suggested that Tyr-394 phosphorylation could override any negative regulation of Lck due to Tyr-505 phosphorylation. However, it was impossible to rule out the possibility that two differentially phosphorylated subpopulations of Lck existed in H2O2-stimulated cells, a relatively inactive population phosphorylated only on Tyr-505 and an activated population phosphorylated only on Tyr-394. Thus, it was formally possible that activation of Lck by Tyr-394 phosphorylation occurred only in the absence of phosphorylation of Tyr-505.Through the use of a phosphorylation state-specific antibody, we have now shown formally that Lck that is phosphorylated on Tyr-505 may be additionally phosphorylated on Tyr-394. If dephosphorylation of Tyr-505 were required for phosphorylation of Tyr-394, we would expect that Lck immunoprecipitated by α-Tyr(P)-416 sera would only be phosphorylated on Tyr-394. This was not seen. The observation that the Tyr(P)-505:Tyr(P)-394 ratio is approximately 1:1 suggests that the molecules phosphorylated on Tyr-394 are also phosphorylated on Tyr-505. Thus, the activating effects of Tyr-394 phosphorylation are dominant over the inhibitory effects of Tyr-505 phosphorylation. Apparently, even when Lck is in a “closed” conformation with Tyr(P)-505 bound to the SH2 domain and the SH3 domain bound to the polyproline type II helix in the linker region between the SH2 and catalytic domains (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar), Tyr-394 is still accessible as a substrate for phosphorylation. Comparison of the crystal structure of Lck phosphorylated at Tyr-394 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar) with that of Src and Hck lacking phosphorylation at this site (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar) suggests that phosphorylation of Tyr-394 allows the formation of hydrogen bonds between Tyr(P)-394 and Arg-387 and Arg-363. These interactions appear to induce the repositioning of Glu-288, Leu-385, and Arg-387 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar), and this in turn allows Lys-273, Glu-288, Asp-364, Asn-369, and Asp-382, residues critical to ATP binding and phosphate transfer, to assume positions characteristic of an active catalytic site (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar). Our data suggest that such repositioning of residues in the catalytic site of Lck can occur while the SH3 domain is still intramolecularly bound to the SH2-kinase linker region.Our results agree with data presented by others who showed that the Src tyrosine kinase retains activity when phosphorylated on Tyr-416 and Tyr-527 (41Boerner R.J. Kassel D.B. Barker S.C. Ellis B. DeLacy P. Knight W.B. Biochemistry. 1996; 35: 9519-9525Crossref PubMed Scopus (84) Google Scholar). 3J. D. Bjorge and D. J. Fujita, personal communication. Previous work in our laboratory as well as kinetic data by other groups suggest that the activating phosphorylation of Tyr-394 in Src family members is an intermolecular event rather than intramolecular reaction (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar,42Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (136) Google Scholar, 43Barker S.C. Kassel D.B. Weigl D. Huang X. Luther M.A. Knight W.B. Biochemistry. 1995; 34: 14843-14851Crossref PubMed Scopus (158) Google Scholar, 44Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar). 4K. Pierno and B. M. Sefton, unpublished results. Intermolecular phosphorylation of Tyr-394 may be carried out by Lck in vivo, but it is quite possible that other Src family members, or non-Src tyrosine kinases, may also act to phosphorylate Tyr-394 and activate Lck. Consistent with this second possibility is our finding that H2O2 stimulates the activity of an as yet unidentified tyrosine kinase in Lck-deficient JCaM1.6 cells that can phosphorylate Lck on Tyr-394.2Our results show that it is possible for the Src family member Lck to be phosphorylated at both the conserved tyrosine in the activation loop and the C-terminal tyrosine simultaneously. Because H2O2 activates Lck while inducing an increase of phosphorylation of both Tyr-394 and Tyr-505, these results suggest that Lck phosphorylated on Tyr-394 and Tyr-505 is catalytically activated. Thus, activation of Lck can occur in the absence of Tyr505 dephosphorylation or SH3 domain disassociation from the SH2-kinase linker. It is reasonable to predict that phosphorylation of the absolutely conserved tyrosine in the activation loop of other Src kinases will also activate them in the absence of dephosphorylation of the conserved carboxyl-terminal tyrosine. The catalytic activity of Lck is greatly influenced by the phosphorylation state of Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 38Shibuya H. Kohu K. Yamada K. Barsoumian E. Perlmutter R. Taniguchi T. Mol. Cell. Biol. 1994; 14: 5812-5819Crossref PubMed Scopus (35) Google Scholar, 39Veillette A. Fournel M. Oncogene. 1990; 5: 1455-1462PubMed Google Scholar). Mutation of Tyr-394 to phenylalanine not only decreases Lck activity in unstimulated cells, but also prevents activation of Lck by oxidative stress. In addition, Lck that has been genetically activated by mutation of Tyr-505 to phenylalanine loses its transforming ability when Tyr-394 is also mutated to phenylalanine (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 40Caron L. Abraham N. Pawson T. Veillette A. Mol. Cell. Biol. 1992; 12: 2720-2729Crossref PubMed Scopus (80) Google Scholar). We previously showed that the extent of Tyr-505 phosphorylation in Lck from H2O2-stimulated cells was at least as great as that of Tyr-394. Thus, H2O2 activation appeared not to require Tyr-505 dephosphorylation. These observations suggested that Tyr-394 phosphorylation could override any negative regulation of Lck due to Tyr-505 phosphorylation. However, it was impossible to rule out the possibility that two differentially phosphorylated subpopulations of Lck existed in H2O2-stimulated cells, a relatively inactive population phosphorylated only on Tyr-505 and an activated population phosphorylated only on Tyr-394. Thus, it was formally possible that activation of Lck by Tyr-394 phosphorylation occurred only in the absence of phosphorylation of Tyr-505. Through the use of a phosphorylation state-specific antibody, we have now shown formally that Lck that is phosphorylated on Tyr-505 may be additionally phosphorylated on Tyr-394. If dephosphorylation of Tyr-505 were required for phosphorylation of Tyr-394, we would expect that Lck immunoprecipitated by α-Tyr(P)-416 sera would only be phosphorylated on Tyr-394. This was not seen. The observation that the Tyr(P)-505:Tyr(P)-394 ratio is approximately 1:1 suggests that the molecules phosphorylated on Tyr-394 are also phosphorylated on Tyr-505. Thus, the activating effects of Tyr-394 phosphorylation are dominant over the inhibitory effects of Tyr-505 phosphorylation. Apparently, even when Lck is in a “closed” conformation with Tyr(P)-505 bound to the SH2 domain and the SH3 domain bound to the polyproline type II helix in the linker region between the SH2 and catalytic domains (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar), Tyr-394 is still accessible as a substrate for phosphorylation. Comparison of the crystal structure of Lck phosphorylated at Tyr-394 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar) with that of Src and Hck lacking phosphorylation at this site (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar) suggests that phosphorylation of Tyr-394 allows the formation of hydrogen bonds between Tyr(P)-394 and Arg-387 and Arg-363. These interactions appear to induce the repositioning of Glu-288, Leu-385, and Arg-387 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar), and this in turn allows Lys-273, Glu-288, Asp-364, Asn-369, and Asp-382, residues critical to ATP binding and phosphate transfer, to assume positions characteristic of an active catalytic site (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar). Our data suggest that such repositioning of residues in the catalytic site of Lck can occur while the SH3 domain is still intramolecularly bound to the SH2-kinase linker region. Our results agree with data presented by others who showed that the Src tyrosine kinase retains activity when phosphorylated on Tyr-416 and Tyr-527 (41Boerner R.J. Kassel D.B. Barker S.C. Ellis B. DeLacy P. Knight W.B. Biochemistry. 1996; 35: 9519-9525Crossref PubMed Scopus (84) Google Scholar). 3J. D. Bjorge and D. J. Fujita, personal communication. Previous work in our laboratory as well as kinetic data by other groups suggest that the activating phosphorylation of Tyr-394 in Src family members is an intermolecular event rather than intramolecular reaction (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar,42Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (136) Google Scholar, 43Barker S.C. Kassel D.B. Weigl D. Huang X. Luther M.A. Knight W.B. Biochemistry. 1995; 34: 14843-14851Crossref PubMed Scopus (158) Google Scholar, 44Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar). 4K. Pierno and B. M. Sefton, unpublished results. Intermolecular phosphorylation of Tyr-394 may be carried out by Lck in vivo, but it is quite possible that other Src family members, or non-Src tyrosine kinases, may also act to phosphorylate Tyr-394 and activate Lck. Consistent with this second possibility is our finding that H2O2 stimulates the activity of an as yet unidentified tyrosine kinase in Lck-deficient JCaM1.6 cells that can phosphorylate Lck on Tyr-394.2 Our results show that it is possible for the Src family member Lck to be phosphorylated at both the conserved tyrosine in the activation loop and the C-terminal tyrosine simultaneously. Because H2O2 activates Lck while inducing an increase of phosphorylation of both Tyr-394 and Tyr-505, these results suggest that Lck phosphorylated on Tyr-394 and Tyr-505 is catalytically activated. Thus, activation of Lck can occur in the absence of Tyr505 dephosphorylation or SH3 domain disassociation from the SH2-kinase linker. It is reasonable to predict that phosphorylation of the absolutely conserved tyrosine in the activation loop of other Src kinases will also activate them in the absence of dephosphorylation of the conserved carboxyl-terminal tyrosine. These experiments would not have been possible without the very generous gift of the α-Tyr(P)-416 serum from Dr. Michael Weber.
Referência(s)