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Transmembrane Phosphoprotein Cbp Positively Regulates the Activity of the Carboxyl-terminal Src Kinase, Csk

2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês

10.1074/jbc.c000326200

1083-351X

Satoru Takeuchi, Yoshiharu Takayama, Akira Ogawa, Keiko Tamura, Masato Okada,

Erythrocyte Function and Pathophysiology

Csk (carboxyl-terminalSrc kinase) is a cytoplasmic tyrosine kinase that phosphorylates a critical tyrosine residue in each of the Src family kinases (SFKs) to inhibit their activities. Recently, we identified a transmembrane protein, Cbp (Csk-binding protein), that, when phosphorylated, can recruit Csk to the membrane where the SFKs are located. The Cbp-mediated relocation of Csk to the membrane may play a role in turning off the signaling events initiated by SFKs. To further characterize the Csk-Cbp interaction, we have generated a reconstituted system using soluble, highly purified proteins. Csk and phosphorylated Cbp were co-purified as a large protein complex consisting of at least four Csk·Cbp units. The addition of the phosphorylated, but not nonphosphorylated, Cbp to an in vitro assay stimulated Csk activity toward Src. Csk was also activated by a phosphopeptide containing the tyrosine in Cbp that binds to Csk (Tyr-314). Kinetic analysis revealed that Cbp or the phosphopeptide induced up to a 6-fold reduction in the Km for Src, indicating that the Csk·Cbp complex has a greater affinity for Src than free Csk. These findings suggest that Cbp is involved in the regulation of SFKs not only by relocating Csk to the membrane but also by directly activating Csk. Csk (carboxyl-terminalSrc kinase) is a cytoplasmic tyrosine kinase that phosphorylates a critical tyrosine residue in each of the Src family kinases (SFKs) to inhibit their activities. Recently, we identified a transmembrane protein, Cbp (Csk-binding protein), that, when phosphorylated, can recruit Csk to the membrane where the SFKs are located. The Cbp-mediated relocation of Csk to the membrane may play a role in turning off the signaling events initiated by SFKs. To further characterize the Csk-Cbp interaction, we have generated a reconstituted system using soluble, highly purified proteins. Csk and phosphorylated Cbp were co-purified as a large protein complex consisting of at least four Csk·Cbp units. The addition of the phosphorylated, but not nonphosphorylated, Cbp to an in vitro assay stimulated Csk activity toward Src. Csk was also activated by a phosphopeptide containing the tyrosine in Cbp that binds to Csk (Tyr-314). Kinetic analysis revealed that Cbp or the phosphopeptide induced up to a 6-fold reduction in the Km for Src, indicating that the Csk·Cbp complex has a greater affinity for Src than free Csk. These findings suggest that Cbp is involved in the regulation of SFKs not only by relocating Csk to the membrane but also by directly activating Csk. Src family kinase protein tyrosine kinase polyacrylamide gel electrophoresis glutathione S-transferase Src homology 2/3 carboxyl-terminalSrc kinase Csk-binding protein detergent-insoluble membrane phenylmethylsulfonyl fluoride phosphotyrosine The Src family kinases (SFKs)1 are nonreceptor protein tyrosine kinases (PTKs) that are associated with the inner surface of plasma membrane through their fatty-acylated amino termini (1Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1079) Google Scholar). SFKs are known to act as molecular switches that regulate a variety of cellular events, including cell growth and division, cell attachment and movement, differentiation, survival, or death (2Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2145) Google Scholar). SFKs are ordinarily present in an inactive state in which the phosphorylated carboxyl-terminal regulatory tyrosine binds to its own SH2 domain (3Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar). In response to an external stimulus, an SFK is activated through dephosphorylation of the carboxyl-terminal tyrosine or through binding to another protein that displaces the intramolecular interaction. The phosphorylation of the regulatory tyrosine of SFK is known to be catalyzed by another PTK, Csk (4Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (509) Google Scholar, 5Nada S. Yagi T. Takeda H. Tokunaga T. Nakagawa H. Ikawa Y. Okada M. Aizawa S. Cell. 1993; 73: 1125-1135Abstract Full Text PDF PubMed Scopus (360) Google Scholar). In contrast, the phosphatases that activate SFKs have not yet been positively identified, although some candidate molecules have been proposed (6Ponniah S. Wang D.Z. Lim K.L. Pallen C.J. Curr. Biol. 1999; 9: 535-538Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Thomas M.J. Brown E.J. Immunol. Today. 1999; 20: 406-411Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). To understand the regulation of SFKs, it is essential to clarify the regulation mechanism controlling the phosphorylation and dephosphorylation of the critical carboxyl-terminal tyrosine.Csk is a cytoplasmic PTK consisting of an SH3, an SH2, and a kinase domain. Because it lacks an amino-terminal acylation signal and a carboxyl-terminal tyrosine, the regulatory mechanisms of Csk itself have remained unknown. A line of evidence has suggested that the SH2 and/or SH3 domain of Csk is essential for SFK regulation (8Sabe H. Hata A. Okada M. Nakagawa H. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3984-3988Crossref PubMed Scopus (212) Google Scholar, 9Cloutier J.F. Chow L.M. Veillette A. Mol. Cell. Biol. 1995; 15: 5937-5944Crossref PubMed Scopus (56) Google Scholar). The relocation of Csk to the membrane, specifically to regions where SFKs are active, was also observed (10Howell B.W. Cooper J.A. Mol. Cell. Biol. 1994; 14: 5402-5411Crossref PubMed Scopus (123) Google Scholar). In addition, a membrane-targeted form of Csk, containing the myristoylation signal of Src, more actively suppressed SFK functions (11Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (236) Google Scholar). These facts suggested the possible existence of a membrane factor that can recruit Csk to the membrane where SFKs are active. The importance of the SH2 domain of Csk further suggested that such a membrane factor might be a tyrosine-phosphorylated protein.To test the hypothesis presented above, we searched for phosphoproteins that can bind tightly to the SH2 domain of Csk and identified a transmembrane phosphoprotein, Cbp (Csk-bindingprotein) (12Kawabuchi M. Satomi Y. Takao T. Shimonishi Y. Nada S. Nagai K. Tarakhovsky A. Okada M. Nature. 2000; 404: 999-1003Crossref PubMed Scopus (458) Google Scholar). Cbp is involved in the membrane localization of Csk as well as in the Csk-mediated inhibition of Src. When phosphorylated on Tyr-314, Cbp can bind to Csk. Within the plasma membrane, Cbp is exclusively localized in the GM1 ganglioside-enriched detergent-insoluble membrane (DIM) domain, which is thought to play an important role in receptor-mediated signaling and where the majority of SFKs are localized (13Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8018) Google Scholar, 14Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2542) Google Scholar, 15Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1715) Google Scholar, 16Xavier R. Brennan T. Li Q. McCormack C. Seed B. Immunity. 1998; 8 (1998): 723-732Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar). These findings suggested that Cbp is a novel component of the regulatory mechanism controlling the activity of SFKs. To further evaluate the role of Cbp in the Csk-mediated regulation of SFKs, we examined the effect of Cbp binding on the kinase activity of Csk. The binding of phosphorylated Cbp or a phosphopeptide containing Tyr-314 could substantially elevate the affinity of Csk for Src. The Src family kinases (SFKs)1 are nonreceptor protein tyrosine kinases (PTKs) that are associated with the inner surface of plasma membrane through their fatty-acylated amino termini (1Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1079) Google Scholar). SFKs are known to act as molecular switches that regulate a variety of cellular events, including cell growth and division, cell attachment and movement, differentiation, survival, or death (2Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2145) Google Scholar). SFKs are ordinarily present in an inactive state in which the phosphorylated carboxyl-terminal regulatory tyrosine binds to its own SH2 domain (3Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar). In response to an external stimulus, an SFK is activated through dephosphorylation of the carboxyl-terminal tyrosine or through binding to another protein that displaces the intramolecular interaction. The phosphorylation of the regulatory tyrosine of SFK is known to be catalyzed by another PTK, Csk (4Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (509) Google Scholar, 5Nada S. Yagi T. Takeda H. Tokunaga T. Nakagawa H. Ikawa Y. Okada M. Aizawa S. Cell. 1993; 73: 1125-1135Abstract Full Text PDF PubMed Scopus (360) Google Scholar). In contrast, the phosphatases that activate SFKs have not yet been positively identified, although some candidate molecules have been proposed (6Ponniah S. Wang D.Z. Lim K.L. Pallen C.J. Curr. Biol. 1999; 9: 535-538Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Thomas M.J. Brown E.J. Immunol. Today. 1999; 20: 406-411Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). To understand the regulation of SFKs, it is essential to clarify the regulation mechanism controlling the phosphorylation and dephosphorylation of the critical carboxyl-terminal tyrosine. Csk is a cytoplasmic PTK consisting of an SH3, an SH2, and a kinase domain. Because it lacks an amino-terminal acylation signal and a carboxyl-terminal tyrosine, the regulatory mechanisms of Csk itself have remained unknown. A line of evidence has suggested that the SH2 and/or SH3 domain of Csk is essential for SFK regulation (8Sabe H. Hata A. Okada M. Nakagawa H. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3984-3988Crossref PubMed Scopus (212) Google Scholar, 9Cloutier J.F. Chow L.M. Veillette A. Mol. Cell. Biol. 1995; 15: 5937-5944Crossref PubMed Scopus (56) Google Scholar). The relocation of Csk to the membrane, specifically to regions where SFKs are active, was also observed (10Howell B.W. Cooper J.A. Mol. Cell. Biol. 1994; 14: 5402-5411Crossref PubMed Scopus (123) Google Scholar). In addition, a membrane-targeted form of Csk, containing the myristoylation signal of Src, more actively suppressed SFK functions (11Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (236) Google Scholar). These facts suggested the possible existence of a membrane factor that can recruit Csk to the membrane where SFKs are active. The importance of the SH2 domain of Csk further suggested that such a membrane factor might be a tyrosine-phosphorylated protein. To test the hypothesis presented above, we searched for phosphoproteins that can bind tightly to the SH2 domain of Csk and identified a transmembrane phosphoprotein, Cbp (Csk-bindingprotein) (12Kawabuchi M. Satomi Y. Takao T. Shimonishi Y. Nada S. Nagai K. Tarakhovsky A. Okada M. Nature. 2000; 404: 999-1003Crossref PubMed Scopus (458) Google Scholar). Cbp is involved in the membrane localization of Csk as well as in the Csk-mediated inhibition of Src. When phosphorylated on Tyr-314, Cbp can bind to Csk. Within the plasma membrane, Cbp is exclusively localized in the GM1 ganglioside-enriched detergent-insoluble membrane (DIM) domain, which is thought to play an important role in receptor-mediated signaling and where the majority of SFKs are localized (13Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8018) Google Scholar, 14Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2542) Google Scholar, 15Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1715) Google Scholar, 16Xavier R. Brennan T. Li Q. McCormack C. Seed B. Immunity. 1998; 8 (1998): 723-732Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar). These findings suggested that Cbp is a novel component of the regulatory mechanism controlling the activity of SFKs. To further evaluate the role of Cbp in the Csk-mediated regulation of SFKs, we examined the effect of Cbp binding on the kinase activity of Csk. The binding of phosphorylated Cbp or a phosphopeptide containing Tyr-314 could substantially elevate the affinity of Csk for Src. We thank T. Hirose for production of recombinant Csk in Sf9 cells, S. Aimoto for peptide synthesis, and J. A. Cooper for critical comments.