14-3-3 Protein Binds to Insulin Receptor Substrate-1, One of the Binding Sites of Which Is in the Phosphotyrosine Binding Domain
1997; Elsevier BV; Volume: 272; Issue: 40 Linguagem: Inglês
10.1074/jbc.272.40.25267
ISSN1083-351X
AutoresTakehide Ogihara, Toshiaki Isobe, Tohru Ichimura, Masato Taoka, Makoto Funaki, Hideyuki Sakoda, Yukiko Onishi, Kouichi Inukai, Motonobu Anai, Yasushi Fukushima, Masatoshi Kikuchi, Yoshio Yazaki, Yoshitomo Oka, Tomoichiro Asano,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoInsulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with32P-labeled recombinant IRS-1 and obtained two isoforms (ε and ζ) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1. Insulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with32P-labeled recombinant IRS-1 and obtained two isoforms (ε and ζ) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1. Protein tyrosine kinases play key roles in transmitting extracellular signals that induce specific cellular events such as proliferation, differentiation, gene expression, and metabolism. These signals are propagated by sequential protein-protein interactions and the resulting protein phosphorylation cascade. Thus, to identify the molecule(s) associated with the some key molecules mediating these events constitutes a strategy for elucidating the signal transduction network. The CORT (cloning ofreceptor target) method is a modification of the expression cloning method, using tyrosine-phosphorylated growth factor receptor as a probe, which was originally established by Skolniket al. (1Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (437) Google Scholar) for the purpose of isolating cDNAs coding for proteins bound to the tyrosine-phosphorylated epidermal growth factor receptor.In the case of insulin signaling, the first action exerted by insulin is activation of insulin receptor tyrosine kinase, which leads to the phosphorylation of several cytosolic substrates including insulin receptor substrate (IRS) 1The abbreviations used are; IRS, insulin receptor substrate; GST, glutathione S-transferase; CHO, Chinese hamster ovary; PH, pleckstrin homology; PTB, phosphotyrosine binding; PAGE, polyacrylamide gel electrophoresis; SH2, Src-homology 2.1The abbreviations used are; IRS, insulin receptor substrate; GST, glutathione S-transferase; CHO, Chinese hamster ovary; PH, pleckstrin homology; PTB, phosphotyrosine binding; PAGE, polyacrylamide gel electrophoresis; SH2, Src-homology 2.-1 (2Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1275) Google Scholar) and IRS-2 (3Sun X.J. Wang L.-M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (763) Google Scholar). IRS-1 possesses 21 potential tyrosine phosphorylation sites and functions as a "docking protein," transmitting insulin signals to several proteins containing Src-homology 2 (SH2) domains (4Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar). Thus, similar methods have been employed successfully to clone cDNAs coding for the proteins that bind to phosphorylated IRS-1. To date, three novel regulatory subunits for phosphatidylinositol 3-kinase (p55γ (5Pons S. Asano T. Glasheen E. Miralpeix M. Zhang Y. Fisher T.L. Myers Jr., M.G. Sun X.J. White M.F. Mol. Cell. Biol. 1995; 15: 4453-4465Crossref PubMed Scopus (230) Google Scholar, 6Inukai K. Anai M. Van Breda E. Hosaka T. Katagiri H. Funaki M. Fukushima Y. Ogihara T. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1996; 271: 5317-5320Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), p55α (6Inukai K. Anai M. Van Breda E. Hosaka T. Katagiri H. Funaki M. Fukushima Y. Ogihara T. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1996; 271: 5317-5320Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 7Antonetti D.A. Algenstaedt P. Kahn C.R. Mol. Cell. Biol. 1996; 16: 2185-2203Crossref Scopus (126) Google Scholar), and 50α (8Inukai K. Funaki M. Ogihara T. Katagiri H. Kanda A. Anai M. Fukushima Y. Hosaka T. Suzuki M. Shin B.-C. Takata K. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 7873-7882Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar)) have been isolated by this method and reported. In addition, Fyn tyrosine kinase (9Sun X.J. Pons S. Asano T. Myers Jr., M.G. Glasheen E. White M.F. J. Biol. Chem. 1996; 271: 10583-10587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) was demonstrated to bind phosphorylated IRS-1.In this study, to identify additional IRS-1-binding proteins, we screened a human heart cDNA library and obtained cDNAs coding for the 14-3-3 family proteins, which function as IRS-1-binding proteins. Recently, the roles of 14-3-3 proteins have been clarified with respect to the regulation of various signal transductions and enzyme activities (10Ichimura T. Isobe T. Okumura T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar, 11Isobe T. Ichimura T. Sunaya T. Okuyama T. Takahashi N. Kuwano R. Takahashi Y. J. Mol. Biol. 1991; 217: 125-132Crossref PubMed Scopus (102) Google Scholar, 12Aitken A. Ellis C.A. Sellers L.A. Toker A. Nature. 1990; 344: 594Crossref PubMed Google Scholar, 13Isobe T. Hiyane Y. Ichimura T. Okuyama T. Takahashi N. Nakajo S. Nakaya K. FEBS Lett. 1992; 308: 121-124Crossref PubMed Scopus (79) Google Scholar, 14Fu H. Coburn J. Collier R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2320-2324Crossref PubMed Scopus (215) Google Scholar, 15Ford J.C. Al-Khodairy F. Fotou E. Sheldrick K.S. Griffiths D.J. Carr A.M. Science. 1994; 265: 533-535Crossref PubMed Scopus (298) Google Scholar, 16Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 17Freed E. Symons M. MacDonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 18Pallas D.C. Fu H. Haehnel L.C. Weller W. Collier R.J. Roberts T.M. Science. 1994; 265: 535-537Crossref PubMed Scopus (148) Google Scholar, 19Reuther G.W. Fu H. Cripe L.D. Collier R.J. Pendergast A.M. Science. 1994; 266: 129-133Crossref PubMed Scopus (209) Google Scholar). These proteins are known to bind to the phosphoserine-containing motifs in several proteins (20Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar) but not to the phosphotyrosine-containing motifs in IRS-1.DISCUSSIONRecently, extensive studies have been done to clarify the mechanism of insulin resistance, which is involved in the pathogenesis of non-insulin-dependent diabetes mellitus, as well as the factors determining insulin sensitivity. A number of reports have demonstrated that serine phosphorylation of IRS-1 affects its tyrosine phosphorylation (27Tanti J.-F. Gremeaux T. Van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 32Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 33Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2177) Google Scholar). When the serine phosphorylation of IRS-1 is augmented by tumor necrosis factor-α, which has been suggested to be a mediator of insulin resistance in obesity, insulin-induced tyrosine phosphorylation of IRS-1 is impaired (32Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Tumor necrosis factor-α was also shown to induce serine phosphorylation of IRS-1 and to convert IRS-1 into an inhibitor of the insulin receptor tyrosine kinase activity in vitro (33Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2177) Google Scholar). The 14-3-3 protein association with the serine-phosphorylated IRS-1, and possibly IRS-2, may contribute to the regulation of insulin sensitivity.In this study, we have not determined whether IRS-1 is actually phosphorylated on the serine residues in the 14-3-3 protein binding motifs. However, if it is assumed that phosphorylation of these motifs is required for the observed association, then the amount of 14-3-3 protein associated with IRS-1 would be determined by the activity of an as yet unknown serine kinase(s) that phosphorylates the serine residue in the 14-3-3 binding motif of the target protein, rather by the level of 14-3-3 protein expression, because these proteins are extremely abundant (reportedly approximately 1% of total brain tissue-soluble proteins (34Boston P.F. Jackson P. Kynoch P.A.M. Thompson R.J. J. Neurochem. 1982; 38: 1466-1474Crossref PubMed Scopus (86) Google Scholar), probably more than 0.1% of cytosolic protein in most cells). Thus, to identify the serine kinase involved in the 14-3-3 protein association is essential for clarifying the regulatory mechanism of the 14-3-3 protein association with IRS-1. In the case of tyrosine/tryptophan hydroxylase, it has been demonstrated that calmodulin kinase II phosphorylates the 14-3-3 binding motif of hydroxylase (10Ichimura T. Isobe T. Okumura T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar, 11Isobe T. Ichimura T. Sunaya T. Okuyama T. Takahashi N. Kuwano R. Takahashi Y. J. Mol. Biol. 1991; 217: 125-132Crossref PubMed Scopus (102) Google Scholar). 14-3-3 proteins bind to phosphorylated hydroxylase, thereby activating the enzyme (35Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar). In the case of BAD, heart muscle kinase (a form of protein kinase A) was shown to phosphorylate the serine residue in the 14-3-3 binding motif of BADin vitro, but the kinase acting in vivo remains unknown (20Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar).We investigated whether PTB domains found in other proteins share a 14-3-3 protein binding motif similar to that of IRS-1 and IRS-2. Sequential alignments of PTB domains identified in various proteins are shown in Fig. 6. The α-helices and β-sheet strands based on a structural study (36Eck M.J. Dhe-Paganon S. Trüb T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) and the putative 14-3-3 binding motif (around Ser-270) are boxed. Based on crystallographic study, β5, β6, β7, and α2 of IRS-1 are important for recognizing phosphotyrosine in the insulin receptor NPXY motif (36Eck M.J. Dhe-Paganon S. Trüb T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Because the putative 14-3-3 binding motif is next to the α2-helix, 14-3-3 protein binding to IRS-1 would presumably influence the binding of its PTB domain to the insulin receptor. Another interesting point is that IRS-1 and IRS-2 contain the insertion in the corresponding regions of other proteins and that the putative 14-3-3 binding motif is in this insertion sequence. Thus, the binding of 14-3-3 protein to the PTB domain may be specific to IRS-1 and IRS-2.Further study is necessary to elucidate the physiological role of the 14-3-3 protein association with IRS-1 and IRS-2 in insulin signaling and the regulation of insulin sensitivity, as well as its possible involvement in the insulin resistance observed in non-insulin-dependent diabetes mellitus. Protein tyrosine kinases play key roles in transmitting extracellular signals that induce specific cellular events such as proliferation, differentiation, gene expression, and metabolism. These signals are propagated by sequential protein-protein interactions and the resulting protein phosphorylation cascade. Thus, to identify the molecule(s) associated with the some key molecules mediating these events constitutes a strategy for elucidating the signal transduction network. The CORT (cloning ofreceptor target) method is a modification of the expression cloning method, using tyrosine-phosphorylated growth factor receptor as a probe, which was originally established by Skolniket al. (1Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (437) Google Scholar) for the purpose of isolating cDNAs coding for proteins bound to the tyrosine-phosphorylated epidermal growth factor receptor. In the case of insulin signaling, the first action exerted by insulin is activation of insulin receptor tyrosine kinase, which leads to the phosphorylation of several cytosolic substrates including insulin receptor substrate (IRS) 1The abbreviations used are; IRS, insulin receptor substrate; GST, glutathione S-transferase; CHO, Chinese hamster ovary; PH, pleckstrin homology; PTB, phosphotyrosine binding; PAGE, polyacrylamide gel electrophoresis; SH2, Src-homology 2.1The abbreviations used are; IRS, insulin receptor substrate; GST, glutathione S-transferase; CHO, Chinese hamster ovary; PH, pleckstrin homology; PTB, phosphotyrosine binding; PAGE, polyacrylamide gel electrophoresis; SH2, Src-homology 2.-1 (2Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1275) Google Scholar) and IRS-2 (3Sun X.J. Wang L.-M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (763) Google Scholar). IRS-1 possesses 21 potential tyrosine phosphorylation sites and functions as a "docking protein," transmitting insulin signals to several proteins containing Src-homology 2 (SH2) domains (4Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar). Thus, similar methods have been employed successfully to clone cDNAs coding for the proteins that bind to phosphorylated IRS-1. To date, three novel regulatory subunits for phosphatidylinositol 3-kinase (p55γ (5Pons S. Asano T. Glasheen E. Miralpeix M. Zhang Y. Fisher T.L. Myers Jr., M.G. Sun X.J. White M.F. Mol. Cell. Biol. 1995; 15: 4453-4465Crossref PubMed Scopus (230) Google Scholar, 6Inukai K. Anai M. Van Breda E. Hosaka T. Katagiri H. Funaki M. Fukushima Y. Ogihara T. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1996; 271: 5317-5320Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), p55α (6Inukai K. Anai M. Van Breda E. Hosaka T. Katagiri H. Funaki M. Fukushima Y. Ogihara T. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1996; 271: 5317-5320Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 7Antonetti D.A. Algenstaedt P. Kahn C.R. Mol. Cell. Biol. 1996; 16: 2185-2203Crossref Scopus (126) Google Scholar), and 50α (8Inukai K. Funaki M. Ogihara T. Katagiri H. Kanda A. Anai M. Fukushima Y. Hosaka T. Suzuki M. Shin B.-C. Takata K. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 7873-7882Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar)) have been isolated by this method and reported. In addition, Fyn tyrosine kinase (9Sun X.J. Pons S. Asano T. Myers Jr., M.G. Glasheen E. White M.F. J. Biol. Chem. 1996; 271: 10583-10587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) was demonstrated to bind phosphorylated IRS-1. In this study, to identify additional IRS-1-binding proteins, we screened a human heart cDNA library and obtained cDNAs coding for the 14-3-3 family proteins, which function as IRS-1-binding proteins. Recently, the roles of 14-3-3 proteins have been clarified with respect to the regulation of various signal transductions and enzyme activities (10Ichimura T. Isobe T. Okumura T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar, 11Isobe T. Ichimura T. Sunaya T. Okuyama T. Takahashi N. Kuwano R. Takahashi Y. J. Mol. Biol. 1991; 217: 125-132Crossref PubMed Scopus (102) Google Scholar, 12Aitken A. Ellis C.A. Sellers L.A. Toker A. Nature. 1990; 344: 594Crossref PubMed Google Scholar, 13Isobe T. Hiyane Y. Ichimura T. Okuyama T. Takahashi N. Nakajo S. Nakaya K. FEBS Lett. 1992; 308: 121-124Crossref PubMed Scopus (79) Google Scholar, 14Fu H. Coburn J. Collier R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2320-2324Crossref PubMed Scopus (215) Google Scholar, 15Ford J.C. Al-Khodairy F. Fotou E. Sheldrick K.S. Griffiths D.J. Carr A.M. Science. 1994; 265: 533-535Crossref PubMed Scopus (298) Google Scholar, 16Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 17Freed E. Symons M. MacDonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 18Pallas D.C. Fu H. Haehnel L.C. Weller W. Collier R.J. Roberts T.M. Science. 1994; 265: 535-537Crossref PubMed Scopus (148) Google Scholar, 19Reuther G.W. Fu H. Cripe L.D. Collier R.J. Pendergast A.M. Science. 1994; 266: 129-133Crossref PubMed Scopus (209) Google Scholar). These proteins are known to bind to the phosphoserine-containing motifs in several proteins (20Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar) but not to the phosphotyrosine-containing motifs in IRS-1. DISCUSSIONRecently, extensive studies have been done to clarify the mechanism of insulin resistance, which is involved in the pathogenesis of non-insulin-dependent diabetes mellitus, as well as the factors determining insulin sensitivity. A number of reports have demonstrated that serine phosphorylation of IRS-1 affects its tyrosine phosphorylation (27Tanti J.-F. Gremeaux T. Van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 32Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 33Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2177) Google Scholar). When the serine phosphorylation of IRS-1 is augmented by tumor necrosis factor-α, which has been suggested to be a mediator of insulin resistance in obesity, insulin-induced tyrosine phosphorylation of IRS-1 is impaired (32Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Tumor necrosis factor-α was also shown to induce serine phosphorylation of IRS-1 and to convert IRS-1 into an inhibitor of the insulin receptor tyrosine kinase activity in vitro (33Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2177) Google Scholar). The 14-3-3 protein association with the serine-phosphorylated IRS-1, and possibly IRS-2, may contribute to the regulation of insulin sensitivity.In this study, we have not determined whether IRS-1 is actually phosphorylated on the serine residues in the 14-3-3 protein binding motifs. However, if it is assumed that phosphorylation of these motifs is required for the observed association, then the amount of 14-3-3 protein associated with IRS-1 would be determined by the activity of an as yet unknown serine kinase(s) that phosphorylates the serine residue in the 14-3-3 binding motif of the target protein, rather by the level of 14-3-3 protein expression, because these proteins are extremely abundant (reportedly approximately 1% of total brain tissue-soluble proteins (34Boston P.F. Jackson P. Kynoch P.A.M. Thompson R.J. J. Neurochem. 1982; 38: 1466-1474Crossref PubMed Scopus (86) Google Scholar), probably more than 0.1% of cytosolic protein in most cells). Thus, to identify the serine kinase involved in the 14-3-3 protein association is essential for clarifying the regulatory mechanism of the 14-3-3 protein association with IRS-1. In the case of tyrosine/tryptophan hydroxylase, it has been demonstrated that calmodulin kinase II phosphorylates the 14-3-3 binding motif of hydroxylase (10Ichimura T. Isobe T. Okumura T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar, 11Isobe T. Ichimura T. Sunaya T. Okuyama T. Takahashi N. Kuwano R. Takahashi Y. J. Mol. Biol. 1991; 217: 125-132Crossref PubMed Scopus (102) Google Scholar). 14-3-3 proteins bind to phosphorylated hydroxylase, thereby activating the enzyme (35Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar). In the case of BAD, heart muscle kinase (a form of protein kinase A) was shown to phosphorylate the serine residue in the 14-3-3 binding motif of BADin vitro, but the kinase acting in vivo remains unknown (20Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar).We investigated whether PTB domains found in other proteins share a 14-3-3 protein binding motif similar to that of IRS-1 and IRS-2. Sequential alignments of PTB domains identified in various proteins are shown in Fig. 6. The α-helices and β-sheet strands based on a structural study (36Eck M.J. Dhe-Paganon S. Trüb T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) and the putative 14-3-3 binding motif (around Ser-270) are boxed. Based on crystallographic study, β5, β6, β7, and α2 of IRS-1 are important for recognizing phosphotyrosine in the insulin receptor NPXY motif (36Eck M.J. Dhe-Paganon S. Trüb T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Because the putative 14-3-3 binding motif is next to the α2-helix, 14-3-3 protein binding to IRS-1 would presumably influence the binding of its PTB domain to the insulin receptor. Another interesting point is that IRS-1 and IRS-2 contain the insertion in the corresponding regions of other proteins and that the putative 14-3-3 binding motif is in this insertion sequence. Thus, the binding of 14-3-3 protein to the PTB domain may be specific to IRS-1 and IRS-2.Further study is necessary to elucidate the physiological role of the 14-3-3 protein association with IRS-1 and IRS-2 in insulin signaling and the regulation of insulin sensitivity, as well as its possible involvement in the insulin resistance observed in non-insulin-dependent diabetes mellitus. Recently, extensive studies have been done to clarify the mechanism of insulin resistance, which is involved in the pathogenesis of non-insulin-dependent diabetes mellitus, as well as the factors determining insulin sensitivity. A number of reports have demonstrated that serine phosphorylation of IRS-1 affects its tyrosine phosphorylation (27Tanti J.-F. Gremeaux T. Van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 32Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 33Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2177) Google Scholar). When the serine phosphorylation of IRS-1 is augmented by tumor necrosis factor-α, which has been suggested to be a mediator of insulin resistance in obesity, insulin-induced tyrosine phosphorylation of IRS-1 is impaired (32Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Tumor necrosis factor-α was also shown to induce serine phosphorylation of IRS-1 and to convert IRS-1 into an inhibitor of the insulin receptor tyrosine kinase activity in vitro (33Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2177) Google Scholar). The 14-3-3 protein association with the serine-phosphorylated IRS-1, and possibly IRS-2, may contribute to the regulation of insulin sensitivity. In this study, we have not determined whether IRS-1 is actually phosphorylated on the serine residues in the 14-3-3 protein binding motifs. However, if it is assumed that phosphorylation of these motifs is required for the observed association, then the amount of 14-3-3 protein associated with IRS-1 would be determined by the activity of an as yet unknown serine kinase(s) that phosphorylates the serine residue in the 14-3-3 binding motif of the target protein, rather by the level of 14-3-3 protein expression, because these proteins are extremely abundant (reportedly approximately 1% of total brain tissue-soluble proteins (34Boston P.F. Jackson P. Kynoch P.A.M. Thompson R.J. J. Neurochem. 1982; 38: 1466-1474Crossref PubMed Scopus (86) Google Scholar), probably more than 0.1% of cytosolic protein in most cells). Thus, to identify the serine kinase involved in the 14-3-3 protein association is essential for clarifying the regulatory mechanism of the 14-3-3 protein association with IRS-1. In the case of tyrosine/tryptophan hydroxylase, it has been demonstrated that calmodulin kinase II phosphorylates the 14-3-3 binding motif of hydroxylase (10Ichimura T. Isobe T. Okumura T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar, 11Isobe T. Ichimura T. Sunaya T. Okuyama T. Takahashi N. Kuwano R. Takahashi Y. J. Mol. Biol. 1991; 217: 125-132Crossref PubMed Scopus (102) Google Scholar). 14-3-3 proteins bind to phosphorylated hydroxylase, thereby activating the enzyme (35Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar). In the case of BAD, heart muscle kinase (a form of protein kinase A) was shown to phosphorylate the serine residue in the 14-3-3 binding motif of BADin vitro, but the kinase acting in vivo remains unknown (20Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). We investigated whether PTB domains found in other proteins share a 14-3-3 protein binding motif similar to that of IRS-1 and IRS-2. Sequential alignments of PTB domains identified in various proteins are shown in Fig. 6. The α-helices and β-sheet strands based on a structural study (36Eck M.J. Dhe-Paganon S. Trüb T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) and the putative 14-3-3 binding motif (around Ser-270) are boxed. Based on crystallographic study, β5, β6, β7, and α2 of IRS-1 are important for recognizing phosphotyrosine in the insulin receptor NPXY motif (36Eck M.J. Dhe-Paganon S. Trüb T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Because the putative 14-3-3 binding motif is next to the α2-helix, 14-3-3 protein binding to IRS-1 would presumably influence the binding of its PTB domain to the insulin receptor. Another interesting point is that IRS-1 and IRS-2 contain the insertion in the corresponding regions of other proteins and that the putative 14-3-3 binding motif is in this insertion sequence. Thus, the binding of 14-3-3 protein to the PTB domain may be specific to IRS-1 and IRS-2. Further study is necessary to elucidate the physiological role of the 14-3-3 protein association with IRS-1 and IRS-2 in insulin signaling and the regulation of insulin sensitivity, as well as its possible involvement in the insulin resistance observed in non-insulin-dependent diabetes mellitus. We are grateful to Dr. Izumu Saito (Institute of Medical Science, University of Tokyo) for the generous gift of recombinant Adex1CALacZ and the cassette cosmid used for constructing the recombinant adenoviruses.
Referência(s)