Cloning and Functional Characterization of Related TC10 Isoforms, a Subfamily of Rho Proteins Involved in Insulin-stimulated Glucose Transport
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m109471200
ISSN1083-351X
AutoresShian-Huey Chiang, June Chunqiu Hou, Joseph Hwang, Jeffrey E. Pessin, Alan R. Saltiel,
Tópico(s)Pancreatic function and diabetes
ResumoInsulin stimulates glucose transport via phosphatidylinositol 3-kinase-dependent and -independent pathways. The phosphatidylinositol 3-kinase-independent pathway involves activation of the G protein TC10. A cDNA encoding the mouse homolog of TC10 was cloned, and its gene was mapped at the distal end of chromosome 17. Additionally, a second gene was discovered with ∼70% sequence identity to TC10. We refer to this gene as TC10β. Both isoforms of TC10 were activated by insulin upon transfection in 3T3L1 adipocytes. Cotransfection of cells with TC10α or β plus a dominant negative form of the c-cbl-associated protein CAP prevented the activation by insulin, implicating the CAP/Cbl pathway. Interestingly, both forms of TC10 were also localized in lipid raft fractions in transfected adipocytes. However, although overexpression of TC10α completely blocked glucose transport, TC10β only partially inhibited this process. Furthermore, TC10α overexpression disrupted adipocyte cortical actin, whereas TC10β had little if any effect. Thus, there are two isoforms of this key signaling intermediate, both of which are activated by insulin, but they may play different roles in initiating downstream effectors that influence glucose transport. Insulin stimulates glucose transport via phosphatidylinositol 3-kinase-dependent and -independent pathways. The phosphatidylinositol 3-kinase-independent pathway involves activation of the G protein TC10. A cDNA encoding the mouse homolog of TC10 was cloned, and its gene was mapped at the distal end of chromosome 17. Additionally, a second gene was discovered with ∼70% sequence identity to TC10. We refer to this gene as TC10β. Both isoforms of TC10 were activated by insulin upon transfection in 3T3L1 adipocytes. Cotransfection of cells with TC10α or β plus a dominant negative form of the c-cbl-associated protein CAP prevented the activation by insulin, implicating the CAP/Cbl pathway. Interestingly, both forms of TC10 were also localized in lipid raft fractions in transfected adipocytes. However, although overexpression of TC10α completely blocked glucose transport, TC10β only partially inhibited this process. Furthermore, TC10α overexpression disrupted adipocyte cortical actin, whereas TC10β had little if any effect. Thus, there are two isoforms of this key signaling intermediate, both of which are activated by insulin, but they may play different roles in initiating downstream effectors that influence glucose transport. c-cbl-associated protein human TC10 TC10βLong hemagglutinin glutathione S-transferase rapid amplification of cDNA ends p21-activated kinase 1 p21 binding domain guanosine 5′-3-O-(thio)triphosphate expressed sequence tag dithiothreitol 4-morpholineethanesulfonic acid enhanced green fluorescent protein mouse forms of TC10 Insulin increases glucose uptake by stimulating the translocation of the GLUT4 glucose transporter isoform from intracellular storage sites to the cell surface (1.Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 2.Fletcher L.M. Tavare J.M. Biochem. Soc. Trans. 1999; 27: 677-683Crossref PubMed Scopus (7) Google Scholar, 3.Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). Although it has been well established that the activation of phosphatidylinositol 3-kinase and the generation of phosphatidylinositol-3,4,5-trisphosphate is essential for this biological response, several lines of evidence indicate that it is not sufficient (4.Guilherme A. Czech M.P. J. Biol. Chem. 1998; 273: 33119-33122Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5.Isakoff S.J. Taha C. Rose E. Marcusohn J. Klip A. Skolnik E.Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10247-10251Crossref PubMed Scopus (140) Google Scholar, 6.Wiese R.J. Mastick C.C. Lazar D.F. Saltiel A.R. J. Biol. Chem. 1995; 270: 3442-3446Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 7.Jiang T. Sweeney G. Rudolf M.T. Klip A. Traynor-Kaplan A. Tsien R.Y. J. Biol. Chem. 1998; 273: 11017-11024Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 8.Krook A. Whitehead J.P. Dobson S.P. Griffiths M.R. Ouwens M. Baker C. Hayward A.C. Sen S.K. Maassen J.A. Siddle K. Tavare J.M. O'Rahilly S. J. Biol. Chem. 1997; 272: 30208-30214Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9.Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). Recent data suggest that the second requisite pathway might involve the insulin-stimulated tyrosine phosphorylation of Cbl (10.Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar, 11.Ribon V. Printen J.A. Hoffman N.G. Kay B.K. Saltiel A.R. Mol. Cell. Biol. 1998; 18: 872-879Crossref PubMed Scopus (190) Google Scholar, 12.Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (553) Google Scholar). Cbl forms a complex with the adapter protein CAP,1 a member of the SoHo family of proteins that contains a flotillin-binding SoHo domain in its amino terminus and three adjacent SH3 domains in its carboxyl terminus (11.Ribon V. Printen J.A. Hoffman N.G. Kay B.K. Saltiel A.R. Mol. Cell. Biol. 1998; 18: 872-879Crossref PubMed Scopus (190) Google Scholar, 12.Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (553) Google Scholar, 13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar). Once phosphorylated, the Cbl/CAP complex is recruited to lipid raft plasma membrane subdomains through the interaction of CAP with flotillin. The expression of dominant-interfering CAP mutants that lack either the SH3 or SoHo domains prevented the localization of this complex to plasma membrane microdomains and inhibited the stimulation of glucose uptake and GLUT4 translocation by insulin (13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar).Following insulin-stimulated tyrosine phosphorylation, Cbl recruits the SH2-containing adapter protein CrkII to lipid raft microdomains along with the guanine nucleotide exchange factor C3G (14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar). Upon its translocation, C3G appears to activate the Rho family protein TC10, a member of the family of small GTP-binding proteins expressed in muscle and adipose tissue (15.Drivas G.T. Shih A. Coutavas E. Rush M.G. D'Eustachio P. Mol. Cell. Biol. 1990; 10: 1793-1798Crossref PubMed Scopus (248) Google Scholar). Upon overexpression in murine 3T3L1 adipocytes, insulin activates hTC10 in a CAP-dependent but PI 3-kinase-independent manner (14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar). Although the physiologically relevant effectors that interact with TC10 are unknown, disruption of its activation blocks insulin-stimulated glucose transport and GLUT4 translocation. Moreover, the mistargeting of TC10 to a non-lipid raft domain by production of a TC10/K-Ras chimera or disruption of lipid raft microdomains via expression of a dominant-interfering mutant form of caveolin-3 also completely prevented the activation of TC10 by insulin (16.Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (146) Google Scholar).Although these experiments suggest that TC10 is a critical player in the hormonal regulation of glucose transport, they relied almost exclusively on the overexpression of the human form of TC10 in mouse cells. To study the endogenous forms of TC10 in the highly insulin-responsive mouse 3T3L1 cell line, we cloned the mouse ortholog of TC10. Interestingly, these efforts led to the identification of a closely related gene, referred to as TC10β, and another variant termed TC10βLong (TC10βL). We describe here the characterization of this gene, the regulation of its gene product by insulin, and the evaluation of its role in glucose transport.DISCUSSIONThe stimulation of glucose transport by insulin requires both phosphatidylinositol 3-kinase-dependent and -independent pathways (9.Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar, 19.Baumann C.A. Saltiel A.R. Bioessays. 2001; 23: 215-222Crossref PubMed Scopus (49) Google Scholar). We recently described a novel signaling pathway that is segregated into a lipid raft subdomain of the plasma membrane. The insulin receptor catalyzes the tyrosine phosphorylation of the protooncogene Cbl, which is recruited to the receptor with the adaptor protein CAP (10.Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar, 11.Ribon V. Printen J.A. Hoffman N.G. Kay B.K. Saltiel A.R. Mol. Cell. Biol. 1998; 18: 872-879Crossref PubMed Scopus (190) Google Scholar, 26.Mastick C.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 20706-20714Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Upon Cbl phosphorylation, the CAP/Cbl complex is translocated to lipid rafts via the interaction of the SoHo domain of CAP with the hydrophobic protein flotillin (12.Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (553) Google Scholar, 13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar). Phospho-Cbl can in turn recruit the SH2/SH3 adapter protein CrkII to these microdomains along with the guanyl nucleotide exchange protein C3G. The insulin-stimulated recruitment of C3G to lipid rafts brings the exchange factor into proximity with the Rho family protein TC10, which appears to reside in lipid rafts because of its unique carboxyl-terminal sequences (14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar, 16.Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (146) Google Scholar). TC10 undergoes activation via the C3G-catalyzed exchange of GTP for GDP, and the blockade of this pathway by overexpression of dominant negative forms of CAP prevents the stimulation of glucose transport by insulin (13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar, 14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar).A key determinant in the activation of TC10 by insulin lies in its localization in lipid raft microdomains. This property of the protein appears to be defined by its carboxyl terminal sequences. A TC10/K-Ras chimera was not activated by insulin and did not target to lipid rafts, whereas a TC10/H-ras chimera was insulin sensitive and localized into lipid rafts (16.Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (146) Google Scholar). Interestingly, the second isoform of TC10 described here (TC10β) is also activated by insulin treatment and is also localized in lipid rafts, providing further support for the functional importance of spatial targeting of this protein. Indeed, TC10α and β are similarly activated by insulin in a CAP-dependent manner and localized in identical fractions by sucrose density gradients as well as by immunolocalization by confocal microscopy.Despite similarities in function, the three isoforms of mTC10 differ in their impact on insulin-stimulated GLUT4 translocation. The overexpression of mTC10α profoundly inhibits GLUT4 translocation in response to insulin, as was observed for the human form of this protein. This inhibitory effect was produced with the wild type, constitutively active, and inactive forms of the protein. In contrast, TC10β only slightly inhibited insulin-stimulated GLUT4 translocation, whereas TC10βL was essentially without effect. Although the underlying reasons for these differences in biological effect remain unknown, the ability to inhibit GLUT4 translocation appears to correlate with the disruption in cortical actin structure. This may also account for the surprising observation that overexpression of the wild type, constitutively active, and dominant-interfering mutants of the TC10α isoforms are all inhibitory. Furthermore, these findings suggest that there are important determinants of effector binding in the amino-terminal sequences, where the TC10 isoforms are the most divergent. Alternatively, the active form of the protein might prevent the interaction of endogenous TC10 with its effectors, perhaps targeting the protein to an incorrect location or dramatically changing the kinetics of the interaction. Indeed, preliminary results suggest that candidate effector molecules show different affinities for TC10α and β in pull-down and immunoprecipitation experiments. In any case, TC10 isoforms appear to serve as signaling intermediates in the CAP/Cbl pathway, and the identification of multiple TC10 proteins in lipid raft microdomains will provide important clues toward understanding how TC10 isoforms mediate the effects of insulin on its target cells. Insulin increases glucose uptake by stimulating the translocation of the GLUT4 glucose transporter isoform from intracellular storage sites to the cell surface (1.Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 2.Fletcher L.M. Tavare J.M. Biochem. Soc. Trans. 1999; 27: 677-683Crossref PubMed Scopus (7) Google Scholar, 3.Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). Although it has been well established that the activation of phosphatidylinositol 3-kinase and the generation of phosphatidylinositol-3,4,5-trisphosphate is essential for this biological response, several lines of evidence indicate that it is not sufficient (4.Guilherme A. Czech M.P. J. Biol. Chem. 1998; 273: 33119-33122Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5.Isakoff S.J. Taha C. Rose E. Marcusohn J. Klip A. Skolnik E.Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10247-10251Crossref PubMed Scopus (140) Google Scholar, 6.Wiese R.J. Mastick C.C. Lazar D.F. Saltiel A.R. J. Biol. Chem. 1995; 270: 3442-3446Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 7.Jiang T. Sweeney G. Rudolf M.T. Klip A. Traynor-Kaplan A. Tsien R.Y. J. Biol. Chem. 1998; 273: 11017-11024Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 8.Krook A. Whitehead J.P. Dobson S.P. Griffiths M.R. Ouwens M. Baker C. Hayward A.C. Sen S.K. Maassen J.A. Siddle K. Tavare J.M. O'Rahilly S. J. Biol. Chem. 1997; 272: 30208-30214Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9.Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). Recent data suggest that the second requisite pathway might involve the insulin-stimulated tyrosine phosphorylation of Cbl (10.Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar, 11.Ribon V. Printen J.A. Hoffman N.G. Kay B.K. Saltiel A.R. Mol. Cell. Biol. 1998; 18: 872-879Crossref PubMed Scopus (190) Google Scholar, 12.Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (553) Google Scholar). Cbl forms a complex with the adapter protein CAP,1 a member of the SoHo family of proteins that contains a flotillin-binding SoHo domain in its amino terminus and three adjacent SH3 domains in its carboxyl terminus (11.Ribon V. Printen J.A. Hoffman N.G. Kay B.K. Saltiel A.R. Mol. Cell. Biol. 1998; 18: 872-879Crossref PubMed Scopus (190) Google Scholar, 12.Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (553) Google Scholar, 13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar). Once phosphorylated, the Cbl/CAP complex is recruited to lipid raft plasma membrane subdomains through the interaction of CAP with flotillin. The expression of dominant-interfering CAP mutants that lack either the SH3 or SoHo domains prevented the localization of this complex to plasma membrane microdomains and inhibited the stimulation of glucose uptake and GLUT4 translocation by insulin (13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar). Following insulin-stimulated tyrosine phosphorylation, Cbl recruits the SH2-containing adapter protein CrkII to lipid raft microdomains along with the guanine nucleotide exchange factor C3G (14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar). Upon its translocation, C3G appears to activate the Rho family protein TC10, a member of the family of small GTP-binding proteins expressed in muscle and adipose tissue (15.Drivas G.T. Shih A. Coutavas E. Rush M.G. D'Eustachio P. Mol. Cell. Biol. 1990; 10: 1793-1798Crossref PubMed Scopus (248) Google Scholar). Upon overexpression in murine 3T3L1 adipocytes, insulin activates hTC10 in a CAP-dependent but PI 3-kinase-independent manner (14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar). Although the physiologically relevant effectors that interact with TC10 are unknown, disruption of its activation blocks insulin-stimulated glucose transport and GLUT4 translocation. Moreover, the mistargeting of TC10 to a non-lipid raft domain by production of a TC10/K-Ras chimera or disruption of lipid raft microdomains via expression of a dominant-interfering mutant form of caveolin-3 also completely prevented the activation of TC10 by insulin (16.Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (146) Google Scholar). Although these experiments suggest that TC10 is a critical player in the hormonal regulation of glucose transport, they relied almost exclusively on the overexpression of the human form of TC10 in mouse cells. To study the endogenous forms of TC10 in the highly insulin-responsive mouse 3T3L1 cell line, we cloned the mouse ortholog of TC10. Interestingly, these efforts led to the identification of a closely related gene, referred to as TC10β, and another variant termed TC10βLong (TC10βL). We describe here the characterization of this gene, the regulation of its gene product by insulin, and the evaluation of its role in glucose transport. DISCUSSIONThe stimulation of glucose transport by insulin requires both phosphatidylinositol 3-kinase-dependent and -independent pathways (9.Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar, 19.Baumann C.A. Saltiel A.R. Bioessays. 2001; 23: 215-222Crossref PubMed Scopus (49) Google Scholar). We recently described a novel signaling pathway that is segregated into a lipid raft subdomain of the plasma membrane. The insulin receptor catalyzes the tyrosine phosphorylation of the protooncogene Cbl, which is recruited to the receptor with the adaptor protein CAP (10.Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar, 11.Ribon V. Printen J.A. Hoffman N.G. Kay B.K. Saltiel A.R. Mol. Cell. Biol. 1998; 18: 872-879Crossref PubMed Scopus (190) Google Scholar, 26.Mastick C.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 20706-20714Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Upon Cbl phosphorylation, the CAP/Cbl complex is translocated to lipid rafts via the interaction of the SoHo domain of CAP with the hydrophobic protein flotillin (12.Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (553) Google Scholar, 13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar). Phospho-Cbl can in turn recruit the SH2/SH3 adapter protein CrkII to these microdomains along with the guanyl nucleotide exchange protein C3G. The insulin-stimulated recruitment of C3G to lipid rafts brings the exchange factor into proximity with the Rho family protein TC10, which appears to reside in lipid rafts because of its unique carboxyl-terminal sequences (14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar, 16.Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (146) Google Scholar). TC10 undergoes activation via the C3G-catalyzed exchange of GTP for GDP, and the blockade of this pathway by overexpression of dominant negative forms of CAP prevents the stimulation of glucose transport by insulin (13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar, 14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar).A key determinant in the activation of TC10 by insulin lies in its localization in lipid raft microdomains. This property of the protein appears to be defined by its carboxyl terminal sequences. A TC10/K-Ras chimera was not activated by insulin and did not target to lipid rafts, whereas a TC10/H-ras chimera was insulin sensitive and localized into lipid rafts (16.Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (146) Google Scholar). Interestingly, the second isoform of TC10 described here (TC10β) is also activated by insulin treatment and is also localized in lipid rafts, providing further support for the functional importance of spatial targeting of this protein. Indeed, TC10α and β are similarly activated by insulin in a CAP-dependent manner and localized in identical fractions by sucrose density gradients as well as by immunolocalization by confocal microscopy.Despite similarities in function, the three isoforms of mTC10 differ in their impact on insulin-stimulated GLUT4 translocation. The overexpression of mTC10α profoundly inhibits GLUT4 translocation in response to insulin, as was observed for the human form of this protein. This inhibitory effect was produced with the wild type, constitutively active, and inactive forms of the protein. In contrast, TC10β only slightly inhibited insulin-stimulated GLUT4 translocation, whereas TC10βL was essentially without effect. Although the underlying reasons for these differences in biological effect remain unknown, the ability to inhibit GLUT4 translocation appears to correlate with the disruption in cortical actin structure. This may also account for the surprising observation that overexpression of the wild type, constitutively active, and dominant-interfering mutants of the TC10α isoforms are all inhibitory. Furthermore, these findings suggest that there are important determinants of effector binding in the amino-terminal sequences, where the TC10 isoforms are the most divergent. Alternatively, the active form of the protein might prevent the interaction of endogenous TC10 with its effectors, perhaps targeting the protein to an incorrect location or dramatically changing the kinetics of the interaction. Indeed, preliminary results suggest that candidate effector molecules show different affinities for TC10α and β in pull-down and immunoprecipitation experiments. In any case, TC10 isoforms appear to serve as signaling intermediates in the CAP/Cbl pathway, and the identification of multiple TC10 proteins in lipid raft microdomains will provide important clues toward understanding how TC10 isoforms mediate the effects of insulin on its target cells. The stimulation of glucose transport by insulin requires both phosphatidylinositol 3-kinase-dependent and -independent pathways (9.Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar, 19.Baumann C.A. Saltiel A.R. Bioessays. 2001; 23: 215-222Crossref PubMed Scopus (49) Google Scholar). We recently described a novel signaling pathway that is segregated into a lipid raft subdomain of the plasma membrane. The insulin receptor catalyzes the tyrosine phosphorylation of the protooncogene Cbl, which is recruited to the receptor with the adaptor protein CAP (10.Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar, 11.Ribon V. Printen J.A. Hoffman N.G. Kay B.K. Saltiel A.R. Mol. Cell. Biol. 1998; 18: 872-879Crossref PubMed Scopus (190) Google Scholar, 26.Mastick C.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 20706-20714Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Upon Cbl phosphorylation, the CAP/Cbl complex is translocated to lipid rafts via the interaction of the SoHo domain of CAP with the hydrophobic protein flotillin (12.Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (553) Google Scholar, 13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar). Phospho-Cbl can in turn recruit the SH2/SH3 adapter protein CrkII to these microdomains along with the guanyl nucleotide exchange protein C3G. The insulin-stimulated recruitment of C3G to lipid rafts brings the exchange factor into proximity with the Rho family protein TC10, which appears to reside in lipid rafts because of its unique carboxyl-terminal sequences (14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar, 16.Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (146) Google Scholar). TC10 undergoes activation via the C3G-catalyzed exchange of GTP for GDP, and the blockade of this pathway by overexpression of dominant negative forms of CAP prevents the stimulation of glucose transport by insulin (13.Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (126) Google Scholar, 14.Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (473) Google Scholar). A key determinant in the activation of TC10 by insulin lies in its localization in lipid raft microdomains. This property of the protein appears to be defined by its carboxyl terminal sequences. A TC10/K-Ras chimera was not activated by insulin and did not target to lipid rafts, whereas a TC10/H-ras chimera was insulin sensitive and localized into lipid rafts (16.Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (146) Google Scholar). Interestingly, the second isoform of TC10 described here (TC10β) is also activated by insulin treatment and is also localized in lipid rafts, providing further support for the functional importance of spatial targeting of this protein. Indeed, TC10α and β are similarly activated by insulin in a CAP-dependent manner and localized in identical fractions by sucrose density gradients as well as by immunolocalization by confocal microscopy. Despite similarities in function, the three isoforms of mTC10 differ in their impact on insulin-stimulated GLUT4 translocation. The overexpression of mTC10α profoundly inhibits GLUT4 translocation in response to insulin, as was observed for the human form of this protein. This inhibitory effect was produced with the wild type, constitutively active, and inactive forms of the protein. In contrast, TC10β only slightly inhibited insulin-stimulated GLUT4 translocation, whereas TC10βL was essentially without effect. Although the underlying reasons for these differences in biological effect remain unknown, the ability to inhibit GLUT4 translocation appears to correlate with the disruption in cortical actin structure. This may also account for the surprising observation that overexpression of the wild type, constitutively active, and dominant-interfering mutants of the TC10α isoforms are all inhibitory. Furthermore, these findings suggest that there are important determinants of effector binding in the amino-terminal sequences, where the TC10 isoforms are the most divergent. Alternatively, the active form of the protein might prevent the interaction of endogenous TC10 with its effectors, perhaps targeting the protein to an incorrect location or dramatically changing the kinetics of the interaction. Indeed, preliminary results suggest that candidate effector molecules show different affinities for TC10α and β in pull-down and immunoprecipitation experiments. In any case, TC10 isoforms appear to serve as signaling intermediates in the CAP/Cbl pathway, and the identification of multiple TC10 proteins in lipid raft microdomains will provide important clues toward understanding how TC10 isoforms mediate the effects of insulin on its target cells.
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