Joseph Hwang

Insulin stimulation of adipocytes resulted in the recruitment of atypical PKC (PKCzeta/lambda) to plasma membrane lipid raft microdomains. This redistribution of PKCzeta/lambda was prevented by Clostridium difficile toxin B and by cholesterol depletion, but was unaffected by inhibition of phosphatidylinositol (PI) 3-kinase activity. Expression of the constitutively active GTP-bound form of TC10 (TC10Q/75L), but not the inactive GDP-bound mutant (TC10/T31N), targeted PKCzeta/lambda to the plasma membrane through an indirect association with the Par6-Par3 protein complex. In parallel, insulin stimulation as well as TC10/Q75L resulted in the activation loop phosphorylation of PKCzeta. Although PI 3-kinase activation also resulted in PKCzeta/lambda phosphorylation, it was not recruited to the plasma membrane. Furthermore, insulin-induced GSK-3beta phosphorylation was mediated by both PI 3-kinase-PKB and the TC10-Par6-atypical PKC signaling pathways. Together, these data demonstrate that PKCzeta/lambda can serve as a convergent downstream target for both the PI 3-kinase and TC10 signaling pathways, but only the TC10 pathway induces a spatially restricted targeting to the plasma membrane.

Previous studies suggest that the stimulation of glucose transport by insulin involves the tyrosine phosphorylation of c-Cbl and the translocation of the c-Cbl/CAP complex to lipid raft subdomains of the plasma membrane. We now demonstrate that Cbl-b also undergoes tyrosine phosphorylation and membrane translocation in response to insulin in 3T3-L1 adipocytes. Ectopic expression of APS facilitated insulin-stimulated phosphorylation of tyrosines 665 and 709 in Cbl-b. The phosphorylation of APS produced by insulin drove the translocation of both c-Cbl and Cbl-b to the plasma membrane. Like c-Cbl, Cbl-b associates constitutively with CAP and interacts with Crk upon insulin stimulation. Cbl proteins formed homo- and heterodimers in vivo, which required the participation of a conserved leucine zipper domain. A Cbl mutant incapable of dimerization failed to interact with APS and to undergo tyrosine phosphorylation in response to insulin, indicating an essential role of Cbl dimerization in these processes. Thus, both c-Cbl and Cbl-b can initiate a phosphatidylinositol 3-kinase/protein kinase B-independent signaling pathway critical to insulin-stimulated GLUT4 translocation. Previous studies suggest that the stimulation of glucose transport by insulin involves the tyrosine phosphorylation of c-Cbl and the translocation of the c-Cbl/CAP complex to lipid raft subdomains of the plasma membrane. We now demonstrate that Cbl-b also undergoes tyrosine phosphorylation and membrane translocation in response to insulin in 3T3-L1 adipocytes. Ectopic expression of APS facilitated insulin-stimulated phosphorylation of tyrosines 665 and 709 in Cbl-b. The phosphorylation of APS produced by insulin drove the translocation of both c-Cbl and Cbl-b to the plasma membrane. Like c-Cbl, Cbl-b associates constitutively with CAP and interacts with Crk upon insulin stimulation. Cbl proteins formed homo- and heterodimers in vivo, which required the participation of a conserved leucine zipper domain. A Cbl mutant incapable of dimerization failed to interact with APS and to undergo tyrosine phosphorylation in response to insulin, indicating an essential role of Cbl dimerization in these processes. Thus, both c-Cbl and Cbl-b can initiate a phosphatidylinositol 3-kinase/protein kinase B-independent signaling pathway critical to insulin-stimulated GLUT4 translocation. Molecular protein adapters have emerged as essential components of signal transduction pathways. The Cbl family of adapters, which comprises c-Cbl, Cbl-b, and Cbl-c/Cbl-3, has been implicated in receptor tyrosine kinase signaling. These related gene products all have a tyrosine kinase-binding (TKB) 1The abbreviations used are: TKB, tyrosine kinase-binding; PI, phosphatidylinositol; ERK, extracellular signal-regulated kinase; SH, Src homology; EGFP, enhanced green fluorescence protein; PKB, protein kinase B; HA, hemagglutinin; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium. domain, a RING finger domain, and a proline-rich region (1Thien C.F. Langdon W.Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 294-305Crossref PubMed Scopus (523) Google Scholar, 2Tsygankov A.Y. Teckchandani A.M. Feshchenko E.A. Swaminathan G. Oncogene. 2001; 20: 6382-6402Crossref PubMed Scopus (106) Google Scholar). The TKB domain, also called the Cbl-N domain, is an integrated phosphopeptide-binding platform composed of a four-helical bundle, a Ca2+-binding EF hand, and an SH2 domain (3Meng W. Sawasdikosol S. Burakoff S.J. Eck M.J. Nature. 1999; 398: 84-90Crossref PubMed Scopus (248) Google Scholar). The Cbl family proteins are tyrosine-phosphorylated in response to a wide variety of stimuli, including epidermal growth factor, platelet-derived growth factor, various antigens, integrins, and cytokines (1Thien C.F. Langdon W.Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 294-305Crossref PubMed Scopus (523) Google Scholar, 2Tsygankov A.Y. Teckchandani A.M. Feshchenko E.A. Swaminathan G. Oncogene. 2001; 20: 6382-6402Crossref PubMed Scopus (106) Google Scholar, 4Liu Y.C. Altman A. Cell. Signal. 1998; 10: 377-385Crossref PubMed Scopus (84) Google Scholar). Moreover, Cbl and Cbl-b interact with critical signaling molecules in both phosphorylation-dependent and -independent fashions. Their binding partners include Src family tyrosine kinases, Zap-70/Syk family tyrosine kinases, the p85 subunit of PI 3-kinase, Vav, Crk, and the Slp-76/BLNK family of linker proteins (5Andoniou C.E. Thien C.B. Langdon W.Y. Oncogene. 1996; 12: 1981-1989PubMed Google Scholar, 6Deckert M. Elly C. Altman A. Liu Y.C. J. Biol. Chem. 1998; 273: 8867-8874Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 7Feshchenko E.A. Langdon W.Y. Tsygankov A.Y. J. Biol. Chem. 1998; 273: 8323-8331Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 8Liu Y.-C. Elly C. Langdon W.Y. Altman A. J. Biol. Chem. 1997; 272: 168-173Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar). Recent studies have also shown that Cbl family proteins negatively regulate protein-tyrosine kinase signaling. This effect may be dependent, at least in part, on the activity of Cbl as an E3 ubiquitinprotein ligase (10Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar, 11Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (916) Google Scholar, 12Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 13Lupher Jr., M.L. Rao N. Lill N.L. Andoniou C.E. Miyake S. Clark E.A. Druker B. Band H. J. Biol. Chem. 1998; 273: 35273-35281Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14Wang Y. Yeung Y.G. Langdon W.Y. Stanley E.R. J. Biol. Chem. 1996; 271: 17-20Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, evidence has also emerged for positive roles of Cbl proteins in cellular signaling processes. For example, c-Cbl facilitates Met-induced activation of c-Jun N-terminal kinase and ERK in HeLa cells via two separate mechanisms. Although binding of tyrosine-phosphorylated c-Cbl to c-Crk is crucial for c-Jun N-terminal kinase activation, the activation of ERK in response to Met is Crk-independent (15Garcia-Guzman M. Larsen E. Vuori K. Oncogene. 2000; 19: 4058-4065Crossref PubMed Scopus (41) Google Scholar). A recent study has also shown that Cbl-b positively regulates the activation of phospholipase C-γ2 by Btk in B cells (16Yasuda T. Tezuka T. Maeda A. Inazu T. Yamanashi Y. Gu H. Kurosaki T. Yamamoto T. J. Exp. Med. 2002; 196: 51-63Crossref PubMed Scopus (44) Google Scholar). Our previous studies demonstrated that c-Cbl plays an important role in insulin action. This function is regulated by two additional adapter proteins, APS (for adapter containing PH and SH2 domains) and CAP (for Cbl-associated protein). Insulin stimulates the tyrosine phosphorylation of c-Cbl in 3T3-L1 adipocytes, inducing its association with Crk (9Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar). The phosphorylation of c-Cbl by the insulin receptor kinase is facilitated by APS. Upon stimulation, the insulin receptor catalyzes the tyrosine phosphorylation of APS on tyrosine 618. Once phosphorylated, APS recruits c-Cbl to the insulin receptor for subsequent phosphorylation of tyrosines 700 and 774 (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). CAP contains three SH3 domains in its C terminus and a region of homology to the gut peptide sorbin (SoHo domain) in its N terminus. CAP constitutively interacts with Cbl via its C-terminal SH3 domain (18Ribon V. Herrera R. Kay B.K. Saltiel A.R. J. Biol. Chem. 1998; 273: 4073-4080Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Upon Cbl phosphorylation, the CAP/Cbl complex migrates to caveolin-enriched lipid rafts, as a result of the interaction of the SoHo domain on CAP with the lipid raft-associated protein flotillin (19Baumann 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 (564) Google Scholar, 20Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (127) Google Scholar). This leads to the recruitment of the Crk/C3G complex to this microdomain of the plasma membrane, where C3G, a guanyl nucleotide exchange factor, activates the small G protein TC10. The activation of TC10 has been shown to occur independently of the PI 3-kinase pathway and, more importantly, to be crucial to insulin-stimulated GLUT4 translocation Baumann C.A. Kanzaki M. Thurmond D.C. Pessin J.E. Saltiel A.R. Nature. 2001; PubMed Scopus Google Scholar). c-Cbl and Cbl-b are in a variety of an conserved N-terminal and both tyrosine phosphorylation at the C-terminal region (1Thien C.F. Langdon W.Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 294-305Crossref PubMed Scopus (523) Google Scholar, 2Tsygankov A.Y. Teckchandani A.M. Feshchenko E.A. Swaminathan G. Oncogene. 2001; 20: 6382-6402Crossref PubMed Scopus (106) Google Scholar, 4Liu Y.C. Altman A. Cell. Signal. 1998; 10: 377-385Crossref PubMed Scopus (84) Google Scholar). 3T3-L1 adipocytes, of the APS mutant insulin-stimulated tyrosine phosphorylation and subsequent Crk binding of c-Cbl, as as the membrane translocation of GLUT4 (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). separate of a CAP mutant in which the SH3 domains the SoHo domain the recruitment of Cbl to lipid and GLUT4 translocation in response to insulin (19Baumann 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 (564) Google Scholar, 20Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (127) Google Scholar). Although these suggest an essential signaling role of c-Cbl in insulin-stimulated glucose to be Cbl-b tyrosine-phosphorylated and as an adapter protein in this and these proteins interact with the insulin We that both c-Cbl and Cbl-b undergo tyrosine phosphorylation and membrane translocation in response to insulin and, that these Cbl c-Cbl Cbl-b and and The The The Crk The Molecular and and by and Stanley c-Cbl by c-Cbl as (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). Cbl by c-Cbl with at the C terminus in in the and of CAP CAP as (19Baumann 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 (564) Google Scholar, 20Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (127) Google Scholar, V. Kay B.K. Saltiel A.R. Mol. Biol. 1998; PubMed Scopus Google Scholar). by CAP in the and of of Cbl and CAP by the to the the The by and cells in essential containing cells in containing 3T3-L1 in with G and to as E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The cells in containing insulin cells for in medium. 3T3-L1 for in glucose with cells and 3T3-L1 by as (19Baumann 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 (564) Google Scholar, J. S. Kanzaki M. Y. Saltiel A.R. Pessin J.E. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). cells in by as J. J. C. S. Biochem. J. 2000; PubMed Scopus Google Scholar). and in with and for at with containing and of The with the for at The with protein for at and with in For Cbl on with of protein for to at proteins by and to proteins with the and by with including plasma membrane, and 3T3-L1 a of and as by and J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). cells on in containing for cells with insulin for various three on with the cells in of and containing and in a The various and the in a protein of by to membrane, and 3T3-L1 on in insulin cells with for with for and with and for and used at in and on with fluorescence for Insulin the of Cbl-b in 3T3-L1 a role of Cbl-b in insulin the tyrosine phosphorylation of this protein in 3T3-L1 adipocytes. cells with insulin, and with The by and tyrosine phosphorylation by with shown in Cbl-b tyrosine-phosphorylated in The of insulin a tyrosine phosphorylation of a Cbl-b. phosphorylation of Cbl-b of insulin stimulation, a by and that of Cbl-b protein at A of c-Cbl tyrosine phosphorylation in response to insulin Thus, insulin stimulation of 3T3-L1 a and tyrosine phosphorylation of both Cbl-b and Cbl-b tyrosine phosphorylation, cells with of kinase of the insulin receptor of cells with the protein kinase to the protein kinase pathway the PI 3-kinase to the pathway the stimulation of Cbl-b tyrosine phosphorylation in response to The of and on the activation of protein kinase and demonstrated by of with adipocytes, c-Cbl phosphorylation to be by the insulin receptor Src family tyrosine as (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). this of cells with the Src kinase the tyrosine phosphorylation of Cbl-b in response to These suggest that the tyrosine phosphorylation of Cbl-b and c-Cbl is by the insulin Cbl-b and c-Cbl to the in to 3T3-L1 adipocytes, c-Cbl is to the plasma membrane in response to insulin (19Baumann 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 (564) Google Scholar). to the Cbl-b protein also its in response to this 3T3-L1 with insulin, and by of protein and plasma membrane by and with and both c-Cbl and Cbl-b at in the plasma membrane However, both proteins in the plasma membrane upon of cells with insulin for a lipid raft its membrane in response to The an of Cbl-b to the plasma membrane to that for of Cbl-b with Crk and tyrosine-phosphorylated c-Cbl to the SH2 domain of Crk (9Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google the insulin-stimulated tyrosine phosphorylation of Cbl-b result in a insulin-stimulated 3T3-L1 with shown by of the insulin the association of Cbl-b with Crk that of Crk and insulin-stimulated We the of Cbl-b to with CAP in 3T3-L1 adipocytes. Previous studies have shown that the SH3 domain of CAP is for its interaction with c-Cbl (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar, V. Herrera R. Kay B.K. Saltiel A.R. J. Biol. Chem. 1998; 273: 4073-4080Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We CAP with Cbl-b in and the of with of the that and the two proteins at to interact with However, of the SH3 domain in CAP the of by of Cbl-b with These that Cbl-b and CAP are of via an interaction the SH3 domain of CAP and the proline-rich of Cbl-b. APS the of Cbl-b by the Insulin is a family protein that is tyrosine-phosphorylated by the insulin receptor (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar, S.A. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). phosphorylation of tyrosine in APS is for its association with c-Cbl and the subsequent tyrosine phosphorylation of c-Cbl by the insulin receptor in 3T3-L1 (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). APS also Cbl-b to the insulin receptor for phosphorylation, Cbl-b with APS in 3T3-L1 of cells with insulin, of by insulin-stimulated tyrosine phosphorylation of with the tyrosine phosphorylation of in response to These that APS is of Cbl-b tyrosine phosphorylation by the insulin Previous studies have shown that tyrosines 700 and 774 are the two in c-Cbl in response to of these two in c-Cbl its interaction with Cbl-b has tyrosines at 709 and 665 in its C-terminal region with that are to and in c-Cbl, C. S. Lipkowitz S. Altman A. Liu Y.C. Oncogene. 1999; PubMed Scopus Google Scholar). the insulin receptor these two in Cbl-b, a Cbl-b with both and to with the Cbl-b the phosphorylation of the mutant in response to insulin Thus, tyrosines 665 and 709 are the by the insulin receptor in the of APS. APS the of Cbl to the in to is constitutively at the plasma membrane in 3T3-L1 (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). the role of APS in the translocation of Cbl proteins to the plasma membrane upon insulin stimulation, in 3T3-L1 in the of by with insulin, and with and that with with previous at the plasma membrane of insulin and both the of of Insulin stimulation this of and However, the of APS to a recruitment of c-Cbl and Cbl-b to the plasma membrane in response to A to in the TKB domain of Cbl-b this membrane translocation by APS These that the interaction tyrosine-phosphorylated APS and the TKB domain of Cbl is crucial for the membrane translocation of Cbl and, that the tyrosine phosphorylation of Cbl its translocation to the plasma membrane in response to and of Cbl-b and has been that c-Cbl its C-terminal leucine zipper domain, and of this domain Cbl tyrosine phosphorylation and its association with the epidermal growth receptor M. A. R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the leucine zipper domain is conserved Cbl-b and c-Cbl, Cbl-b heterodimers with c-Cbl in 3T3-L1 adipocytes. with and the for the of Cbl-b. can be in a protein of which to the of Cbl-b, in cells Cbl-b be in cells Cbl proteins also c-Cbl and Cbl-b of 3T3-L1 with the by Cbl-b with of cells with insulin this c-Cbl to with Cbl-b an the N-terminal region of Cbl-b used for However, c-Cbl in the Cbl-b an the C-terminal region of Cbl-b that of Cbl-b with both these that Cbl-b constitutively with c-Cbl in and, that this dimerization may the C-terminal region of Cbl-b. the role of the leucine zipper domain in the of Cbl proteins, a mutant of c-Cbl in which the leucine zipper domain the c-Cbl cells with in the of shown in all proteins at Although with both Cbl proteins, with c-Cbl Cbl-b. This result that the leucine zipper domain is required for the and to the region containing the and leucine zipper domains is to of c-Cbl, the interaction of which contains the and leucine zipper with c-Cbl and Cbl-b proteins in 3T3-L1 to c-Cbl Cbl-b. Cbl-b the and leucine zipper domains are to of Cbl However, additional may be in the c-Cbl and Cbl-b. for of c-Cbl to APS and of c-Cbl Insulin stimulation of the insulin receptor the association of c-Cbl with APS and the recruitment of c-Cbl to insulin receptor for tyrosine phosphorylation (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). these are by the dimerization of cells with with of cells with insulin, of by with previous insulin the association of with of the leucine zipper domain in c-Cbl its interaction with in response to of that of c-Cbl and in all the role of Cbl dimerization in its insulin-stimulated tyrosine phosphorylation, proteins by and the with shown in insulin stimulation in a in tyrosine phosphorylation of APS However, phosphorylation the Thus, the leucine dimerization of c-Cbl to be required for the insulin-stimulated association of c-Cbl with APS and for the tyrosine phosphorylation of the effect of dimerization on the of c-Cbl 3T3-L1 with cells with by Although c-Cbl the to be to the plasma membrane. Insulin of cells the of protein This result that Cbl dimerization is required for its of a C-terminal of c-Cbl GLUT4 critical role of Cbl in insulin-stimulated GLUT4 translocation has been implicated by in which c-Cbl tyrosine phosphorylation by of For example, in 3T3-L1 of a CAP mutant an APS mutant in Cbl binding insulin-stimulated tyrosine phosphorylation of c-Cbl and membrane translocation of GLUT4 (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar, 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 (564) Google Scholar). additional evidence that Cbl phosphorylation is an essential in insulin-stimulated glucose the effect of expression of a C-terminal mutant of c-Cbl in 3T3-L1 adipocytes. Although the C-terminal tyrosine phosphorylation for Crk binding and for the proline-rich region for CAP a complex with c-Cbl with in 3T3-L1 of that and at by that CAP with an to that with the The result that the of the C-terminal the of c-Cbl to with We expression of insulin-stimulated GLUT4 translocation. 3T3-L1 with with a an enhanced green fluorescence protein of GLUT4 The cells with insulin, and the of by fluorescence The Cbl proteins by Although c-Cbl the to be constitutively at the plasma membrane. insulin the translocation of to the plasma membrane in with c-Cbl Cbl-b have effect on the insulin-stimulated translocation of of in of insulin-stimulated translocation of of these demonstrated that the of the cells green fluorescence in response to insulin by with the of in with cells and with of effect on the membrane of the insulin-stimulated of at the plasma membrane, of activation of the PI 3-kinase phosphorylation-dependent adapter and proteins important roles in tyrosine kinase signaling pathways. Upon insulin stimulation of adipocytes, c-Cbl on tyrosines 700 and its interaction with signaling molecules as Crk (9Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar, J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). phosphorylation of c-Cbl by the insulin receptor is by the adapter protein APS. APS is an insulin receptor and c-Cbl to the insulin receptor upon phosphorylation on tyrosine (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). Moreover, insulin translocation of the c-Cbl/CAP complex to lipid raft subdomains of the plasma membrane, where CAP to the protein flotillin (19Baumann 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 (564) Google Scholar, 20Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (127) Google Scholar). of mutant of CAP both translocation of to lipid rafts, as as the membrane of GLUT4 in response to insulin, indicating a critical role of c-Cbl in insulin-stimulated glucose transport (19Baumann 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 (564) Google Scholar, 20Kimura A. Baumann C.A. Chiang S.H. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9098-9103Crossref PubMed Scopus (127) Google Scholar). Cbl-b is Cbl gene with proline-rich and tyrosine phosphorylation Although is Cbl-b tyrosine phosphorylation, is that Cbl-b is tyrosine-phosphorylated in and cells in response to as and C. S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, K. C. I. T. A. S. A. A. J. I. H. Lipkowitz S. Nature. 2000; PubMed Scopus (564) Google Scholar). We demonstrate that Cbl-b tyrosine-phosphorylated in response to insulin in 3T3-L1 adipocytes. This phosphorylation of Cbl-b is of PI 3-kinase and protein kinase and is also to the of Src kinase indicating that the insulin receptor catalyzes this Moreover, also demonstrate that tyrosine-phosphorylated APS is the adapter that Cbl-b to the insulin a Cbl-b is on tyrosines 665 and 665 and 709 have been as binding C. S. Lipkowitz S. Altman A. Liu Y.C. Oncogene. 1999; PubMed Scopus Google the tyrosine phosphorylation of Cbl-b been to this Although studies that c-Cbl of its by various proteins to is the role of Cbl-b as an adapter that Cbl-b interacts with CAP and Crk in 3T3-L1 adipocytes. Like c-Cbl, Cbl-b to a complex with CAP an SH3 proline-rich The interaction of Cbl-b with Crk is to insulin stimulation and is on the of Cbl-b to undergo tyrosine to its tyrosine phosphorylation, Cbl-b to the plasma membrane in a to that with c-Cbl (19Baumann 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 (564) Google Scholar). Like c-Cbl, Cbl-b to to the plasma membrane upon insulin stimulation its interaction with APS. This on the of APS to the membrane of both c-Cbl and Cbl-b in response to Moreover, of the TKB domain of Cbl proteins this translocation. be that the of Cbl proteins to the plasma membrane is to as a result of the of APS. For the translocation of Cbl proteins in the of the of APS. The leucine zipper domain is an formed by of as leucine and The C-terminal of c-Cbl and Cbl-b a conserved leucine which in c-Cbl to M. A. R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Our demonstrate for the that Cbl-b is of a as as heterodimers with of both to the participation of the leucine zipper domain, of this domain to both homo- and a of in the leucine zipper proteins containing the and leucine zipper domains of c-Cbl and Cbl-b the and leucine zipper domains are for Cbl may be in the of Cbl has in insulin signaling. the of c-Cbl, that the of the leucine zipper and the of Cbl dimerization to insulin-stimulated association and tyrosine phosphorylation of this APS also to have the to undergo Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). Thus, be to dimerization of APS is to Cbl to the insulin Moreover, Cbl dimerization also to an important role in its cellular of the leucine zipper in the association of c-Cbl with the plasma membrane, that dimerization is for the of Cbl the interaction of Cbl with Insulin the these at the of membrane translocation of of the leucine zipper domain effect on the of Cbl to interact with indicating that important for Cbl tyrosine phosphorylation, may be required for its to lipid previous of of APS CAP tyrosine phosphorylation of Cbl and translocation of GLUT4 in response to The demonstrate a critical role of Cbl as an signaling in insulin-stimulated glucose The of a c-Cbl mutant in which a C-terminal containing the tyrosine phosphorylation and the and leucine zipper domains has been insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. the hand, the of c-Cbl Cbl-b of 700 activation by insulin, of GLUT4 translocation. We that the effect of on GLUT4 translocation is the result of the of to interact with and proteins in the of insulin signaling protein is to CAP with an to of CAP by in the translocation of Cbl to the lipid rafts, a shown to be critical for insulin-stimulated GLUT4 translocation.

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. 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 forms of the protein. TC10β only inhibited insulin-stimulated GLUT4 translocation, whereas was effect. Although the for these in biological effect unknown, the to GLUT4 translocation appears to with the disruption in cortical This may also for the that overexpression of the wild type, constitutively active, and dominant-interfering mutants of the TC10α isoforms are Furthermore, these suggest that there are of binding in the the TC10 isoforms are the the form of the protein might the interaction of endogenous TC10 with its targeting the protein to or the of the Indeed, suggest that different for TC10α and β in and any TC10 isoforms to as signaling in the CAP/Cbl and the identification of TC10 proteins in lipid raft microdomains TC10 isoforms the 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). 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. 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 forms of the protein. TC10β only inhibited insulin-stimulated GLUT4 translocation, whereas was effect. Although the for these in biological effect unknown, the to GLUT4 translocation appears to with the disruption in cortical This may also for the that overexpression of the wild type, constitutively active, and dominant-interfering mutants of the TC10α isoforms are Furthermore, these suggest that there are of binding in the the TC10 isoforms are the the form of the protein might the interaction of endogenous TC10 with its targeting the protein to or the of the Indeed, suggest that different for TC10α and β in and any TC10 isoforms to as signaling in the CAP/Cbl and the identification of TC10 proteins in lipid raft microdomains TC10 isoforms the 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 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 forms of the protein. TC10β only inhibited insulin-stimulated GLUT4 translocation, whereas was effect. Although the for these in biological effect unknown, the to GLUT4 translocation appears to with the disruption in cortical This may also for the that overexpression of the wild type, constitutively active, and dominant-interfering mutants of the TC10α isoforms are Furthermore, these suggest that there are of binding in the the TC10 isoforms are the the form of the protein might the interaction of endogenous TC10 with its targeting the protein to or the of the Indeed, suggest that different for TC10α and β in and any TC10 isoforms to as signaling in the CAP/Cbl and the identification of TC10 proteins in lipid raft microdomains TC10 isoforms the of insulin on its target cells.