Stephanie M. DeYoung

Although recent studies show that adipose tissue macrophages (ATMs) participate in the inflammatory changes in obesity and contribute to insulin resistance, the properties of these cells are not well understood. We hypothesized that ATMs recruited to adipose tissue during a high-fat diet have unique inflammatory properties compared with resident tissue ATMs. Using a dye (PKH26) to pulse label ATMs in vivo, we purified macrophages recruited to white adipose tissue during a high-fat diet. Comparison of gene expression in recruited and resident ATMs using real-time RT-PCR and cDNA microarrays showed that recruited ATMs overexpress genes important in macrophage migration and phagocytosis, including interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and C-C chemokine receptor 2 (CCR2). Many of these genes were not induced in ATMs from high-fat diet-fed CCR2 knockout mice, supporting the importance of CCR2 in regulating recruitment of inflammatory ATMs during obesity. Additionally, expression of Apoe was decreased, whereas genes important in lipid metabolism, such as Pparg, Adfp, Srepf1, and Apob48r, were increased in the recruited macrophages. In agreement with this, ATMs from obese mice had increased lipid content compared with those from lean mice. These studies demonstrate that recruited ATMs in obese animals represent a subclass of macrophages with unique properties.

Obesity leads to a proinflammatory state with immune responses that include infiltration of adipose tissue with macrophages. These macrophages are believed to alter insulin sensitivity in adipocytes, but the mechanisms that underlie this effect have not been characterized. We have explored the interaction between macrophages and adipocytes in the context of both indirect and direct coculture. Macrophage-secreted factors blocked insulin action in adipocytes via downregulation of GLUT4 and IRS-1, leading to a decrease in Akt phosphorylation and impaired insulin-stimulated GLUT4 translocation to the plasma membrane. GLUT1 was upregulated with a concomitant increase in basal glucose uptake. These changes recapitulate those seen in adipose tissue from insulin-resistant humans and animal models. TNF-alpha-neutralizing antibodies partially reversed the insulin resistance produced by macrophage-conditioned media. Peritoneal macrophages and macrophage-enriched stromal vascular cells from adipose tissue also attenuated responsiveness to insulin in a manner correlating with inflammatory cytokine secretion. Adipose tissue macrophages from obese mice have an F4/80(+)CD11b(+)CD68(+)CD14(-) phenotype and form long cellular extensions in culture. Peritoneal macrophages take on similar characteristics in direct coculture with adipocytes and induce proinflammatory cytokines, suggesting that macrophage activation state is influenced by contact with adipocytes. Thus both indirect/secreted and direct/cell contact-mediated factors derived from macrophages influence insulin sensitivity in adipocytes.

Flotillin-1 is a lipid raft-associated protein that has been implicated in various cellular processes. We examined the subcellular distribution of flotillin-1 in different cell types and found that localization is cell type-specific. Flotillin-1 relocates from a cytoplasmic compartment to the plasma membrane upon the differentiation of 3T3-L1 adipocytes. To delineate the structural determinants necessary for its localization, we generated a series of truncation mutants of flotillin-1. Wild type flotillin-1 has two putative hydrophobic domains and is localized to lipid raft microdomains at the plasma membrane. Flotillin-1 fragments lacking the N-terminal hydrophobic stretch are excluded from the lipid raft compartments but remain at the plasma membrane. On the other hand, mutants with the second hydrophobic region deleted fail to traffic to the plasma membrane but are instead found in intracellular granule-like structures. Flotillin-1 specifically interacts with the adaptor protein CAP, the Src family kinase Fyn, and cortical F-actin in lipid raft microdomains in adipocytes. Furthermore, CAP and Fyn associate with different regions in the N-terminal sequences of flotillin-1. These results furthered our understanding for how flotillin-1 can function as a molecular link between lipid rafts of the plasma membrane and a multimeric signaling complex at the actin cytoskeleton. Flotillin-1 is a lipid raft-associated protein that has been implicated in various cellular processes. We examined the subcellular distribution of flotillin-1 in different cell types and found that localization is cell type-specific. Flotillin-1 relocates from a cytoplasmic compartment to the plasma membrane upon the differentiation of 3T3-L1 adipocytes. To delineate the structural determinants necessary for its localization, we generated a series of truncation mutants of flotillin-1. Wild type flotillin-1 has two putative hydrophobic domains and is localized to lipid raft microdomains at the plasma membrane. Flotillin-1 fragments lacking the N-terminal hydrophobic stretch are excluded from the lipid raft compartments but remain at the plasma membrane. On the other hand, mutants with the second hydrophobic region deleted fail to traffic to the plasma membrane but are instead found in intracellular granule-like structures. Flotillin-1 specifically interacts with the adaptor protein CAP, the Src family kinase Fyn, and cortical F-actin in lipid raft microdomains in adipocytes. Furthermore, CAP and Fyn associate with different regions in the N-terminal sequences of flotillin-1. These results furthered our understanding for how flotillin-1 can function as a molecular link between lipid rafts of the plasma membrane and a multimeric signaling complex at the actin cytoskeleton. The plasma membrane of most cell types contains specialized subdomains that are highly enriched in cholesterol and sphingolipids, referred to as lipid raft microdomains (1Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 2Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8048) Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5133) Google Scholar, 4Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1719) Google Scholar). Lipid rafts are highly organized, dynamic structures connected to the cytoskeleton and are enriched in growth factor receptors, integrins, Src family kinases, glycosylphosphatidylinositol-linked proteins, and adaptor proteins (5Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar, 6Harris T.J. Siu C.H. BioEssays. 2002; 24: 996-1003Crossref PubMed Scopus (84) Google Scholar, 7Pike L.J. Biochem. J. 2004; 378: 281-292Crossref PubMed Scopus (612) Google Scholar). The selective enrichment of key signaling molecules in these regions suggests that they could function as organization centers for signaling via the formation of multicomponent complexes. Lipid raft domains are insoluble in nonionic detergents (Triton X-100) (5Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar). Depending on the tissue and cell type, protein complexes found in these fractions often contain the proteins caveolin and/or flotillin (8Bickel P.E. Scherer P.E. Schnitzer J.E. Oh P. Lisanti M.P. Lodish H.F. J. Biol. Chem. 1997; 272: 13793-13802Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 9Lang D.M. Lommel S. Jung M. Ankerhold R. Petrausch B. Laessing U. Wiechers M.F. Plattner H. Stuermer C.A. J. Neurobiol. 1998; 37: 502-523Crossref PubMed Scopus (202) Google Scholar, 10Stuermer C.A. Lang D.M. Kirsch F. Wiechers M. Deininger S-O. Plattner H. Mol. Biol. Cell. 2001; 12: 3031-3045Crossref PubMed Scopus (184) Google Scholar, 11Solomon S. Masilamani M. Rajendran L. Bastmeyer M. Stuermer C.A. Illges H. Immunobiology. 2002; 205: 108-119Crossref PubMed Scopus (48) Google Scholar). The insertion of caveolin-1 into lipid rafts results in the formation of caveolae, flask-shaped invaginations that are abundant in glial, epithelial, endothelial, muscle, and adipose cells (3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5133) Google Scholar). The flotillin/reggie proteins are ubiquitously expressed (8Bickel P.E. Scherer P.E. Schnitzer J.E. Oh P. Lisanti M.P. Lodish H.F. J. Biol. Chem. 1997; 272: 13793-13802Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 12Schulte T. Paschke K.A. Laessing U. Lottspeich F. Stuermer C.A. Development (Camb.). 1997; 124: 577-587Crossref PubMed Google Scholar). Reggie-1 and -2 were originally identified in developing neurons of goldfish optic nerves (12Schulte T. Paschke K.A. Laessing U. Lottspeich F. Stuermer C.A. Development (Camb.). 1997; 124: 577-587Crossref PubMed Google Scholar). The same proteins were subsequently identified from endothelial cells in low density detergent-insoluble complexes that also contained caveolin, where they were named flotillin-2 and -1 (8Bickel P.E. Scherer P.E. Schnitzer J.E. Oh P. Lisanti M.P. Lodish H.F. J. Biol. Chem. 1997; 272: 13793-13802Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). Both isoforms were shown to interact and co-localize with the Src family kinase Fyn in lipid rafts derived from neurons and astrocytes, although in Jurkat lymphoma cells, flotillin proteins were found to concentrate in endolysosomal compartments (10Stuermer C.A. Lang D.M. Kirsch F. Wiechers M. Deininger S-O. Plattner H. Mol. Biol. Cell. 2001; 12: 3031-3045Crossref PubMed Scopus (184) Google Scholar). As well as its postulated role in axon regeneration in retinal ganglion cells (12Schulte T. Paschke K.A. Laessing U. Lottspeich F. Stuermer C.A. Development (Camb.). 1997; 124: 577-587Crossref PubMed Google Scholar), flotillin-1 has also been implicated in phagosome maturation (13Dermine J.F. Duclos S. Garin J. St-Louis F. Rea S. Parton R.G. Desjardins M. J. Biol. Chem. 2001; 276: 18507-18512Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) in macrophages and in NF-κB in Jurkat cells D. J. T. P.E. J. 2004; PubMed Scopus Google Scholar). to a family of proteins that plasma CAP, density low density plasma CAP, density low density M. Biochem. 24: Full Text Full Text PDF PubMed Scopus Google Scholar). The function of domains most family are membrane proteins with a region the flotillin-1 has two hydrophobic sequences that are in the (8Bickel P.E. Scherer P.E. Schnitzer J.E. Oh P. Lisanti M.P. Lodish H.F. J. Biol. Chem. 1997; 272: 13793-13802Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). has been that is membrane protein with a and a cytoplasmic B. E. D. Li K. Lottspeich F. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). a that flotillin-1 a protein but is to the plasma membrane via of the Rea S. S. R. J.F. Parton R.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We in flotillin-1 we identified the protein as a for in 3T3-L1 C.A. M. S. S. P.E. J.E. Nature. 2000; PubMed Scopus Google Scholar). CAP is a adaptor protein with domains in its and a region of to the in its is highly expressed in and Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). We that in flotillin-1 to the complex to lipid that for the to the of C.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The organization of CAP is also found in other proteins, and S. T. T. K. S. J. Biol. PubMed Scopus Google Scholar, H. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that these proteins a adaptor protein can function in the of cell and growth factor signaling K. T. 2002; PubMed Scopus Google Scholar). and CAP were shown to at in of protein the formation of actin and S. T. T. K. S. J. Biol. PubMed Scopus Google Scholar, R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. H. K. K. H. J. Biol. PubMed Scopus Google Scholar). a the of the complex to lipid rafts to for actin in of cells K. J. 2004; PubMed Scopus Google Scholar). we domains in flotillin-1 that to its subcellular localization and protein in 3T3-L1 adipocytes. and CAP Fyn and were from The from The and were from The caveolin and the and were from were from The and were from and were from The and were from The protein were from from and for different CAP isoforms and caveolin-1 were as M. Mol. PubMed Google Scholar, S. S. J.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). flotillin-1 derived from C.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). flotillin-1 flotillin-1 with at the in the and of mutants of flotillin-1 were generated a of and of the second hydrophobic were the to the The and were of different flotillin-1 in the is shown in and cells were in and cells were in 3T3-L1 were in with and of 3T3-L1 cells to with and as The cells were in were in with of cells to the cells in for 3T3-L1 were as cells in were as J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of a M. of to flotillin-1 into different cell The flotillin-1 at the with into the and of the The into cells in a with the from The at and to The cells were in a and were subsequently in for and cells in 3T3-L1 in were with and were for at with and of The were with the for at from were with of protein from 3T3-L1 adipocytes. The complexes were with protein for at and were with in proteins were and to proteins were with the and with fractions plasma membrane density and low density were from 3T3-L1 and a of and as and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells on were in for cells with for various were on with the cells in of and and were in a The cell were into various and the were in a protein of and to and were on in the with for cells were with for and with and for and were at in and the were on with were 3T3-L1 with the of were on The cells were to for and were for with the and on as were in for with the cells were for on in and The The insoluble in and as C.A. M. S. S. P.E. J.E. Nature. 2000; PubMed Scopus Google Scholar). of Flotillin-1 in the cellular in flotillin-1 has been we to and its intracellular localization in different cell To flotillin-1 at the with a and expressed in a series of cell via that flotillin-1 at membrane with a intracellular distribution in and cells, the protein localized at the plasma membrane with cytoplasmic in cells, flotillin-1 intracellular granule-like with a at the plasma membrane flotillin-1 can in plasma membrane and intracellular and the distribution between these two compartments to cell type-specific. The of Flotillin-1 in 3T3-L1 flotillin-1 is localized in we the distribution of flotillin-1 subcellular As shown in flotillin-1 in and fractions with a in fractions in 3T3-L1 and adipocytes. a of flotillin-1 in the to that in upon differentiation of differentiation the subcellular distribution of caveolin, a lipid raft protein enriched in the of To these we examined the localization of flotillin-1 in 3T3-L1 cells flotillin-1. As shown in flotillin-1 found in intracellular structures in with of these with the actin cytoskeleton. differentiation into flotillin-1 found to localized at the plasma where cortical F-actin also To the cellular compartments in that contain proteins of the and were As shown in the structures flotillin-1 in were but that flotillin-1 is in to cellular To differentiation also a of we expressed in and these cells to of growth in the that flotillin-1 localized in intracellular in and of flotillin-1 is also cell type-specific. To the flotillin-1 protein lipid raft localization in we 3T3-L1 and 3T3-L1 with low from and insoluble fractions were with different of flotillin-1 protein in the of the protein in the differentiation into flotillin-1 protein found in the Fyn, a Src family kinase that shown to interact with flotillin-1 in and cells, also a lipid raft As a caveolin found to localized to lipid rafts of the differentiation of the shown that lipid raft subdomains in the plasma membrane can as for actin cytoskeleton structures (3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5133) Google Scholar, 6Harris T.J. Siu C.H. BioEssays. 2002; 24: 996-1003Crossref PubMed Scopus (84) Google Scholar). To the role of the actin cytoskeleton in flotillin localization, we cells with the actin to the As shown in of actin cytoskeleton on the lipid raft localization of flotillin-1 caveolin in and adipocytes. The N-terminal for the Lipid of Flotillin-1 in the structural determinants for the localization of flotillin-1 to the lipid raft domains of we generated a series of truncation mutants of flotillin-1 at the with a We examined the of these on the flotillin-1 localization in 3T3-L1 As of flotillin-1 in a of the N-terminal that contain the hydrophobic stretch the the second hydrophobic stretch on of a that the N-terminal hydrophobic in a cytoplasmic localization with a of protein at the N-terminal that the second hydrophobic in intracellular The results that the second but the hydrophobic is for the of flotillin-1 to the plasma membrane of 3T3-L1 adipocytes. We examined the localization of and of flotillin-1 to the lipid raft microdomains of the We protein with various flotillin proteins in 3T3-L1 adipocytes. were with at low and the membrane were is that proteins in the are lipid the of flotillin-1 and caveolin from these structures cholesterol that is a for lipid raft localization of flotillin-1 As shown in the protein to the in a in the with its in lipid the flotillin to and in the membrane the with and from the membrane and were they also were these that the hydrophobic to the of flotillin-1 role in the protein to the lipid raft To the results from the cells were also to subcellular into and The distribution of various flotillin mutants between these two fractions examined As shown in although of the protein found in the of and of were localized in the same is with the results from as the that in the intracellular compartments as found to in the the hydrophobic is necessary for flotillin-1 to lipid raft microdomains in and intracellular membrane structures. that of is in flotillin-1 with the in cells Rea S. S. R. J.F. Parton R.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). To of also is for the localization of flotillin in we 3T3-L1 with type of flotillin-1 with As the type flotillin with the in cells and localized with in in intracellular that also were of to on the subcellular localization of flotillin and has been in cells, the of to a role in the lipid raft localization of flotillin-1 in adipocytes. of the as the of of localization of and at the cell of to that the second hydrophobic between and flotillin-1 to the plasma membrane. To delineate the for localization, we various N-terminal of flotillin to the and expressed protein in 3T3-L1 adipocytes. As flotillin and were localized to and intracellular generated of the second hydrophobic stretch to in intracellular of from the protein the same on the localization as and of region On the other hand, and that the second hydrophobic well with the from the protein of the hydrophobic from its these that the second but the hydrophobic of flotillin-1 is for the localization and for the of the protein from intracellular structures. CAP at the of 3T3-L1 is a adaptor protein highly expressed in and Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). to a protein at the in K. H. K. K. H. J. Biol. PubMed Scopus Google Scholar) and a lipid raft in C.A. M. S. S. P.E. J.E. Nature. 2000; PubMed Scopus Google Scholar), we to CAP associate with the cortical actin cytoskeleton at the of adipocytes. on 3T3-L1 a to to for F-actin the of the cells, that CAP to the cortical F-actin with intracellular the plasma membrane CAP found to well with F-actin at the cell and the structures. CAP at the and the of actin at the plasma membrane we also found that CAP of to and F-actin in in and S. M. M. L. H. and R. of Flotillin-1 with that flotillin-1 CAP to plasma membrane lipid rafts in C.A. M. S. S. P.E. J.E. Nature. 2000; PubMed Scopus Google Scholar). To the of CAP with were cell from 3T3-L1 adipocytes. CAP proteins were with the of As were and As shown in a of flotillin-1 with CAP of CAP were The same of and were to CAP a between CAP and flotillin-1 in of the results in isoforms of CAP We M. Mol. PubMed Google Scholar) that were isoforms of CAP protein expressed in adipose To the of CAP to flotillin-1 is we cells with and a of the CAP with As shown in specifically with but with the other and the same the of flotillin with CAP is of with Flotillin-1 and that flotillin-1 and actin to these are in To we and in well that with actin and at at the of the cells, where found to at the of the to well with in the We with in 3T3-L1 Both CAP proteins to in cells were with we that the of with in the of cells with to cortical F-actin from the as well as the membrane On the other hand, the of cortical actin on the localization of flotillin in the lipid These results that is with flotillin-1 in the lipid raft is on the of the cortical F-actin of and Fyn in to the region of flotillin-1 for to and with with the same as the of the that the hydrophobic from to a protein that to a that a of the protein in the various These that the hydrophobic of flotillin-1 is for its with shown that flotillin-1 and flotillin-2 are with the Src family kinase Fyn in fractions of that flotillin role in the formation of centers (10Stuermer C.A. Lang D.M. Kirsch F. Wiechers M. Deininger S-O. Plattner H. Mol. Biol. Cell. 2001; 12: 3031-3045Crossref PubMed Scopus (184) Google Scholar). To we cells with type kinase mutants of Fyn with flotillin-1 with of the that of Fyn with flotillin-1. Fyn in the lacking flotillin-1. that the of Fyn the Fyn kinase a complex with flotillin-1 of its kinase To the region in we Fyn and various truncation mutants of flotillin-1. that of the N-terminal the of the second hydrophobic Fyn that these regions are for the N-terminal to the of the second hydrophobic in flotillin Fyn These results that the region between the two hydrophobic domains in flotillin the of the protein to To Fyn the of to cells were also with and in the of were with that the of with type kinase Fyn of on the between Fyn and flotillin in a These results that the regions in flotillin-1 for and Fyn with these proteins of in a multimeric and of and -2 are ubiquitously expressed lipid raft proteins that been with cellular from Stuermer and D.M. Lommel S. Jung M. Ankerhold R. Petrausch B. Laessing U. Wiechers M.F. Plattner H. Stuermer C.A. J. Neurobiol. 1998; 37: 502-523Crossref PubMed Scopus (202) Google Scholar, 10Stuermer C.A. Lang D.M. Kirsch F. Wiechers M. Deininger S-O. Plattner H. Mol. Biol. Cell. 2001; 12: 3031-3045Crossref PubMed Scopus (184) Google Scholar) that flotillin proteins in at the of and also shown that flotillin-1 localized to the cell of the cell and Rea S. S. R. J.F. Parton R.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Jurkat lymphoma cells, were with and Fyn in intracellular that (10Stuermer C.A. Lang D.M. Kirsch F. Wiechers M. Deininger S-O. Plattner H. Mol. Biol. Cell. 2001; 12: 3031-3045Crossref PubMed Scopus (184) Google Scholar). flotillin-1 found to on J. R. S. J.F. Duclos S. E. R. Desjardins M. J. 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The of the between our results and the Rea S. S. R. J.F. Parton R.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) with cells is is that the of the of in the localization also cell type-specific. role for the second hydrophobic in the subcellular distribution of flotillin-1 the truncation of hydrophobic the the proteins were in the intracellular These results that the second hydrophobic region as a localization as well as a to flotillin-1 from the membrane to the second hydrophobic stretch of flotillin-1 is in the a hydrophobic has been to to membrane for most proteins in family M. Biochem. 24: Full Text Full Text PDF PubMed Scopus Google Scholar). we the that a in flotillin-1 for membrane the with a a flotillin-1 between the two hydrophobic to associate with the membrane the of a in On the other hand, flotillin-2 contain hydrophobic but membrane localization to that flotillin-1 in a of cells D.M. Lommel S. Jung M. Ankerhold R. Petrausch B. Laessing U. 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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.