title: Requirements for pYXXM motifs in Cbl for binding to the p85 subunit of phosphatidylinositol 3-kinase and Crk, and activation of atypical protein kinase C and glucose transport during insulin action in 3T3/L1 adipocytes.
abstract: Cbl is phosphorylated by the insulin receptor and reportedly functions within the flotillin/CAP/Cbl/Crk/C3G/TC10 complex during insulin-stimulated glucose transport in 3T3/L1 adipocytes. Cbl, via pYXXM motifs at tyrosine-371 and tyrosine-731, also activates phosphatidylinositol (PI) 3-kinase, which is required to activate atypical protein kinase C (aPKC) and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes [Miura et al. (2003) Biochemistry 42, 14335-14341]. Presently, we have examined the importance of Cbl in activating PI 3-kinase and aPKC during insulin action in 3T3/L1 adipocytes by expressing Y371F and Y731F Cbl mutants, which nullify pYXXM binding of Cbl to SH2 domains of downstream effectors. Interestingly, these mutants inhibited insulin-induced increases in (a) binding of Cbl to both Crk and the p85 subunit of PI 3-kinase, (b) activation of Cbl-dependent PI 3-kinase, (c) activation and translocation of aPKC to the plasma membrane, (d) translocation of Glut4 to the plasma membrane, (e) and glucose transport. Importantly, coexpression of wild-type Cbl reversed the inhibitory effects of Cbl mutants. In contrast to Cbl-dependent PI 3-kinase, Cbl mutants did not significantly inhibit the activation of PI 3-kinase by IRS-1, which is also required during insulin action. Our findings suggest that (a) Cbl uses pYXXM motifs to simultaneously activate PI 3-kinase and Crk/C3G/TC10 pathways and (b) Cbl, along with IRS-1, functions upstream of PI 3-kinase and aPKCs during insulin-stimulated glucose transport in 3T3/L1 adipocytes.
authors.affiliation: Research Service, James A. Haley Veterans Medical Center, and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Standaert
authors.forename: Mini P
authors.initials: MP
authors.lastname: Sajan
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miura
authors.forename: Gautam
authors.initials: G
authors.lastname: Bandyopadhyay
authors.forename: Robert V
authors.initials: RV
authors.lastname: Farese
mesh_terms: D041721:3T3-L1 Cells; D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007329:Insulin Antagonists / Q000378:metabolism / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D050722:Oncogene Protein v-cbl; D010649:Phenylalanine / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D015682:Retroviridae Proteins, Oncogenic / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta
chemical_list: D005947:Glucose; D007328:Insulin; D007329:Insulin Antagonists; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D050722:Oncogene Protein v-cbl; D010649:Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D011493:Protein Kinase C; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D015682:Retroviridae Proteins, Oncogenic; D014443:Tyrosine; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta; C526292:IRS1 protein, human; C526295:Irs1 protein, mouse