title: RNAi-based analysis of CAP, Cbl, and CrkII function in the regulation of GLUT4 by insulin.
abstract: Stimulation of glucose transport by insulin in cultured adipocytes through translocation of intracellular GLUT4 glucose transporters to the plasma membrane has been suggested to require phosphatidylinositol (PI) 3-kinase-dependent and independent mechanisms. To test the involvement of a PI 3-kinase-independent pathway leading to activation of the TC10 GTPase, the putative intermediates CAP, c-Cbl, Cbl-b, and CrkII were selectively depleted in 3T3-L1 adipocytes using highly efficient small interfering (si) RNAs. Simultaneous depletion of the ubiquitination factors c-Cbl plus Cbl-b in cultured adipocytes had the expected effect of delaying dephosphorylation of EGF receptors upon removal of EGF. However, siRNA-mediated gene silencing of both Cbl isoforms or CAP or CrkII in these cells failed to attenuate insulin-stimulated deoxyglucose transport or Myc-tagged GLUT4-GFP translocation at either sub-maximal or maximal concentrations of insulin. The dose-response relationship for insulin stimulation of deoxyglucose transport in primary adipocytes derived from c-Cbl knock-out mice was also identical to insulin action on adipocytes from wild type mice. These data are consistent with the hypothesis that CAP, Cbl iso-forms, and CrkII are not required components of insulin signaling to GLUT4 transporters.
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: Prasenjit
authors.initials: P
authors.lastname: Mitra
authors.forename: Xuexiu
authors.initials: X
authors.lastname: Zheng
authors.forename: Michael P
authors.initials: MP
authors.lastname: Czech
mesh_terms: D016475:3T3 Cells; D000818:Animals; D003598:Cytoskeletal Proteins / Q000502:physiology*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009124:Muscle Proteins / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D050737:Proto-Oncogene Proteins c-crk; D034622:RNA Interference; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000502:physiology*
chemical_list: D003598:Cytoskeletal Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; C495693:Slc2a4 protein, mouse; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C493849:Cbl protein, mouse