Asb6, an Adipocyte-specific Ankyrin and SOCS Box Protein, Interacts with APS to Enable Recruitment of Elongins B and C to the Insulin Receptor Signaling Complex

Abstract

The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.Insulin promotes glucose transport into target tissues by causing the exocytosis of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (1). There are thought to be two pathways required for insulin-stimulated glucose transport: an insulin receptor substrate-PI 1 3-kinase pathway and a PI 3-kinase-independent pathway involving APS, CAP, and c-Cbl. The PI 3-kinase-independent pathway is initiated by the binding of APS to the activation loop of the insulin receptor (2, 3). APS is constitutively bound to CAP and undergoes tyrosine phosphorylation on Tyr 618 allowing it to bind to the variant SH2 domain of c-Cbl (3, 4). This is followed by the tyrosine phosphorylation of c-Cbl on tyrosines 700 and 774 resulting in the phosphorylated c-Cbl binding to the SH2 domain of Crk (3, 5). Following the insulin-stimulated phosphorylation of c-Cbl, the CAP/Cbl complex migrates to the caveolin-rich lipid rafts, a movement facilitated by the interaction of the CAP SOHO domain with flotillin, a protein in lipid rafts (6). This allows the Crk/C3G complex to be recruited to this microdomain, where C3G activates the small G protein TC10 (7-10). The activation of TC10 occurs independently of PI 3-kinase and is crucial for insulin-stimulated Glut4 translocation (1).The domain structure of APS includes proline-rich regions and a n...

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