Primary and Essential Role of the Adaptor Protein APS for Recruitment of Both c-Cbl and Its Associated Protein CAP in Insulin Signaling

Abstract

APS (adapter protein with Pleckstrin homology and Src homology 2 domains) is recruited by the autophosphorylated insulin receptor and is essential for Glut4 translocation. Although both APS and CAP (c-Cbl-associated protein) interact with c-Cbl during insulin signaling, the relative importance of each protein in recruiting c-Cbl has not been clear. We performed a side-by-side comparison by ectopic expression of APS or Src homology 2-Balpha (SH2-Balpha) and CAP in Chinese hamster ovary (CHO) cells. In cells co-expressing insulin receptor and CAP, without APS, no association of the insulin receptor and CAP could be detected and no insulin-stimulated phosphorylation of Cbl was observed. Insulin-stimulated Cbl phosphorylation was reconstituted when APS was co-expressed with insulin receptor, with or without CAP. APS or SH2-Balpha and CAP interacted in the basal state, and in the case of APS this interaction was mediated by the C terminus of APS. Insulin stimulation resulted in the dissociation of APS and CAP. Similarly, insulin stimulation also resulted in the dissociation of SH2-Balpha and CAP in CHO cells. CAP was localized to the membrane in the presence of APS. Insulin stimulation resulted in the re-localization of CAP to the cytosol only when APS was co-expressed. In 3T3-L1 adipocytes, small interfering RNA-mediated knockdown of the mouse APS gene abolished the insulin-stimulated phosphorylation of c-Cbl. Taken together, these results indicate that APS plays a central role in recruiting both CAP and c-Cbl to the insulin receptor after insulin stimulation and is necessary and sufficient for the insulin-stimulated phosphorylation of c-Cbl, whereas SH2-Balpha may provide an alternative pathway for the recruitment of CAP.