Primary and essential role of the adaptor protein APS for recruitment of both c-Cbl and its associated protein CAP in insulin signaling.
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.
Ahn
Mee-Young
MY
Institute of Cell Signaling and School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.
Katsanakis
Kostas D
KD
Bheda
Farheen
F
Pillay
Tahir S
TS
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D017667:Adipocytes / Q000502:physiology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D007328:Insulin; D008840:Microfilament Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C488161:Sh2b2 protein, mouse; C119201:ponsin; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C067419:CBL protein, human; C493849:Cbl protein, mouse