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

• Published in: The Journal of biological chemistry Vol. 279; no. 37; pp. 38881 - 38888
• Main Authors: Wilcox, Andrew, Katsanakis, Kostas D., Bheda, Farheen, Pillay, T.S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.09.2004; American Society for Biochemistry and Molecular Biology
• Subjects: 3T3-L1 Cells; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport - chemistry; Adaptor Proteins, Vesicular Transport - metabolism; Adipocytes - metabolism; Amino Acid Sequence; Animals; Ankyrins - chemistry; Ankyrins - metabolism; Ankyrins - physiology; Base Sequence; Biological Transport; CHO Cells; Cricetinae; DNA, Complementary - metabolism; Elongin; Epitopes; Gene Library; Glucose - metabolism; Glutathione Transferase - metabolism; Insulin - metabolism; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; Molecular Sequence Data; Phosphorylation; Plasmids - metabolism; Proline - chemistry; Protein Binding; Protein Structure, Tertiary; Rabbits; Receptor, Insulin - metabolism; Recombinant Fusion Proteins - metabolism; Signal Transduction; Suppressor of Cytokine Signaling Proteins; Transcription Factors - metabolism; Transfection; Two-Hybrid System Techniques; Tyrosine - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M406101200

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.