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.
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.
Wilcox
Andrew
A
Institute of Cell Signaling & School of Biomedical Sciences, University of Nottingham Medical School, Nottingham NG7 2UH, United Kingdom.
Katsanakis
Kostas D
KD
Bheda
Farheen
F
Pillay
T S
TS
D041721:3T3-L1 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000378:metabolism; D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D001483:Base Sequence; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D000075065:Elongin; D000939:Epitopes; D015723:Gene Library; D005947:Glucose / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011972:Receptor, Insulin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D017487:Ankyrins; C492435:Asb6 protein, mouse; D018076:DNA, Complementary; D000075065:Elongin; D000939:Epitopes; D007328:Insulin; D011993:Recombinant Fusion Proteins; C488161:Sh2b2 protein, mouse; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D014443:Tyrosine; D011392:Proline; D005982:Glutathione Transferase; D011972:Receptor, Insulin; D005947:Glucose