A novel insulin receptor substrate protein, xIRS-u, potentiates insulin signaling: functional importance of its pleckstrin homology domain.
A novel Xenopus insulin receptor substrate cDNA was isolated by hybridization screening using the rat insulin receptor substrate-1 (IRS-1) cDNA as a probe. The xIRS-u cDNA encodes an open reading frame of 1003 amino acids including a putative amino-terminal pleckstrin homology (PH) domain and phosphotyrosine-binding (PTB) domain. The carboxy terminus of xIRS-u contains several potential Src homology 2 (SH2)-binding sites, five of which are in the context of YM/LXM (presumptive binding sites for phosphatidylinositol 3-kinase). It also contains a putative binding site for Grb2 (YINID). Pair-wise amino acid sequence comparisons with the previously identified xIRS-1 and the four members of the mammalian IRS family (1 through 4) indicated that xIRS-u has similar overall sequence homology (33-45% identity) to all mammalian IRS proteins. In contrast, the previously isolated xIRS-1 is particularly similar (67% identical) to IRS-1 and considerably less similar (31-46%) to the other IRS family members (2 through 4). xIRS-u is also distinct from xIRS-1, having an overall sequence identity of 47%. These sequence analyses suggest that xIRS-u is a novel member of the IRS family rather than a Xenopus homolog of an existing member. Microinjection of mRNA encoding a Myc-tagged xIRS-u into Xenopus oocytes resulted in the expression of a 120-kDa protein (including 5 copies of the 13-amino acid Myc tag). The injection of xIRS-u mRNA accelerated insulin-induced MAP kinase activation with a concomitant acceleration of insulin-induced oocyte maturation. An aminoterminal deletion of the PH domain (xIRS-u deltaPH) significantly reduced the ability of xIRS-u to potentiate insulin signaling. In contrast to the full-length protein, injection of xIRS-u (1-299), which encoded the PH and PTB domain, or xIRS-u (1-170), which encoded only the PH domain, blocked insulin signaling in Xenopus oocytes. Finally, xIRS-u (119-299), which had a truncated PH domain and an intact PTB domain, had no effect on insulin signaling. This is the first report that the PH domain of an IRS protein can function in a dominant negative manner to inhibit insulin signaling.
Ottawa Civic Hospital Loeb Research Institute, University of Ottawa, Canada.
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Ohan
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Bayaa
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Kumar
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Zhu
X J
XJ
Liu
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003001:Cloning, Molecular; D005260:Female; D051380:GRB2 Adaptor Protein; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D011506:Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C526294:irs1 protein, Xenopus; C114973:irs2-A protein, Xenopus; C039077:platelet protein P47; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases