Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called ppl85, were originally found in anti-phosphotyrosine antibody (αPY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to aPY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X.J., et al. (1991) Nature 352, 73-77]. IRS-1 and ppl85 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of ppl85. The ppl85 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp 185 (HMW-pp 185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-ppl85, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-ppl85. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-ppl85, which may play unique roles in insulin signal transmission.
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