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The phosphotyrosine (Tyr(P)) form of insulin receptor substrate 1 (IRS-1) is a key component in insulin signaling. Our previous study revealed that Tyr(P) IRS-1 binds to the widely distributed tyrosine phosphatase PTP2C through the src homology 2 (SH2) domains of the latter. In the present study, we examined the activity of this enzyme and of a truncated form lacking the SH2 domains (delta PTP2C) toward IRS-1 and also toward the cytoplasmic domain of the insulin receptor. Tyr(P) IRS-1 was prepared by phosphorylation of recombinant IRS-1 with recombinant cytoplasmic insulin receptor kinase (CIRK). PTP2C rapidly dephosphorylated Tyr(P) IRS-1; dephosphorylation by delta PTP2C was approximately one-third as fast. Other substrates, including Tyr(P) CIRK, were not dephosphorylated as rapidly by PTP2C; moreover, delta PTP2C was at least 10 times more active than PTP2C toward CIRK and other substrates. These results indicate that the binding of Tyr(P) residues on IRS-1 to the SH2 domain(s) of PTP2C enhances its activity toward IRS-1 and suggest that PTP2C is the phosphatase responsible for the dephosphorylation of IRS-1 in vivo. In addition, with the expectation that a PTP2C-resistant form of IRS-1 will be useful in investigations of IRS-1 function, we determined that IRS-1 can be thiophosphorylated with adenosine 5'-O-(3-thiotriphosphate) and CIRK and that this form of IRS-1 is resistant to PTP2C.

The insulin and insulin-like growth factor-I (IGF-I) receptors are tyrosine kinases. Consequently, an approach to investigating signaling pathways from these receptors is to characterize proteins rapidly phosphorylated on tyrosine in response to insulin and IGF-I. In many cell types the most prominent phosphotyrosine (Ptyr) protein, in addition to the receptors themselves, is a protein of approximately 160 kD, now known as the insulin receptor substrate 1 (IRS-1). We have purified IRS-1 from mouse 3T3-L1 adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA based on this information. Mouse IRS-1 is a protein of 1,231 amino acids. It contains 12 tyrosine residues in sequence contexts typical for tyrosine phosphorylation sites. Six of these begin the sequence motif YMXM and two begin the motif YXXM. Recent studies have shown that the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) binds tightly to the activated platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1) receptors, through interaction of the src homology 2 (SH2) domains on the 85 kD subunit of PI 3-kinase with Ptyr in one of these motifs on the receptors. We have found that, upon insulin treatment of 3T3-L1 adipocytes, a portion of the Ptyr form of IRS-1 becomes tightly complexed with PI 3-kinase. Since IRS-1 binds to fusion proteins containing the SH2 domains of PI 3-kinase, association most likely occurs through this domain. The association of IRS-1 with PI 3-kinase activates the enzyme about fivefold.(ABSTRACT TRUNCATED AT 250 WORDS)

The insulin receptor substrate 1 (IRS1) is a protein that is rapidly phosphorylated on tyrosine by the activated insulin receptor. Syp is a recently discovered, broadly expressed phosphotyrosine (Tyr(P)) phosphatase that contains two Src homology 2 (SH2) domains. We have found that insulin treatment of 3T3-L1 adipocytes leads to complex formation between IRS1 and Syp. Syp was detected in immunoadsorbates of IRS1 from extracts of insulin-treated but not basal cells by both immunoblotting and Tyr(P) phosphatase activity. The association of Syp with IRS1 apparently occurs between the SH2 domains of Syp and Tyr(P)-containing sequences of IRS1, since a fusion protein containing only the SH2 domains of Syp bound the Tyr(P) form of IRS1. Unlike the receptors for epidermal and platelet-derived growth factors, which in their activated state bind to the SH2 domains of Syp and elicit phosphorylation of Syp on tyrosine in intact cells, the Tyr(P) form of the insulin receptor did not bind to the SH2 domains of Syp, and no phosphorylation of Syp on tyrosine was detected in insulin-treated 3T3-L1 adipocytes. In combination with other findings these results indicate that IRS1 functions as a docking protein for SH2 domain-containing proteins participating in signaling from the insulin receptor.

The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.