Abstract
We have previously shown that phosphatidylinositol (PtdIns) 3'-kinase is activated by the binding of proteins or peptides containing the phosphorylated motif Y(P)XXM. In the present study, we examine interactions between PtdIns 3'-kinase and the human insulin receptor, which contains a C-terminal phosphorylation site in the sequence Y1322THM. Partially purified insulin receptors bound tightly to bacterial fusion proteins containing the N- or C-terminal SH2 domains from PtdIns 3'-kinase regulatory subunit (p85). In contrast, a mutant insulin receptor, truncated by 43 amino acids at the C terminus (IR delta CT), bound poorly to the SH2 domains; these mutant receptors have normal kinase activity but lack the Y1322THM motif. Similarly, incubation with wild-type receptors increased the activity of immunopurified PtdIns 3'-kinase, whereas incubation with IR delta CT receptors did not affect PtdIns 3'-kinase activity. Activation of PtdIns 3'-kinase by the wild-type receptor was mimicked by a tyrosyl phosphopeptide derived from the insulin receptor C terminus and containing the Y1322THM motif; non-phosphorylated peptide did not affect activity. Thus, the insulin receptor C terminus activates PtdIns 3'-kinase in vitro by binding to the SH2 domains of the 85-kDa regulatory subunit. These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p85 SH2 domains is a general mechanism for PtdIns 3'-kinase activation, and they suggest that direct interactions between the insulin receptor and PtdIns 3'-kinase may provide an alternative pathway for the activation of this enzyme by insulin.
Highlights
Binding of tyrosyl-phosphorylated IRS-1 to wild-type receptors increased the activity of immunoPptduI-ns 3”kinase activates thliepid kinase i n vitro and in intact rified PtdIns 3’-kinase, whereas incubation withIRAcT cells [17]
From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461 and the wesearchDivision, Joslin Diabetes Center, Department of Medicine, Brigham and Women’s
These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p86 SI32 domains is a general mechanism for PtdIns 3”kinase activation, and they which aredriven by SH2 domaibninding to phosphorylated YXXM motifs
Summary
Binding of tyrosyl-phosphorylated IRS-1 to wild-type receptors increased the activity of immunoPptduI-ns 3”kinase activates thliepid kinase i n vitro and in intact rified PtdIns 3’-kinase, whereas incubation withIRAcT cells [17]. Tyrodomains from p85; SH2-bound receptors were eluted and ana- syl phosphorylation of p85 was directly examined by incubating lyzed by SDS-PAGE (Fig. l).While no binding to GST alone immunopurified PtdIns 3'kinase with wild-type and IRAcT rewas detected, approximately 15% ofthe total applied wild-type ceptors under the same conditions used for the activation exreceptors bound to the N- and N+C-terminal constructs; bind- periments.
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