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Interaction of Wild Type and Dominant-Negative p55^PIK Regulatory Subunit of Phosphatidylinositol 3-Kinase with Insulin-Like Growth Factor-1 Signaling Proteins

Institut National de la Santé et de la Recherche Médicale (INSERM) U145 (I.M., L.D., C.F., E.V.O.) 06107 Nice Cédex 2, France Joslin Diabetes Center (S.P., M.F.W.) and Department of Medicine Harvard Medical School Boston, Massachusetts 02215

In a first series of experiments done in the yeast two-hybrid system, we investigated the nature of protein-protein interaction between the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), p55^PIK, and several of its potential signaling partners. The region between the Src homology 2 (SH2) domains of p55^PIK bound to the NH₂ terminus region of p110, as previously shown for p85. Moreover, we found that the insulin-like growth factor-1 receptor (IGF-IR) bound to p55^PIK; the interaction occurred at the receptor tyrosine 1316 and involved both p55^PIK SH2 domains. Interaction between p55^PIK and IGF-IR was seen not only in the yeast two-hybrid system, but also using in vitro binding and coimmunoprecipitation of lysates from IGF-1 stimulated 293 cells overexpressing p55^PIK. Further, IGF-I stimulation of these cells led to tyrosine phosphorylation of p55^PIK. In 293 cells association of p55^PIK with insulin receptor substrate- 1 and with IGF-IR was dependent on PI 3-kinase, since it was increased by wortmannin, an inhibitor of PI 3-kinase. Further, by deleting amino acids 203–217 of p55^PIK inter-SH2 domain, we engineered a p55^PIK mutant unable to bind to the p110 catalytic subunit of PI 3-kinase. This mutant had a dominant-negative action on insulin-stimulated glucose transport, since insulin’s effect on Glut 4 myc translocation was inhibited in adipocytes expressing mutant p55^PIK. Importantly, this dominant-negative mutant was more efficient than wild type p55^PIK in associating to IGF-IR and insulin receptor substrate-1 in 293 cells. Taken together, our results show that p55^PIK interacts with key elements in the IGF-I signaling pathway, and that these interactions are negatively modulated by PI 3-kinase itself, providing circuitry for regulatory feedback control.

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