Interaction of wild type and dominant-negative p55(PIK) regulatory subunit of phosphatidylinositol 3-kinase with insulin-like growth factor-1 signaling proteins

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 alpha, as previously shown for p85 alpha. 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 alpha 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, 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.