MICROINJECTION OF THE SH2 DOMAIN OF THE 85-KILODALTON SUBUNIT OF PHOSPHATIDYLINOSITOL 3-KINASE INHIBITS INSULIN-INDUCED DNA-SYNTHESIS AND C-FOS EXPRESSION

We have investigated the functional role of the SH2 domain of the 85-k Da subunit (p85) of the phosphatidylinositol 3-kinase in the insulin s ignal transduction pathway. Microinjection of a bacterial fusion prote in containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein exp ression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experimen ts, which showed that the SH2 domain of p85 can independently associat e with both insulin receptor substrate 1 and the insulin receptor itse lf in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-p urified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mu tant insulin receptors which lack the cytoplasmic tyrosine residues wi th which p85 interacts. Microinjection of the SH2 domain fusion protei n also inhibited insulin signaling in these cells, suggesting that ass ociation of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of p urified p21(ras) protein with the p85 fusion protein or the antibody r estored DNA synthesis, suggesting that ras function is either downstre am or independent of p85 SH2 domain interaction.