GENETIC-ANALYSIS OF A PHOSPHATIDYLINOSITOL 3-KINASE SH2 DOMAIN REVEALS DETERMINANTS OF SPECIFICITY

Phosphatidylinositol 3-kinase is an important element in both normal a nd oncogenic signal transduction. Polyomavirus middle T antigen transf orms cells in a manner depending on association of its tyrosine 315 ph osphorylation site with Src homology 2 (SH2) domains on the p85 subuni t of the phosphatidylinositol 3-kinase. Both nonselective and site-dir ected mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed incre ased binding were also found. Comparison of middle T binding to that o f the platelet-derived growth factor receptor showed that some mutatio ns altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-393, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide lib rary. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position af ter the phosphotyrosine.