PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATION IS MEDIATED BY HIGH-AFFINITYINTERACTIONS BETWEEN DISTINCT DOMAINS WITHIN THE P110 AND P85 SUBUNITS

Domains of interaction between the p85 and p110 subunits of phosphatid ylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybr id expression system. A gene fusion between the GALA transactivation d omain and p85 activated transcription from a GAL1-lacZ reporter gene w hen complemented with a gene fusion between the GAL4 DNA binding domai n and p110. To define subdomains responsible for this interaction, a s eries of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH 2 (IS) fragment (residues 429 to 621) was the smallest determinant ide ntified that specifically associated with p110. In analogous experimen ts, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residu es. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-len gth p85 and p110. Although the p85(IS) domain conferred a strong inter action with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity . In contrast, coexpression of the p110 subunit with full-length p85 o r with constructs containing the IS sequences flanked by both SH2 doma ins of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85( nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was ac tivated by a phosphotyrosine peptide. These data suggest that phosphot yrosine peptide binding to either SH2 domain generates an intramolecul ar signal propagated through the IS region to allosterically activate p110.