A BIOSENSOR APPROACH TO PROBE THE STRUCTURE AND FUNCTION OF THE P85-ALPHA-SUBUNIT OF THE PHOSPHATIDYLINOSITOL 3-KINASE COMPLEX

Phosphatidylinositol 3-kinase, which generates putative novel second m essenger phospholipids, is a heterodimer composed of regulatory adapto r 85-kDa and catalytic 110-kDa subunits. The p85alpha subunit contains a NH2-terminal src homology (SH) 3 domain, a region with homology to the product of the breakpoint cluster region (bcr) gene, and a COOH-te rminal portion of the molecule which contains two SH2 domains, separat ed by a spacer region. In this study a panel of monoclonal antibodies (mAb) was raised against recombinant bovine p85alpha to probe its mult idomain structure in relation to function. These mAbs were characteriz ed using a BIAcore biosensor instrument. Epitopes for nine mAbs were m apped in relation to the domain structure of p85alpha using recombinan t protein fragments expressed in bacteria. These mAbs were then used t o map the sites on p85alpha which are involved in growth factor recept or binding. Two interesting classes of functional mAbs were identified . First, mAb U14, whose epitope lies within the NH2-terminal SH2 domai n of p85alpha, blocked the interaction of p85alpha with activated prot ein-tyrosine kinase receptors. Second, real-time binding experiments u sing phospholipid-containing vesicles showed that p85alpha by itself c ould specifically bind certain phospholipids. Two mAbs (U9 and U15) wi th epitopes located in the inter-SH2 spacer region blocked the binding of lipids to this site. The relevance of these observations to unders tanding the relationship of structure to function of p85 and the phosp hatidylinositol 3-kinase are discussed.