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Abstract
Phosphatidylinositol 3-kinase, which generates putative novel second messenger phospholipids, is a heterodimer composed of regulatory adaptor 85-kDa and catalytic 110-kDa subunits. The p85 alpha 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-terminal portion of the molecule which contains two SH2 domains, separated by a spacer region. In this study a panel of monoclonal antibodies (mAb) was raised against recombinant bovine p85 alpha to probe its multidomain structure in relation to function. These mAbs were characterized using a BIAcore biosensor instrument. Epitopes for nine mAbs were mapped in relation to the domain structure of p85 alpha using recombinant protein fragments expressed in bacteria. These mAbs were then used to map the sites on p85 alpha which are involved in growth factor receptor binding. Two interesting classes of functional mAbs were identified. First, mAb U14, whose epitope lies within the NH2-terminal SH2 domain of p85 alpha, blocked the interaction of p85 alpha with activated protein-tyrosine kinase receptors. Second, real-time binding experiments using phospholipid-containing vesicles showed that p85 alpha by itself could specifically bind certain phospholipids. Two mAbs (U9 and U15) with epitopes located in the inter-SH2 spacer region blocked the binding of lipids to this site. The relevance of these observations to understanding the relationship of structure to function of p85 and the phosphatidylinositol 3-kinase are discussed.
Highlights
The NH2-terminal SH2 domain of p85a was expressed as a glutathione transferase-p85fusion protein and immobilizedon the sensorchip at 13,100 RU.All mAbs raised against p85awere injected successively and their binding analyzed
TimeIs1 have demonstrated that an 85-kDa subunit is not essential for PI-3-kinase catalytic activity, it has not been ruled outthatitmightenhance or regulatethecatalytic activity intrinsic to the 110-kDa subunit (Hileset al., 1992)
Taken together these observationssuggest that in the heterodimeric PI-3-kinase complex the p85 component acts as both an adaptor and regulatory subunit, facilitating the coupling the catalytic component to upstreasimgnaling molecules such asactivatedreceptorsand possiblyalso to regulatory Gproteins. In this reportwe describe the evaluation of a panel of mAbs raised against the p85a subunfiot r use in functional studies of the role of p85a, and of the defined structural domains of p85, in the PI-3-kinasecomplex
Summary
60 plates(pretreated with 1%oxalic acid, 1 mM EDTA in water/ Characterization of Monoclonal Antibodies Raised against methanol (64)) anddeveloped in chloroform, methanol, 4N ammonia p85a"mAbs which had been selected for subcloning by en-. Kinetic parameters were determined for the binding of the mAbs to purified recombinant p85a. The procedure used to calculate both the association anddissociation rate constants from theBIAcore data is described in detail under "Materials and Methods." The mAbs to be analyzed were bound toa Fcspecific, rat anti-mouse polyclonal antibody (RamFc), which was immobilized on the matrix. This approach has thme ajor advantagethatit avoids the necessity of determiningthe concentration of the active antibody population in the crude timeIs1 antibody sample. Note that mAbs U2 and U5 do not compete in their binding to p85a while the prior binding of mAb U5 to p85a lM00 0 200 400 r(e0bg.1oe%oneSraDtSi8o)0n0 lo00 1200 completely abolishes any subsequent binding of mAb U10
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