BINDING TO THE PLATELET-DERIVED GROWTH-FACTOR RECEPTOR TRANSIENTLY ACTIVATES THE P85-ALPHA-P110-ALPHA PHOSPHOINOSITIDE 3-KINASE COMPLEX IN-VIVO

Ligand stimulation of the platelet-derived growth factor (PDGF) recept or results in its association with phosphoinositide 3-kinase activity and a corresponding synthesis of 3'-phosphorylated lipids, Early studi es that examined this interaction in vivo employed anti-phosphotyrosin e antiserum or antiserum against the PDGF receptor. The recent identif ication of multiple isoforms of both the regulatory and the catalytic subunit of the enzyme have led us to utilize antisera against p85 alph a and p110 alpha to characterize the association of this particular ph osphoinositide 3-kinase complex with the PDGF receptor following ligan d stimulation of murine fibroblasts. Both the p85 alpha and p110 alpha subunits rapidly associated with the ligand-activated receptor result ing in a transient, 2-fold increase in the total pool of p110 alpha li pid kinase activity. This association was stable for 15 min after init ial stimulation. Subsequently, both subunits began to dissociate from the receptor with similar kinetics. By 60 min this process was complet e, demonstrating that p85 alpha and p110 alpha both associate with the receptor and dissociate from the receptor as a dimeric complex. At th is time, marked PDGF receptor down-regulation was observed. Immunoprec ipitation from metabolically labeled cells revealed that p85 alpha is constitutively phosphorylated on serine residues in quiescent cultures . Upon PDGF stimulation, this phosphorylation upon serine residues was maintained in addition to tyrosine phosphorylation of this subunit. N o phosphorylation of the p110 alpha subunit was detected in either qui escent or PDGF-stimulated cells. Quantitation of Western blot analysis demonstrated that only 5% of the total pool of p85 alpha associated w ith the PDGF receptor upon ligand stimulation. The 2-fold increase in the lipid kinase activity measured in immunoprecipitates using either anti-p85 alpha or anti-p110 alpha) antiserum therefore reflects a far greater increase inn the specific activity of the enzyme upon its asso ciation with the PDGF receptor.