Volume 271, Number 35, Issue of August 30, 1996 pp. 21614-21621
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Binding to the Platelet-derived Growth Factor Receptor Transiently Activates the p85-p110 Phosphoinositide 3-Kinase Complex in Vivo

(Received for publication, December 6, 1995, and in revised form, June 6, 1996)

From the Ludwig Institute for Cancer Research, London, W1P 8BT and the ^¶ Department of Biochemistry and Molecular Biology, University College, London, WCE 6BT, United Kingdom

Ligand stimulation of the platelet-derived growth factor (PDGF) receptor results in its association with phosphoinositide 3-kinase activity and a corresponding synthesis of 3-phosphorylated lipids. Early studies that examined this interaction in vivo employed anti-phosphotyrosine antiserum or antiserum against the PDGF receptor. The recent identification of multiple isoforms of both the regulatory and the catalytic subunit of the enzyme have led us to utilize antisera against p85 and p110 to characterize the association of this particular phosphoinositide 3-kinase complex with the PDGF receptor following ligand stimulation of murine fibroblasts. Both the p85 and p110 subunits rapidly associated with the ligand-activated receptor resulting in a transient, 2-fold increase in the total pool of p110 lipid kinase activity. This association was stable for 15 min after initial stimulation. Subsequently, both subunits began to dissociate from the receptor with similar kinetics. By 60 min this process was complete, demonstrating that p85 and p110 both associate with the receptor and dissociate from the receptor as a dimeric complex. At this time, marked PDGF receptor down-regulation was observed. Immunoprecipitation from metabolically labeled cells revealed that p85 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. No phosphorylation of the p110 subunit was detected in either quiescent or PDGF-stimulated cells. Quantitation of Western blot analysis demonstrated that only 5% of the total pool of p85 associated with the PDGF receptor upon ligand stimulation. The 2-fold increase in the lipid kinase activity measured in immunoprecipitates using either anti-p85 or anti-p110 antiserum therefore reflects a far greater increase in the specific activity of the enzyme upon its association with the PDGF receptor.

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