Binding to the Platelet-derived Growth Factor Receptor Transiently Activates the p85α-p110α Phosphoinositide 3-Kinase Complex in Vivo

Abstract

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.Platelet derived growth factor (PDGF) 1 potently stimulates the proliferation of cells of mesenchymal origin. It has been reported to play a role in the physiology of early development, wound healing, and inflammation in addition to the etiology of atherosclerosis, rheumatoid arthritis, and oncogenesis, (1, 2). PDGF consists of a disulfide-linked dimer of two related polypeptide chains assembled either as homodimers or as a heterodimer. The binding of PDGF to its cell surface receptors causes receptor dimerization and transphosphorylation of specific residues along the PDGF receptor polypeptide chain (1, 3, 4). These phosphorylated tyrosine residues then serve as specific attachment sites for many cytoplasmic effector proteins (5). Substrates for the PDGF receptor include phospholipase C␥, ras GAP, members of the pp60 src family of protein tyrosine kinases, Nck, Grb2, and the phosphotyrosine phosphatase Syp (6 -9). Once bound to the receptor, many of these substrates are themselves phosphorylated on tyrosine residues by the intrinsic receptor kinase ac...

Platelet-derived Growth Factor Stimulation of Monocyte Chemoattractant Protein-1 Gene Expression Is Mediated by Transient Activation of the Phosphoinositide 3-Kinase Signal Transduction Pathway

“…Activity for 10 min is sufficient to stimulate the downstream cascade of events that culminate in MCP-1 stimulation. This temporal window for PI 3-kinase function in MCP-1 gene expression fits very well with previous studies from other workers showing that the association of PI 3-kinase with activated PDGF receptors and the accumulation of in vivo lipid products is transient in nature (39). PI 3-kinase activity appears to be sufficient for MCP-1 mRNA accumulation.…”

“…Only basal levels of activity could be observed at the 2 h time point (data not shown). This is in good agreement with the results of Domin et al (39) for the examination of PI 3-kinase activity following growth factor treatment.…”

Class II Phosphoinositide 3-Kinases Are Downstream Targets of Activated Polypeptide Growth Factor Receptors

“…1). PI3K-C2␤ accumulated rapidly with a time course similar to that previously reported for class I PI3K enzyme heterodimers (13). Similar kinetics were observed with HEK293 and Cos 7 cells (Fig.…”

Kinetic Analysis of Platelet-derived Growth Factor Receptor/Phosphoinositide 3-Kinase/Akt Signaling in Fibroblasts

“…This finding is consistent with previous studies, in which p110␣ but not p110␤ was required for PDGF-stimulated cell responses, whereas the isoform specificity was reversed for insulin stimulation in the same cells (62,63). It was also previously established that PDGF stimulation significantly increases the PI 3-kinase activity of the p110␣ isoform in fibroblasts (64). The extreme differences predicted in both the recruitment and activation of the two PI 3-kinase isoforms are consistent with differential regulation of p110␣ and p110␤, perhaps through differences in their interactions with p85 subunit isoforms and/or Ras-GTP (61).…”