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Abstract
In the present study, we have identified several proteins in Swiss 3T3 cells that are phosphorylated on tyrosine in response to platelet-derived growth factor (PDGF) and exhibit an unusual bell-shaped dose-response curve with a maximum at 5 ng/ml platelet-derived growth factor (PDGF). These proteins include two that are associated with focal adhesions, namely the focal adhesion kinase (p125FAK), a novel cytosolic tyrosine kinase, and paxillin. At low concentrations of PDGF (1-5 ng/ml), these proteins are the predominant tyrosine-phosphorylated species. At 30 ng/ml PDGF, however, there was no stimulation of their phosphorylation over control levels. In contrast, tyrosine phosphorylation of previously described substrates of the PDGF receptor tyrosine kinase, namely the p21ras GTPase-activating protein, p120, phosphatidyl inositol 3' kinase, and phospholipase C gamma exhibited sigmoidal dose-response curves with PDGF and were all efficiently phosphorylated on tyrosine at 30 ng/ml PDGF. Cytochalasin D, which disrupts the actin cytoskeleton, completely inhibited the tyrosine phosphorylation of p125FAK and paxillin by PDGF. Examination of the actin cytoskeleton after stimulation of cells with different concentrations of PDGF revealed that at 5 ng/ml PDGF, actin appears in stress fibers and in membrane ruffles, while at 30 ng/ml, PDGF disrupts the actin cytoskeleton. Bombesin stimulates actin stress fiber formation with no evidence of disruption of stress fibers at high concentrations. When cells were stimulated with bombesin (10 nM) in the presence of 30 ng/ml PDGF, however, the actin cytoskeleton was completely disrupted. Further, the tyrosine phosphorylation of both p125FAK and paxillin induced by bombesin (10 nM) was completely prevented when cells were stimulated with bombesin in the presence of 30 ng/ml PDGF. We propose that the inhibitory limb in the bell-shaped dose-response curve of PDGF and the novel cross-talk between PDGF and bombesin on tyrosine phosphorylation may be explained by the ability of PDGF at 30 ng/ml to disrupt the actin cytoskeleton.
Highlights
Cytochalasin D, which disrupts the actincytoskeleton, completely inhibited the tyrosinephosphorylation of p12SFAKand paxillinby platelet-derived growth factor (PDGF)
When cells were stimu- act as potentgrowth factors through G protein-linkedreceplated with bombesin (10 IIM) in the presence of 30 ng/ tors that do not possess an intrinsic tyrosine kinase activity ml PDGF, the actin cytoskeleton was com- [25, 26]
We propose that theinhibitory limb in thebell- phosphorylated substrates in neuropeptide-stimulated Swiss shaped dose-response curve of PDGF and the novel 3T3 cells [34,35]. ~ 1 2is 5a no~vel~cyt~osolic tyrosine kinase cross-talk between PDGF and bombesin on tyrosine that does not possess an SH2 domain [31, 32]
Summary
Cytochalasin D, which disrupts the actincytoskeleton, completely inhibited the tyrosinephosphorylation of p12SFAKand paxillinby PDGF. Cells cultured and treated as described abovewere (A and C) or 60 min ( B )at 37 “C,with the indicated concentrations immunoprecipitated with anti-Tyr(P) mAb. PI kinase activity in the of PDGFBB,and were lysed.Tyrosine phosphorylation of ~ 1 2wa5s ~ ~ ~
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