Abstract
Platelet-derived growth factor (PDGF) stimulates protein kinase D (PKD) in a time- and dose-dependent manner. We have used a series of PDGF receptor mutants that display a selective impairment of the binding of SH2-containing proteins (GTPase-activating protein, SHP-2, phospholipase Cgamma (PLCgamma), or phosphatidylinositol 3'-kinase (PI3K)) to show that Tyr-1021, the PLCgamma-binding site, is essential for PKD stimulation by PDGF in A431 cells. We next investigated whether any one of these four binding sites could mediate PKD activation in the absence of the other three sites. F5, a receptor mutant that lacks all four binding sites for GTPase-activating protein, PLCgamma, PI3K, and SHP-2, fails to activate PKD. A panel of single add-back mutants was used to investigate if any one of these four sites could restore signaling to PKD. Of the four sites, only the PLCgamma+ single add-back receptor restored PDGF-mediated activation of PKD, and only this add-back receptor produced diacylglycerol (DAG) in a PDGF-dependent manner. 1,2-Dioctanoyl-sn-glycerol, a membrane-permeant DAG analog, was found to be sufficient for activation of PKD. Taken together, these data indicate that PLCgamma activation is not only necessary, but also sufficient to mediate PDGF-induced PKD activation. Although the presence of a pleckstrin homology domain makes PKD a potential PI3K target, PKD was not stimulated by selective PI3K activation, and wortmannin, an inhibitor of PI3K, did not inhibit PDGF signaling to PKD. The activation of PKD by DAG or by the wild-type and PLCgamma+ add-back PDGF receptors was inhibited by GF109203X, suggesting a role for protein kinase C in the stimulation of PKD by PDGF. PDGF induced a time-dependent phosphorylation of PKD that closely correlated with activation. The PDGF-induced activation and phosphorylation of PKD were reversed by in vitro incubation of PKD with protein phosphatase 1 or 2A, indicating that PDGF signaling to PKD involves the Ser/Thr phosphorylation of PKD. Taken together, these results conclusively show that PDGF activates PKD through a pathway that involves activation of PLCgamma and, subsequently, protein kinase C.
Highlights
The production of lipid second messengers is a common theme in the signal transduction of growth factors [1,2,3,4,5]
This work is the first report of a dissection of specific growth factor signaling pathways that activate protein kinase D (PKD)
PKD is stimulated by Platelet-derived growth factor (PDGF) doses as low as 5 ng/ml, with a maximum at 30 ng/ml (Fig. 1, A and B), which correlates well with the concentration of PDGF required for a variety of cellular responses such as PLC␥, phosphatidylinositol 3Ј-kinase (PI3K), and GTPase-activating protein (GAP) tyrosine phosphorylation [28]
Summary
Materials—PDGF was purchased from Upstate Biotechnology, Inc. GF109203X was obtained from Calbiochem, and G418, Lipofectin, Glutamax, and Opti-MEM from were from Life Technologies, Inc. Plasmids containing -PDGFR mutants (in the pLXSN vector, carrying a neomycin resistance gene) were introduced using Lipofectin into an NIH3T3 packaging cell line (⌿2). Protein Phosphatase Incubations—PKD eluates were incubated for 30 min at 30 °C with 50 units/ml PP1C or PP2AC in the presence or absence of 1 M microcystin After this incubation, a PKD kinase assay was performed as indicated above in the presence of 1 M microcystin. Immunoprecipitated PKD was eluted from the immunocomplexes and incubated for 30 min at 30 °C with 50 units/ml PP1C or PP2AC in the presence or absence of 1 M microcystin
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