Stimulation of the Platelet-Derived Growth Factor β Receptor Signaling Pathway Activates Protein Kinase C-δ

Abstract

The murine myeloid progenitor cell line 32D was recently shown to undergo monocytic differentiation when protein kinase C-δ (PKC-δ) was overexpressed and activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) (H. Mischak, J.H. Pierce, J. Goodnight, M.G. Kazanietz, P.M. Blumberg, and J.F. Mushinski, J. Biol. Chem. 268:20110-20115, 1993). Tyrosine phosphorylation of PKC-δ occurred when PKC-δ-transfected 32D cells were stimulated by TPA (W. Li, H. Mischak, J.-C. Yu, L.-M. Wang, J.F. Mushinski, M.A. Heidaran, and J.H. Pierce, J. Biol. Chem. 269:2349-2352, 1994). In order to elucidate the role played by PKC-δ in response to activation of a receptor tyrosine kinase, we transfected platelet-derived growth factor β receptor (PDGF-β R) alone (32D/PDGF-β R) or together with PKC-δ (32D/PDGF-β R/PKC-δ) into 32D cells. NIH 3T3 cells which endogenously express both PDGF-alpha R and PDGF-β R were also transfected with PKC-δ (NIH 3T3/PKC-δ). Like TPA treatment, PDGF-BB stimulation caused striking phosphorylation of PKC-δ in vivo and translocation of some PKC-δ from the cytosol fraction to the membrane fraction in both cell systems. Some of the phosphorylation induced by PDGF-BB treatment was found to be on a tyrosine residue(s). Tyrosine-phosphorylated PKC-δ was observed only for the membrane fraction after stimulation with PDGF-BB or TPA. The enzymatic activity of PKC-δ in the membrane fraction also increased after stimulation with TPA or PDGF, providing a positive correlation between PKC-δ tyrosine phosphorylation and its activation. Overnight treatment of 32D/PDGF-β R/PKC-δ cells with PDGF-BB induced monocytic differentiation as judged by an increase in expression of cell surface macrophage differentiation markers. PDGF-BB had much weaker effects on 32D/PDGF-β R cell differentiation, suggesting that increased PKC-δ expression enhanced monocytic differentiation. These results indicate that PKC-δ is a downstream molecule in the PDGFR signaling pathway and may play a pivotal role in PDGF-β R-mediated cell differentiation.