To further elucidate the role played by protein kinase C-d (PKC-d) in PDGF-mediated signal transduction, an adenosine triphosphate (ATP) binding mutant of PKC-d was generated by converting the invarient lysine to an arginine at amino acid 376. The mutant, PKC-dK376R, did not possess autophosphorylation activity or the ability to transphosphorylate an exogenous substrate when expressed in 32D cells. Unlike 32D cells overexpressing wild type PKC-d (PKC-dWT), 32D cells transfected with PKC- dK376R did not undergo monocytic differentiation in response to ATP stimulation. Moreover, PKC-dK376R competitively inhibited PKC-dWT activity in an in vitro PKC-d activity assay. This last result suggests that the ATP binding mutant might act in a dominant negative manner and block PKC-d-related biological functions. Cotransfection of vectors containing PKC-dK376R and the protooncogene, sis, into NIH/3T3 cells severely impaired sis-induced focus formation; whereas cotransfection of PKC-dWT or vector alone with sis had no effect on sis-mediated transformation. Tumor development by PKC-dK376R/sis cotransfectants was delayed by 10 days when compared to those induced by PKC-dWT/sis or vector/sis cotransfectants. PKC-dK376R expression also inhibited PKGF-BB mediated anchorage-independent colony formation. Expression of PKC- dK376R did not inhibit PDGF-bR autophosphorylation, but severely inhibited PDGF-induced early response gene activation. The results clearly indicate that PKC-d lies downstream of PDGF-bR activation and blockage of PKC-d activation severely inhibits PDGF-mediated cellular transformation.
