Antagonistic Effects of Protein Kinase C α and δ on Both Transformation and Phospholipase D Activity Mediated by the Epidermal Growth Factor Receptor

Abstract

Downregulation of protein kinase C δ (PKC δ) by treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) transforms cells that overexpress the non-receptor class tyrosine kinase c-Src (Z. Lu et al., Mol. Cell. Biol. 17:3418–3428, 1997). We extended these studies to cells overexpressing a receptor class tyrosine kinase, the epidermal growth factor (EGF) receptor (EGFR cells); like c-Src, the EGF receptor is overexpressed in several human tumors. In contrast with expectations, downregulation of PKC isoforms with TPA did not transform the EGFR cells; however, treatment with EGF did transform these cells. Since TPA downregulates all phorbol ester-responsive PKC isoforms, we examined the effects of PKC δ- and PKC α-specific inhibitors and the expression of dominant negative mutants for both PKC δ and α. Consistent with a tumor-suppressing function for PKC δ, the PKC δ-specific inhibitor rottlerin and a dominant negative PKC δ mutant transformed the EGFR cells in the absence of EGF. In contrast, the PKC α-specific inhibitor Go6976 and expression of a dominant negative PKC α mutant blocked the transformed phenotype induced by both EGF and PKC δ inhibition. Interestingly, both rottlerin and EGF induced substantial increases in phospholipase D (PLD) activity, which is commonly elevated in response to mitogenic stimuli. The elevation of PLD activity in response to inhibiting PKC δ, like transformation, was dependent upon PKC α and restricted to the EGFR cells. These data demonstrate that PKC isoforms α and δ have antagonistic effects on both transformation and PLD activity and further support a tumor suppressor role for PKC δ that may be mediated by suppression of tyrosine kinase-dependent increases in PLD activity.

ACKNOWLEDGMENTS

A.H. and Z.L. contributed equally to this work.

We thank Sergey Beychenok and Henghe Tian for comments on the manuscript. We thank Renato Baserga and Stuart Decker for providing EGF receptor expression plasmids and Shigeo Ohno for providing the vectors expressing the PKC α and PKC δ kinase-dead mutants.

This investigation was supported by grants from the National Institutes of Health (CA46677) and from the American Cancer Society (BE-243) (to D.A.F.) and by a Research Centers in Minority Institutions (RCMI) award from the Division of Research Resources, National Institutes of Health (RR-03037), to Hunter College.