Antagonistic effects of protein kinase C alpha and delta on both transformation and phospholipase D activity mediated by the epidermal growth factor receptor - PubMed (original) (raw)

Antagonistic effects of protein kinase C alpha and delta on both transformation and phospholipase D activity mediated by the epidermal growth factor receptor

A Hornia et al. Mol Cell Biol. 1999 Nov.

Abstract

Downregulation of protein kinase C delta (PKC delta) 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 delta- and PKC alpha-specific inhibitors and the expression of dominant negative mutants for both PKC delta and alpha. Consistent with a tumor-suppressing function for PKC delta, the PKC delta-specific inhibitor rottlerin and a dominant negative PKC delta mutant transformed the EGFR cells in the absence of EGF. In contrast, the PKC alpha-specific inhibitor Go6976 and expression of a dominant negative PKC alpha mutant blocked the transformed phenotype induced by both EGF and PKC delta 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 delta, like transformation, was dependent upon PKC alpha and restricted to the EGFR cells. These data demonstrate that PKC isoforms alpha and delta have antagonistic effects on both transformation and PLD activity and further support a tumor suppressor role for PKC delta that may be mediated by suppression of tyrosine kinase-dependent increases in PLD activity.

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Figures

FIG. 1

Establishment of 3Y1 EGFR cells. 3Y1 cells were transfected with pPEGFr, which expresses the EGF receptor from the simian virus 40 promoter and contains a puromycin resistance marker (5). Several puromycin-resistent colonies were picked and analyzed for levels of expression of the EGF receptor. Western blot analysis was performed on lysates from the parental 3Y1 cells (3Y1) and the puromycin-resistant colonies with an antibody raised against the EGF receptor.

FIG. 2

EGFR cells display a transformed phenotype upon treatment with EGF but not TPA. (a) Parental 3Y1 or EGFR cells were either left untreated or treated with TPA (400 nM) or EGF (100 ng/ml) for 24 h, at which time the morphology of the cells was examined. (b) Anchorage-independent growth of the EGFR cells (clone 2) was examined in the presence or absence of either TPA (400 nM) or EGF (100 ng/ml) as shown. TPA and EGF were replenished every 4 days. A total of 103 cells were suspended in soft agar, and the percentage of cells that formed colonies was determined 3 weeks later. (c) Anchorage-independent growth of 3Y1 cells and 3Y1 EGFR cells (clones 2, 3, and 4) was examined in the presence of EGF (100 ng/ml) as described for panel b.

FIG. 3

Effects of PKC α- and δ-specific inhibitors on EGFR cells. EGFR cells were treated with EGF (100 ng/ml) and either Go6976 (0.5 μM) or rottlerin (15 μM) and then examined for the ability to form colonies in soft agar as described for Fig. 2b. (c) The EGFR cells were treated with EGF (100 ng/ml), rottlerin (15 μM), Go6976 (0.5 μM), or TPA (400 nM) as shown, and the morphology of the cells was examined 24 h later as for Fig. 2a. (d) The ability of rottlerin to stimulate colony formation in EGFR clones 2, 3, and 4 was determined as described for panel a.

FIG. 3

FIG. 4

Effects of dominant negative (DN) mutants of PKC α and δ on the ability of EGFR cells to form colonies in soft agar. (a) EGFR cells were cotransfected with plasmids expressing either a dominant negative PKC α or δ mutant and pCEF4 (Invitrogen), which expresses a hygromycin resistance marker gene. One clone expressing the PKC α mutant and two clones expressing different levels of the PKC δ mutant were analyzed for expression of the mutants by Western blot analysis before and after treatment with TPA (400 nM, 24 h). TPA treatment downregulates the endogenous wild-type PKC α and δ, but not the kinase-dead mutants (24). (b) The ability of the EGFR cells and the EGFR cell lines expressing the dominant negative PKC α and δ mutants to form colonies in soft agar was determined as for Fig. 3 in the presence and absence of EGF (100 ng/ml) as shown.

FIG. 4

FIG. 5

Downregulation of PKC δ elevates PLD activity in EGFR cells. (a) Parental 3Y1 and EGFR cells were treated with EGF (100 ng/ml, 5 min) and/or TPA (400 nM, 24 h) in the presence of 1% butanol, and PLD activity was determined by examining the levels of the PLD-generated transphosphatidylation product phosphatidylbutanol as described in Materials and Methods. The relative PLD activity was normalized to the PLD activity in the untreated 3Y1 cells. Error bars represent the standard deviations for two independent experiments performed in duplicate. (b) The effects of the PKC α- and δ-specific inhibitors Go6976 (0.5 μM) and rottlerin (15 μM) on EGF-induced PLD activity were determined as for panel a. The relative PLD activity was normalized to the PLD activity in the untreated EGFR cells. (c) EGFR cells expressing dominant negative (DN) mutants of PKC α and δ were examined for the effect on PLD activity in the presence and absence of EGF as shown. The relative PLD activity was normalized to the PLD activity in the untreated EGFR cells. Error bars represent the standard deviations for two independent experiments performed in duplicate, where duplicates varied by less than 10%.

FIG. 5

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Antagonistic effects of protein kinase C alpha and delta on both transformation and phospholipase D activity mediated by the epidermal growth factor receptor - PubMed (original) (raw)