Abstract
Protein kinase Cepsilon (PKCepsilon), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCdelta-mediated release of death factors, the involvement of PKCepsilon in this response is not known. PKCepsilon depletion by RNAi or expression of a dominant negative kinase-dead PKCepsilon mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFalpha. On the other hand, overexpression of PKCepsilon by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCepsilon RNAi depletion significantly enhanced the release of TNFalpha in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser(112) in PKCepsilon-depleted LNCaP cells, whereas PKCepsilon overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser(112) in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCepsilon accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCepsilon in vitro. Our results established that PKCepsilon modulates survival in prostate cancer cells via multiple pathways.
Highlights
We have recently demonstrated that PKC␦-mediated prostate cancer cell death involves the activation of an apoptotic autocrine loop through the release of death factors, and the subsequent activation of the extrinsic apoptotic cascade and the JNK pathways [20, 25, 26]
In the present study we used a series of gain- and loss-of-function approaches to demonstrate that PKC⑀ is implicated in survival signaling in prostate cancer cells by modulating the secretion of TNF␣ and by acting as an effector of the death factor response
We identified a major role for PKC⑀ in the control of Bad phosphorylation, pointing to multiple mechanisms implicated in the prosurvival response of PKC⑀ in prostate cancer cells
Summary
Materials—PMA was purchased from LC Laboratories (Woburn, MA). DAPI (4Ј,6-diamidino-2-phenylindole) was AUGUST 20, 2010 VOLUME 285 NUMBER 34. Of siRNAs into C4 and DU145 cells was achieved with Oligofectamine (Invitrogen), following the manufacturer’s protocol [27]. The targeting sequences were as follows: PKC⑀. Experiments were carried out 48 h after transfection. DAPI staining, as previously described [20, 26, 27]
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