Invasive Salmonella induces macrophage apoptosis via the activation of caspase-1 by the bacterial protein SipB. Here we show that infection of macrophages with Salmonella causes the activation and degradation of Raf-1, an important intermediate in macrophage proliferation and activation. Raf-1 degradation is SipB- and caspase-1–dependent, and is prevented by proteasome inhibitors. To study the functional significance of Raf-1 in this process, the c-raf-1 gene was inactivated by Cre-loxP–mediated recombination in vivo. Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis. Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) and nuclear factor κB pathways is normal in Raf-1–deficient macrophages, and mitochondrial fragility is not increased. Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.

Protein kinase C alpha (PKCa) has been implicated in tumor development with high levels of PKCa expression being associated with various malignancies including glioblastomas and tumors of the breast and prostate. To account for its upregulation in these cancers, studies have suggested that PKCa plays a role in promoting cell survival. Here we show by siRNA depletion in U87MG glioma cells that a critical threshold level of PKCa protein expression is essential for their growth in the presence of serum and for their survival following serum deprivation. Derivation of PKCa wt and KO mouse embryo fibroblast cell lines confirms a role for PKCa in protecting cells from apoptosis induced by serum deprivation. Notably, PKCa was found to mediate chemo-protection in these fibroblastic cell lines. In U87MG cells PKCa does not confer chemoprotection though this likely reflects growth arrest associated with its depletion. To determine the requirements for catalytic function, comparison was made between distinct classes of PKC inhibitors. In contrast to loss of PKCa protein, inhibition of PKC kinase activity in glioma cell lines does not significantly inhibit growth or survival. Conversely, inhibition with calphostin C, which targets the regulatory domain of PKC, potently inhibits proliferation and induces apoptosis. Evidence is presented that it is the fully phosphorylated, folded form of PKCa that confers this activity-independent behaviour. These results indicate an essential pro-proliferative and pro-survival role for PKCa in glioma but question the use of ATP competitive inhibitors as therapeutics, either alone, or in combination with chemotoxic agents. ' 2008 Wiley-Liss, Inc. Key words: PKC alpha; glioblastoma; chemotherapeuticsThe protein kinase C (PKC) family of ser/thr protein kinases has been implicated in the regulation of multiple cell processes pertinent to transformation including proliferation, apoptosis, migration and invasion. They also constitute the largest group of targets for the phorbol ester class of tumor promoters. As such there has long been interest in elucidating the various functions of PKC with the expectation that they will represent useful targets in the treatment of malignancies.1-4 The PKC family comprises multiple isoforms, displaying differential regulatory inputs 5,6 and have been associated with malignant transformation. 7-10Regulation of apoptosis by PKCa has been the focus of extensive study with the weight of evidence supporting a pro-survival role in transformed cells. Expression of a PKCa dominant negative mutant triggers an apoptotic response in multiple cell types. 11,12 Anti-sense experiments have also placed PKCa in a pro-survival role in cell culture and in vivo. [13][14][15] In contrast, a recent in vivo study using the Apc Min/1 model for intestinal cancer has revealed a tumour suppressor role for PKCa. 16 In this model, deletion of PKCa is associated with enhanced tumorogenesis indicative of cell context differences.Over-expressed or hyperactive PKC is a distinguishing charact...

Classical and novel protein kinase C (PKC) isoforms are down-regulated as a result of chronic activation by certain tumour promoters and physiological stimuli; however, the mechanisms leading to down-regulation are not fully understood. In the present study, we have studied the PMA ('TPA')-induced degradation of PKCdelta in NIH 3T3 cells under culture conditions where PKCdelta displays cell-cycle-dependent down-regulation. In contrast with previous studies, a hyperphosphorylated form of this PKC isoform, promoted by calyculin A, was rapidly degraded in PMA-treated cells. Similarly, the presence of calyculin A enhanced the down-regulation of PKCdelta observed on G(1)/S-phase progression through the cell cycle. Analysis of phosphorylation-site mutants indicated that the T-loop Thr(505) phosphorylation site was critical for induced degradation.