Protein Kinase C Regulates FADD Recruitment and Death-inducing Signaling Complex Formation in Fas/CD95-induced Apoptosis

Mireia Gómez-Angelats,John A Cidlowski

doi:10.1074/jbc.m104919200

Abstract

Activation of protein kinase C (PKC) triggers cellular signals that inhibit Fas/CD95-induced cell death in Jurkat T-cells by poorly defined mechanisms. Previously, we have shown that one effect of PKC on Fas/CD95-dependent cell death occurs through inhibition of cell shrinkage and K(+) efflux (Gómez-Angelats, M., Bortner, C. D., and Cidlowski, J. A. (2000) J. Biol. Chem. 275, 19609-19619). Here we report that PKC alters Fas/CD95 signaling from the plasma membrane to the activation of caspases by exerting a profound action on survival/cell death decisions. Specific activation of PKC with 12-O-tetradecanoylphorbol-13-acetate or bryostatin-1 induced translocation of PKC from the cytosol to the membrane and effectively inhibited cell shrinkage and cell death triggered by anti-Fas antibody in Jurkat cells. In contrast, inhibition of classical PKC isotypes with Gö6976 exacerbated the effect of Fas activation on both apoptotic volume decrease and cell death. PKC activation/inhibition did not affect anti-Fas antibody binding to the cell surface, intracellular levels of FADD (Fas-associated protein with death domain), or c-FLIP (cellular FLICE-like inhibitory protein) expression. However, processing/activation of both caspase-8 and caspase-3 and BID cleavage were markedly blocked upon PKC activation and, conversely, were augmented during PKC inhibition, suggesting a role for PKC upstream of caspase-8 processing and activation. Analysis of death-inducing signaling complex (DISC) formation was carried out to examine the influence of PKC on recruitment of both FADD and procaspase-8 to the Fas receptor. PKC activation blocked FADD recruitment and caspase-8 activation and thus DISC formation in both type I and II cells. In contrast, inhibition of classical PKCs promoted the opposite effect on the Fas pathway by rapidly increasing FADD recruitment, caspase-8 activation, and DISC formation. Together, these data show that PKC finely modulates Fas/CD95 signaling by altering the efficiency of DISC formation.

Highlights

There is a massive removal of CD4ϩ helper T-cells
Given that Go6976 has been reported to be highly selective for the protein kinase C (PKC)␣ and PKC␤ isotypes [37], these results suggest that inhibition of the ␣ and/or ␤ isotype may likely be involved in altering the effectiveness of anti-Fas antibody in inducing cell shrinkage in Jurkat cells
We show for the first time that activation of PKC leads to inhibition of Fas-induced apoptosis in Jurkat T-cells by controlling the most apical intracellular signaling in the Fas pathway: recruitment of FADD to the receptor

Summary

Introduction

There is a massive removal of CD4ϩ helper T-cells. In contrast, defective apoptosis can have detrimental effects on the regulation of the immune response or on the emergence of hyperplasia among immune system cell lineages [1]. Resistance to apoptosis arises from constitutive or up-regulated expression of an array of proteins and signaling molecules that counteract death signals initiated by activation of the Fas/CD95 receptor. FLIP contains two N-terminal death effector domain motifs and has been found to be expressed as two different spliced variants designated as short and long FLIP (FLIPS and FLIPL, respectively) [25, 26] Both molecules are structurally homologous, except that the long form of FLIP has a proteolytic caspase-8-like domain that lacks the conserved cysteine residue present in all caspases and that is required for catalytic protease activity [30]. A number of studies support the notion that both forms of FLIP can prevent Fas/CD95-mediated apoptosis by interacting with either FADD and/or procaspase-8 [25, 26]

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Conclusion