Protein Kinase C θ and ε Promote T-cell Survival by a Rsk-dependent Phosphorylation and Inactivation of BAD

Abstract

Both MAPK and protein kinase C (PKC) signaling pathways promote cell survival and protect against cell death. Here, we show that 12-O-tetradecanoylphorbol-13-acetate (TPA) prevents Fas-induced apoptosis in T lymphocytes. The effect of TPA was specifically abolished by the PKC inhibitor GF109203X and by dominant negative PKC, PKC⑀, and PKC␣, suggesting that novel and conventional PKC isoforms mediate phorbol ester action. Moreover, TPA stimulated phosphorylation of BAD at serine 112, an effect abrogated by GF109203X but not by the MEK inhibitor PD98059. Expression of constitutively active PKC increased the phosphorylation of BAD at serine 112 but not at serine 136. Additionally, Fas-mediated cell death was enhanced by overexpression of a catalytically inactive form of p90Rsk (Rsk2-KN). Finally, Rsk2-KN abolished the protective effect of constitutively active PKC and totally blocked phosphorylation of BAD on serine 112. Thus, novel PKC and PKC⑀ rescue T lymphocytes from Fas-mediated apoptosis via a p90Rsk-dependent phosphorylation and inactivation of BAD.In several cell lines, apoptosis is antagonized by growth factors and hormones and, more generally, by stimuli that promote cell survival. Interleukin 3 and insulin-like growth factor 1 exert their antiapoptotic effect through activation of phosphatidylinositol 3-kinase, which, in turn, leads to activation of the serine/threonine protein kinase B (PKB/Akt), which promotes cell survival by phosphorylating BAD at Ser 136 (1-6). Interestingly, interleukin 3, through activation of a mitochondrial membrane based protein kinase A, also stimulates phosphorylation of BAD at Ser 112 (6, 7 (8,9). Brain-derived neurotrophic factor exerts its antiapoptotic effect in a mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase pathway (8). The mechanism of action of TPA remains unclear, although MAPKdependent (10 -13) and -independent pathways have been described (9). TPA is a tumor promoter that binds and activates members of a family of serine/threonine protein kinases termed protein kinase C (PKC). PKC comprised at least 12 isotypes that have been classified into three groups according to their structure and cofactor requirement: (a) conventional PKCs (PKC␣, PKC␤I, PKC␤II, and PKC␥) are diacylglycerol-and calcium-dependent, (b) novel PKCs (PKC␦, PKC⑀, PKC, PKC, and PKC) are diacylglycerol-dependent but calciumindependent, and (c) atypical PKCs (PKC, PKC, and PKC) are not activated by phorbol esters but can bind diacylglycerol (14, 15). Overexpression of PKC⑀, PKC␣, or PKC increases the resistance of cells to apoptosis, and PKC inhibitors are known to sensitize cells to apoptosis (16 -20). Additionally, Fas ligation-induced apoptosis in Jurkat T cells resulted in a blockade of cellular PKC activity, suggesting a link between the two events (21). Although involvement of PKC in the suppression of apoptosis has been demonstrated recently, the mechanisms by which PKC promotes cell survival remain to be elucidated. EXPERIMENTAL PROCEDURESReporter Plasmids, Tr...

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis

“…Surprisingly, higher doses of SB202190, which drastically increased JNK activity, were nevertheless found to inhibit megakaryocytic differentiation of K562 and to induce cell death more especially in the presence of PMA. The effect of the combination of PMA and high doses of SB may appear somewhat surprising since PMA has been consistently shown to protect various cell lines against apoptosis (Bertolotto et al, 2000;Villalba et al, 2001;Herrant et al, 2002). However, we have previously established that inhibition of the NFkB pathway, for example, can convert PMA from a death-protecting to a death-inducing function (Busuttil et al, 2002).…”

Protein kinase Cε makes the life and death decision

“…Overexpression of constitutively active PKCε increased phosphorylation of Bad at Ser112 [130,136] but not Ser136 and inhibited CH11 (a FAS ligand mimic)-induced apoptosis in Jurkat T cells [130]. An interaction between PKCε and Bax was identified in CWR22 prostate tumor xenografts.…”

“…Data supporting isoform-specific regulation of Bcl-2 family members are sparse, but quite consistent in that antiapoptotic PKCε enhances antiapoptotic Bcl-2 members [34,129] while inhibiting the proapoptotic members of the family [31,34,130,131]. Suzuki et al first reported that intense expression of Bcl-2 during the development of pregnancy-dependent mammary tumors (PDMTs) to malignant tumors was associated with overexpression of PKCε but not of other isozymes, such as PKCα, -δ, -η, -ζ or -λ [132].…”

Alterations of intracellular pH homeostasis in apoptosis: origins and roles

“…A clue may come from the existence of the 90 kDa Na þ /H þ exchanger 1 (NHE1) kinase (also known as p90 RSK ) that has been suggested to represent a common NHE1 regulator for diverse signaling pathways (reviewed by Karmazyn et al 12 ) and whose activation is blocked by tyrosine kinase inhibitors. 16 In this regard, it is worth noting that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) has been recently reported to rescue T lymphocytes from Fas-mediated apoptosis via activation of p90 RSK , resulting in phosphorylation and inactivation of the proapoptotic protein BAD; 17 moreover, TPA has long been known to induce intracellular alkalinization via NHE activation in various cells (reviewed by Putney et al 11 and Karmazyn et al 12 ). In this context, one might then speculate that p90 RSK would be inactivated upon Fas ligation, possibly due to an SHP-1-dependent dephosphorylation step, leading to downregulation of NHE1 and hence development of acidification and apoptosis.…”