Synergistic action of protein kinase Cθ and calcineurin is sufficient for Fas ligand expression and induction of a crmA-sensitive apoptosis pathway in Jurkat T cells

Abstract

Deletion of activated peripheral T cell clones by apoptosis requires the regulated expression of Fas ligand (FasL) and sensitization of these cells to CD95-mediated signaling. To investigate the signaling pathways responsible for FasL expression in T cells, we tested - besides subfamily-selective protein kinase C (PKC) inhibitors - the effect of constitutively active mutants of representatives of all PKC subfamilies, i.e. PKCα,ε,θ,ι, on FasL luciferase promoter reporter constructs, in synergy with a constitutively active form of protein phosphatase 2B calcineurin (CaN), only PKCθ, but not PKCα,ε,ι, preferentially induced FasL promoter reporter activity and, consequently, FasL protein expression in Jurkat T cells. Activation of an inducible PKCθ AE-estrogen receptor fusion mutant led to a CaN-dependent and rapid FasL reporter activity detected as early as 4 h after addition of 4-hydroxytamoxifen, incidating a direct effect of PKCθ action on FasL expression. Consistently, in Jurkat T cells, expression of PKCθ AE/CaN significantly enhanced FasL protein expression and apoptosis in a CD95-dependent manner since cell death was not observed in T cells co-expressing the caspase-8 inhibitor crmA. Taken together, our results support the notion that PKCθ and CaN are sufficient to regulate apoptosis through FasL expression.