The role of protein kinase C isoenzymes in the regulation of calcineurin activity in human peripheral blood mononuclear cells

It is known that PMA (phorbol-12-myristate-13-acetate) can activate the classical and novel protein kinase C isoenzymes (cPKC α, β, γ and nPKC δ, ε, η, ), while the calcium ion can induce only the activity of cPKC. Calcineurin binding protein (Cabin 1) belongs to the group of endogenous inhibitors of calcineurin. Cabin 1 becomes hyperphosphorylated in response to PKC activation and may play a negative role in calcineurin signalling. It was observed that both PMA treatment and the increase in intracellular Ca2+ contributed to the reduction of calcineurin activity in human peripheral blood mononuclear cells without modulating the mRNA and the protein levels of calcineurin. PMA and Ca-ionophore (A23187), the activating agents of PKC, applied alone or in combination, significantly increased the phosphorylation state of Cabin 1 as revealed by immunoprecipitation of Cabin 1 detecting its phospho-Ser content by specific antibodies. GF109203X, an inhibitor of the classic and the novel protein kinase C isoenzymes, and Gö6976, the selective inhibitor of the classical cPKC isoenzymes were able to abolish the effect of PMA or/and Ca-ionophore on the calcineurin activity with concomitant reversal of the hyperphosphorylation of Cabin 1. The calcineurin/Cabin 1 system was not influenced by Rottlerin, an inhibitor of PKC δ isoenzyme either in the absence or in the presence of Ca-ionophore and PMA. We presented evidence for the prominent role of cPKC α, β, γ isoenzymes in the inhibition of calcineurin as induced by PMA and Ca-ionophore. We demonstrated also that hyperphosphorylation of Cabin 1 by PMA/Ca2+-activated cPKC isoenzymes resulted in a simultaneous inhibition of calcineurin in peripheral blood mononuclear cells. These results suggest a negative regulatory role for Cabin 1 in calcineurin signalling and provide a possible mechanism of feedback inhibition through cross-talk between PKC and calcineurin.