The control of phosphorylation of CK2 target sites has long been a matter of controversy. We have demonstrated that phosphorylation of the ribosomal P protein CK2 sites is reliably measurable by 2D western blotting of whole cell lysates. Physiologically, phosphorylation of the CK2 sites of the P proteins is necessary for the elongation phase of protein translation, which can be used as an indirect parameter of P protein function. Based on previous data showing that crosslinking of CD95 and hyperthermia lead to the dephosphorylation of the ribosomal P protein CK2 phosphorylation sites and decreased protein synthesis, which was associated with the induction of apoptosis in Jurkat cells, we examined whether similar mechanisms are initiated when apoptosis is induced by inhibition of the PKC pathway. In Jurkat cells and freshly isolated peripheral blood T cells, rapid dephosphorylation of the ribosomal P proteins P0, P1 and P2 was induced by low concentrations of chelerythrine, a specific inhibitor of PKC. Neither of the specific PKC activators thymeleatoxin or PMA were able to prevent the dephosphorylation. Inhibition of intracellular Ca2+ release by TMB-8 also induced dephosphorylation of the P proteins, which is compatible with the requirement of intracellular Ca2+ for classical PKC isozyme activity. Chelerythrine also induced apoptosis in Jurkat cells, which was prevented by zVAD-fmk and ZnCL2, though these agents did not inhibit the dephosphorylation of the P proteins. The effects of chelerythrine were not due to altered CK2 activity, and there was no evidence that the cAMP-dependent PKA, ornithine decarboxylase, or protein phosphatase 2A pathways were involved in signaling leading to P protein dephosphorylation. The dephosphorylation of the P proteins was accompanied by markedly reduced whole cell protein synthesis, which, in parallel with dephosphorylation of the P proteins, was not affected by zVAD-fmk or ZnCl2. Freshly isolated peripheral blood T cells showed the same pattern of responses as Jurkat cells, with the exception that chelerythrine did not induce apoptosis in resting T cells.
Our results demonstrate that inhibition of Ca2+ dependent PKC activity decreases the phosphorylation of the P protein CK2 sites and protein synthesis. The failure of caspase inhibition to prevent the dephosphorylation and decreased protein synthesis due to PKC inhibition indicates early divergence of PKC and caspase-dependent signaling in T cells and Jurkat cells.