The catalytic domain limits the translocation of protein kinase Cα in response to increases in Ca2+ and diacylglycerol

Translocation of protein kinase C (PKC) alpha, betaII, delta and epsilon fused to enhanced green fluorescent protein (EGFP) was studied in living neuroblastoma cells by confocal microscopy. Exposure to carbachol elicited transient translocation of PKCalpha-EGFP and betaII-EGFP in most of the cells, PKCdelta-EGFP in a few cells and induced sustained translocation of PKCepsilon-EGFP. To monitor levels of Ca2+ and diacylglycerol and the translocation of PKC in the same cell, the Ca2+-sensitive C2 domain, diacylglycerol-sensitive C1 domains and full-length PKC were fused to red, cyan and yellow fluorescent proteins respectively. PKCalpha was translocated a few seconds after the C2 domain, which represents an increase in Ca2+. This delay was insensitive to removal of the pseudosubstrate in PKCalpha, but the isolated regulatory domain translocated simultaneously with the C2 domain. Translocation of PKCepsilon coincided with the increase in diacylglycerol. Ionomycin induced translocation of PKCalpha and the C2 domain, whereas 1,2-dioctanoylglycerol caused translocation of the C1 domains and PKCepsilon, but not PKCalpha. Experiments with individual C1 domains showed that treatment with carbachol or phorbol 12,13-dibutyrate elicited translocation of PKCalphaCla, PKCepsilonCla and PKCepsilonClb, whereas PKCalphaClb was largely insensitive to these agents. In contrast with full-length PKCalpha, the regulatory domain of PKCalpha and pseudosubstrate-devoid PKCalpha responded to the carbachol-stimulated increase in diacylglycerol.