Translocation of protein kinase C (PKC) α, βII, Δ and ∊ fused to enhanced green fluorescent protein (EGFP) was studied in living neuroblastoma cells by confocal microscopy. Exposure to carbachol elicited transient translocation of PKCα-EGFP and βII-EGFP in most of the cells, PKCΔ-EGFP in a few cells and induced sustained translocation of PKC∊-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. PKCα 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 PKCα, but the isolated regulatory domain translocated simultaneously with the C2 domain. Translocation of PKC∊ coincided with the increase in diacylglycerol. Ionomycin induced translocation of PKCα and the C2 domain, whereas 1,2-dioctanoylglycerol caused translocation of the C1 domains and PKC∊, but not PKCα. Experiments with individual C1 domains showed that treatment with carbachol or phorbol 12,13-dibutyrate elicited translocation of PKCαC1a, PKC∊C1a and PKC∊C1b, whereas PKCαC1b was largely insensitive to these agents. In contrast with full-length PKCα, the regulatory domain of PKCα and pseudosubstrate-devoid PKCα responded to the carbachol-stimulated increase in diacylglycerol.
Abbreviations used: BAPTA, bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid; BAPTA/AM, BAPTA tetrakis(acetoxymethyl ester); DAG, 1,2-diacylglycerol; DOG, 1,2-dioctanoylglycerol; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced GFP; EYFP, enhanced yellow fluorescent protein; GFP, green fluorescent protein; PDBu, phorbol 12,13-dibutyrate; PKC, protein kinase C; RD, regulatory domain.