DIRECT VISUALIZATION OF THE TRANSLOCATION OF THE GAMMA-SUBSPECIES OF PROTEIN-KINASE-C IN LIVING CELLS USING FUSION PROTEINS WITH GREEN FLUORESCENT PROTEIN

We expressed the gamma-subspecies of protein kinase C (gamma-PKC) fuse d with green fluorescent protein (GFP) in various cell lines and obser ved the movement of this fusion protein in living cells under a confoc al laser scanning fluorescent microscope. gamma-PKC-GFP fusion protein had enzymological properties very similar to that of native gamma-PKC . The fluorescence of gamma-PKC-GFP was observed throughout the cytopl asm in transiently transfected COS-7 cells. Stimulation by an active p horbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by a n inactive phorbol ester (4 alpha-phorbol 12, 13-didecanoate) induced a significant translocation of gamma-PKC-GFP from cytoplasm to the pla sma membrane, A23187, a Ca2+ ionophore, induced a more rapid transloca tion of gamma-PKC-GFP than TPA. The A23187-induced translocation was a bolished by elimination of extracellular and intracellular Ca2+., TPA- induced translocation of gamma-PKC-GFP was unidirected, while Ca2+ ion ophore-induced translocation was reversible; that is, gamma-PKC-GFP tr anslocated to the membrane returned to the cytosol and finally accumul ated as patchy dots on the plasma membrane. To investigate the signifi cance of C1 and C2 domains of gamma-PKC in translocation, we expressed mutant gamma-PKC-GFP fusion protein in which the two cysteine rich re gions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ i onophore but not by TPA. In contrast, BS 239 mutant was translocated b y TPA but not by Ca2+ ionophore, To examine the translocation of gamma -PKC-GFP under physiological conditions, we expressed it in NG-108 cel ls, N-methyl-D-aspartate (NMDA) receptor-transfected COS-7 cells, or C HO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cell s). In NG-108 cells,K+ depolarization induced rapid translocation of g amma-PKC-GFP., In NMDA receptor-transfected COS-7 cells, application o f NMDA plus glycine also translocated gamma-PKC-GFP., Furthermore, rap id translocation and sequential retranslocation of gamma-PKC-GFP were observed in CHO/mGluR1 cells on stimulation with the receptor, Neither cytochalasin D nor colchicine affected the translocation of gamma-PKC -GFP, indicating that translocation of gamma-PKC was independent of ac tin and microtubule, gamma-PKC-GFP fusion protein is a useful tool for investigating the molecular mechanism of gamma-PKC translocation and the role of gamma-PKC in the central nervous system.