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© The Rockefeller University Press, 0021-9525/1997//1465 $5.00
The Journal of Cell Biology, Volume 139, Number 6, , 1997 1465-1476

Article

Direct Visualization of the Translocation of the {gamma}-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein



Norio Sakai*, Keiko Sasaki*, Natsu Ikegaki*, Yasuhito Shirai*, Yoshitaka Ono{ddagger}, and Naoaki Saito*

* Laboratory of Molecular Pharmacology, Biosignal Research Center, {ddagger} Department of Biology, Faculty of Science, Kobe University, Kobe 657, Japan

We expressed the {gamma}-subspecies of protein kinase C ({gamma}-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal 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 cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4{alpha}-phorbol 12, 13-didecanoate) induced a significant translocation of {gamma}-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of {gamma}-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of {gamma}-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, {gamma}-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of {gamma}-PKC in translocation, we expressed mutant {gamma}-PKC–GFP fusion protein in which the two cysteine rich regions 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+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of {gamma}-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-D-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of {gamma}-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated {gamma}-PKC–GFP. Furthermore, rapid 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 actin 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.

Abbreviations used in this paper: 4{alpha}-PDD, 4{alpha}-phorbol 12, 13-didecanoate; DG, diacylglycerol; DO, diolein; GFP, green fluorescent protein; NMDA, N-methyl-D-aspartate; MCPG, {alpha}-methyl-4-carboxyphenyl-glycine; mGluR1, metabotropic glutamate receptor 1; PKC, protein kinase C; PS, phosphatidylserine; TPA, 12-O-tetradecanoylphorbol 13-acetate; trans-ACPD, (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid.

Address all correspondence to Naoaki Saito, Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657, Japan. Tel.: 81-78-803-1251. Fax: 81-78-803-0993. E-mail: naosaito{at}inherit.biosig.kobe-u.ac.jp


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