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) 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 (4alpha-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.
Sakai
N
N
Laboratory of Molecular Pharmacology, Biosignal Research Center, Faculty of Science, Kobe University, Kobe 657, Japan.
Sasaki
K
K
Ikegaki
N
N
Shirai
Y
Y
Ono
Y
Y
Saito
N
N
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D015638:Cytochalasin D / Q000494:pharmacology; D005910:Glioma; D049452:Green Fluorescent Proteins; D006822:Hybrid Cells; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D011493:Protein Kinase C / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D007527:Isoenzymes; D008164:Luminescent Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D000001:Calcimycin; C087857:protein kinase C gamma; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium