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-Subspecies of Protein Kinase C in Living Cells Using
Fusion Proteins with Green Fluorescent Protein
* Laboratory of Molecular Pharmacology, Biosignal Research Center, We expressed the Department of Biology, Faculty of Science, Kobe
University, Kobe 657, Japan
-subspecies of protein kinase C (-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. -PKC-GFP fusion protein had enzymological properties very similar to that of native -PKC. The fluorescence of -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
-phorbol
12, 13-didecanoate) induced a significant translocation
of -PKC-GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more
rapid translocation of -PKC-GFP than TPA. The
A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA-
induced translocation of -PKC-GFP was unidirected,
while Ca2+ ionophore-induced translocation was reversible; that is, -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 -PKC in
translocation, we expressed mutant -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
-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 -PKC-GFP. In
NMDA receptor-transfected COS-7 cells, application
of NMDA plus glycine also translocated -PKC-GFP.
Furthermore, rapid translocation and sequential retranslocation of -PKC-GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither
cytochalasin D nor colchicine affected the translocation
of -PKC-GFP, indicating that translocation of -PKC
was independent of actin and microtubule. -PKC-GFP
fusion protein is a useful tool for investigating the molecular mechanism of -PKC translocation and the role
of -PKC in the central nervous system.
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