Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Dec 15;139(6):1465-76.
doi: 10.1083/jcb.139.6.1465.

Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein

N Sakai  1 , K Sasaki, N Ikegaki, Y Shirai, Y Ono, N Saito
Affiliations

Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein

N Sakai et al. J Cell Biol. .

Abstract

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Constructs of γ-PKC–GFP fusion protein and its mutants. BS 186 was a γ-PKC–GFP fusion protein in which γ-PKC (BS 55) and GFP were bound at the COOH terminus of γ-PKC. In the C1 region of γ-PKC, there are two cysteine-rich sequences that interact with DG or phorbol esters, such as TPA. To produce BS 238 (C1 mutant), one cysteine residue in each of the two cysteine-rich regions was substituted with serine by site-directed mutagenesis. The C1 region of BS 238 no longer displayed activity for binding phorbol esters (Ono et al., 1989). The C2 region of γ-PKC, the Ca2+ binding domain, was deleted for the production of BS 239 (C2 deletion) as described in the text.
Figure 2
Figure 2
Enzymological property of γ-PKC and γ-PKC–GFP transiently expressed in COS-7 cells. (A) Immunoblotting analysis by anti–γ-PKC antibody revealed that expressed γ-PKC (BS 55) and γ-PKC–GFP (BS 186) were proteins with molecular sizes of 80 and 110 kD, respectively. Treatment with 5 μM TPA for 90 min increased the amount of both γ-PKC and γ-PKC–GFP associated with the particulate fraction. p, pellet (particulate fraction); s, supernatant (cytosol fraction). (B) Kinase activity of expressed γ-PKC and γ-PKC–GFP in cytosol and particulate fractions. Kinase activities of both γ-PKC and γ-PKC–GFP were translocated from the cytosol to particulate fraction after treatment with 5 μM TPA for 90 min. ppt, pellet (particulate fraction) sup, supernatant (cytosol fraction). (C) Enzymological property of γ-PKC and γ-PKC–GFP immunoprecipitated by anti–γ-PKC antibody. Kinase activities of expressed γ-PKC and γ-PKC–GFP, which were immunoprecipitated by anti–γ-PKC antibody, were measured in the presence or absence of activators of γ-PKC. The kinase activity of γ-PKC–GFP maximized in the presence of PS, DO, and Ca2+, as did that of γ-PKC. The enzymological properties of γ-PKC and γ-PKC–GFP were very similar.
Figure 3
Figure 3
Phorbol ester– induced translocation of γ-PKC–GFP in COS-7 cells. (A) Change in the fluorescence of γ-PKC–GFP expressed in COS-7 cells by 5 μM TPA at room temperature. γ-PKC–GFP fusion protein was observed throughout the cytoplasm in transfected COS-7 cells. Activation of PKC by 5 μM TPA induced the obvious translocation of γ-PKC–GFP fluorescence from cytosol to membrane. Translocation was almost completed within 60 min after the treatment with TPA. The same view was taken before the stimulation under Nomarski interference microscope and shown at the upper left corner. (B) Change in the fluorescence of γ-PKC–GFP expressed in COS-7 cells by 200 nM TPA and 500 nM 4α-PDD at 37°C. Lower concentration of TPA (200 nM) induced the translocation of γ-PKC–GFP from cytosol to membrane when examined at 37°C (upper trace). The translocation occurred more rapidly, and the complete translocation was observed at 10 min after the treatment with TPA. In contrast, 4α-PDD, an inactive phorbol ester, at 500 nM failed to induce the translocation of γ-PKC–GFP even at 37°C. Bars, 10 μm.
Figure 4
Figure 4
Ca2+ ionophore– induced translocation of γ-PKC–GFP in COS-7 cells. (A) Change in the fluorescence of γ-PKC–GFP expressed in COS-7 cells by 80 μM A23187, Ca2+ ionophore. A23187 also translocated the fluorescence of γ-PKC–GFP from cytosol to membrane; however, the time course of the translocation was significantly different from the TPA-induced one. Ca2+ ionophore–induced translocation was rapid and reversible (upper and middle traces). In the lower trace, profiles of the GFP intensity on the same line across the cell were shown. (The measured line is between the arrows in the upper left picture.) The translocation was expressed as the increase in the fluorescence at the fringe of the cell at 30 s and 45 min. Comparing the profile of the GFP intensity, very similar profiles were obtained at 0 and 5 min, and the fading of the fluorescence is negligible. (B) The γ-PKC– GFP translocation by A23187 was not always reversible. The left cell reveals unidirected translocation, while the right cell shows the reversible translocation, as seen in A. Unidirected translocation was more common than reversible translocation. γ-PKC–GFP eventually accumulated as patchy dots on the plasma membrane and in neighboring cytoplasm. Bars, 10 μm.
Figure 5
Figure 5
Thapsigargin- induced translocation of γ-PKC–GFP. 5 μM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+- ATPase, also induced rapid translocation of γ-PKC–GFP. Fluorescence of γ-PKC–GFP accumulated as patchy dots as in A23187-induced translocation. Bar, 10 μm.
Figure 6
Figure 6
Effects of Ca2+ chelators on A23187-induced translocation of γ-PKC–GFP. Pretreatment with 2.5 mM EGTA and 15 μM BAPTA-AM completely blocked A23187 (50 μM)–induced γ-PKC–GFP translocation (upper trace). Treatment with 2.5 mM EGTA and 15 μM BAPTA-AM retranslocated γ-PKC–GFP from membrane to cytosol, even after A23187- induced translocation had occurred (lower trace). Bars, 10 μm.
Figure 7
Figure 7
Comparison of the fluorescence of γ-PKC–GFP with the γ-PKC–like immunoreactivity. Immunostaining of γ-PKC– GFP–transfected COS-7 cells by anti–γ-PKC antibody showed that GFP fluorescence and γ-PKC–like immunoreactivity had very similar localizations, even after the TPA- or A23187-induced translocation was completed. Bar, 10 μm.
Figure 8
Figure 8
Translocation of mutant γ-PKC–GFPs (BS 238 and BS 239) expressed in COS-7 cells. (A) Translocation of BS 238 (C1 mutant γ-PKC–GFP). TPA at 5 μM did not induce the translocation of BS 238, while A23187 at 50 μM did. A23187- induced translocation of BS 238 was insufficient compared with that of BS 186 (control γ-PKC–GFP). (B) Translocation of BS 239 (C2 deletion γ-PKC–GFP). In contrast to BS 238, BS 239 was translocated by 5 μM TPA but not by 50 μM A23187. Bars, 10 μm.
Figure 9
Figure 9
K+ depolarization– induced translocation of γ-PKC–GFP expressed in NG 108-15 cells. Replacing the external solution with a high K+–containing one rapidly induced translocation of γ-PKC–GFP. The fluorescence of γ-PKC–GFP accumulated as patchy dots on the plasma membrane and in neighboring cytoplasm, as in Ca2+ ionophore–induced translocation. Bar, 10 μm.
Figure 10
Figure 10
NMDA-induced translocation of γ-PKC– GFP in COS-7 cells coexpressing NMDA receptors. NMDARζ1 and NMDARε1 subunits were cotransfected with γ-PKC–GFP into COS-7 cells by electroporation. NMDA at 1 mM was applied to cells in the absence of Mg2+ and presence of 10 μM glycine. NMDA induced faint but significant translocation of γ-PKC–GFP. Simultaneous application of 100 μM AP-5 with 1 mM NMDA blocked NMDA- induced γ-PKC–GFP translocation. Bars, 10 μm.
Figure 11
Figure 11
mGluR1-mediated translocation of γ-PKC–GFP. γ-PKC–GFP was transfected into CHO cells stably expressing mGluR1 by lipofection as described in the text. (Upper trace) Application of 1 mM trans-ACPD rapidly induced the translocation of γ-PKC–GFP from cytosol to membrane. The fluorescence was retranslocated from membrane to cytosol within 20 min. (Lower trace) Simultaneous application of 500 μM MCPG with 1 mM trans-ACPD completely blocked mGluR1-mediated translocation of γ-PKC–GFP. Bar, 10 μm.
Figure 12
Figure 12
Involvement of γ-PKC–GFP translocation in cytoskeleton. (A) Effects of 10 μM cytochalasin D on γ-PKC–GFP translocation. Treatment with 10 μM γ-PKC–GFP affected neither TPA- nor A23187-induced translocation of γ-PKC–GFP. (B) Effects of 100 μM colchicine on γ-PKC–GFP translocation. Treatment with 100 μM colchicine did not affect TPA- or A23187-induced translocation. In these experiments, γ-PKC–GFP was transfected into NIH3T3 cells by lipofection as described in the text. The concentrations of TPA and A23187 applied were 1 and 50 μM, respectively. Bars, 10 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abeliovich A, Chen C, Goda Y, Silva AJ, Stevens CF, Tonegawa S. Modified hippocampal long-term potentiation in PKC γ-mutant mice. Cell. 1993a;75:1253–1262. - PubMed
    1. Abeliovich A, Paylor R, Chen C, Kim JJ, Wehner JM, Tonegawa S. PKCγ mutant mice exhibit mild deficits in spatial and contextual learning. Cell. 1993b;75:1263–1271. - PubMed
    1. Atlas D, Adler M. α-Adrenergic antagonist as possible calcium channel inhibitors. Proc Natl Acad Sci USA. 1981;78:1237–1241. - PMC - PubMed
    1. Bloom GS. Motor proteins for cytoplasmic microtubules. Curr Opin Cell Biol. 1992;4:66–73. - PubMed
    1. Chen CS, Poenie M. New fluorescent probes for protein kinase C. Synthesis, characterization, and application. J Biol Chem. 1993;268:15812–15822. - PubMed

Publication types

MeSH terms