* Laboratory of Molecular Pharmacology, Biosignal Research Center, Department of Biology, Faculty of Science, Kobe
University, Kobe 657, Japan
We expressed the -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.
PROTEIN kinase C (PKC),1 a family of phospholipid-dependent serine/threonine kinases and of which
there are at least 12 subspecies, plays an important
role in various cellular signal transductions (Nishizuka,
1984 PKC isozymes are divided into three subfamilies based
on differences in the regulatory domain: conventional PKC
(cPKC), novel PKC (nPKC), and atypical PKC (aPKC).
Conventional PKCs have two common regions in the regulatory domain, C1 and C2. The C1 region has two cysteine-rich loops (zinc finger-like motifs) that interact with
diacylglycerol (DG) or phorbol esters (Nishizuka, 1988 Conventional PKCs and nPKCs, whose regulatory domains contain C1, are known to be translocated from the
cytosol to particulate fraction when activated by DG or
phorbol esters (Kraft et al., 1982 To resolve these problems and to directly observe the
translocation of Materials
A23187 was purchased from Calbiochem (La Jolla, CA). N-methyl-D-aspartate (NMDA), 12-O-tetradecanoylphorbol 13-acetate (TPA), and
4 Cell Culture
COS-7 and NIH3T3 cells were purchased from Riken cell bank (Tsukuba,
Japan). Mouse neuroblastoma × glioma hybrid cells (NG 108-15 cells)
were obtained from Dr. T. Amano (The Mitsubishi-Kasei Institute of Life
Science, Tokyo, Japan). The CHO cells, stably expressing metabotropic
glutamate receptor 1 (CHO/mGluR1 cells) (Tanabe et al., 1992 Construct of Plasmids Encoding A plasmid containing GFP cDNA (pGFP 10.1) was donated by Dr. D.C.
Prasher (Columbia University, New York) (Prasher et al., 1992
We next produced a construct encoding mutant Expression of Transient transfection into COS-7 cells was performed by electroporation.
Plasmids (~32 µg) encoding each type of Coexpression of NMDA Receptor and Mouse cDNAs encoding NMDAR Immunoblotting, Kinase Assay, and
Immunoprecipitation of For immunoblotting, the samples of each fraction were subjected to
10% SDS-PAGE in the same volume, and the separated proteins were
electorophoretically transferred onto polyvinylidine difluoride (PVDF)
filters (Millipore Corp., Bedford, MA). Nonspecific binding sites on the
PVDF filters were blocked by incubation with 5% gelatine for 18 h. The
PVDF filters were then incubated with anti-PKC- Kinase assays of For immunoprecipitation of Observation of The fluorescence of Immunostaining of The Characteristics of To examine whether
Translocation of Intense fluorescence of
At 80 µM, A23187, the Ca2+ ionophore, also produced
The Effects of Thapsigargin on Translocation
of To examine the influence of Ca2+ released from the intracellular Ca2+ store on
Effects of Ca2+ Chelators on
To elucidate whether Ca2+ ionophore-induced translocation depends on the increase in the intracellular Ca2+
concentration ([Ca2+]i), we examined the effects of Ca2+
chelators on A23187-induced translocation. Ca2+ ionophore-induced translocation was completely blocked by
the pretreatment with Ca2+ chelators, 2.5 mM EGTA, and
15 µM BAPTA-AM (Fig. 6, upper trace). In addition,
A23187-induced translocation was reversed by the treatment with Ca2+ chelators after the stimulation of A23187.
The fluorescence of
Immunostaining of Translocated PKCs are known to be digested between the regulatory
and catalytic domains by protease such as calpain (Kishimoto et al., 1983
Translocation of Mutant To examine the significance of the C1 and C2 region of
We also examined the kinase character of BS 238 and BS
239. The kinase activity of BS 238 was dependent on Ca2+
(157.8 ± 9.1% of the control; control kinase activity was
measured in the presence of EGTA), but was not activated
by TPA (94.5 ± 5.6% of the control ). In contrast, the kinase
activity of BS 239 was not activated by neither Ca2+ (100.4 ± 5.6% of the control ) nor TPA (81.2 ± 7.2% of the control). The kinase activity of BS239, however, was reduced
to 27.2% ± 0.4% of the control by the treatment with 1 µM staurosporine, while the activity of BS238 was slightly
inhibited by staurosporine (70.9 ± 14.5% of the control).
K+ Depolarization Induced Translocation of
The translocation of
NMDA Receptor-mediated Translocation
of The translocation of
mGluR1-mediated Translocation of We transfected
Effects of Cytochalasin D and Colchicine on
To investigate the involvement of filamentous actin in
We first examined the enzymological property of PKCs are reported to be proteolysed by protease such
as calpain, a Ca2+-dependent neutral protease (Kishimoto
et al., 1983 Immunohistochemical and enzymological analyses revealed that TPA-induced translocation was completed
within 5 min (Kraft et al., 1982 In contrast with TPA-induced translocation, Ca2+ ionophore-induced translocation was rapid and reversible. Even
the TPA-induced translocation of When translocation was completed, the localization of
The treatment with TPA caused subtype-specific subcellular distribution in cardiac myocytes (Disatnik et al., 1994 In our present study, To elucidate whether the translocation of Although PKC translocation is a well-known phenomenon, the molecular mechanism and significance of PKC
translocation have not yet been clarified. Phorbol esters
translocated some subtypes of PKCs from cytosol to particulate fractions including cytoskeleton (Zalewski et al.,
1988 In conclusion, a GFP fusion protein with , 1988
, 1992
). Regardless of ubiquitous expression of
PKCs in various tissues, the central nervous system abundantly contains several unique PKCs. In particular, the
-subspecies of PKC (
-PKC) is present only in the central
nervous system and is thought to be involved in many neuronal functions including the formation of neural plasticity
and memory (Nishizuka, 1986
; Abeliovich et al., 1993a
,b;
Tanaka and Nishizuka, 1994
).
;
Ono et al., 1989
). The C2 region mediates calcium binding
(Ono et al., 1989
) and is only present in cPKCs (Ono et al.,
1988b
), although a region related to C2 region was recently
reported in nPKC, a calcium-independent PKC (Parker
and Dekker, 1997
). Full activation of cPKCs, including
-PKC, requires DG and calcium. The C1 region is also
present in nPKC, and one of the cysteine-rich loops is found in aPKCs.
). Therefore, the translocation of PKCs is a good marker of whether these enzymes
are activated. Although this phenomenon is well known,
the mechanism and physiological significance of PKC translocation have not yet been clarified. By conventional enzymological or immunohistochemical methods, it is impossible to observe the translocation of PKC in real time, in the
same cells, and in living states, except in the investigation
using fluorescent probes that directly bind PKC (Chen and
Poenie, 1993
). In addition, these fluorescent compounds
are suggested to inhibit the activity of PKC itself at high
concentration.
-PKC in living cells, we produced a fusion protein of
-PKC and green fluorescent protein (GFP).
The GFP, isolated from jellyfish Aequorea victoria, has fluorescence without additional substrates and cofactors (Cubitt et al., 1995
). Recent studies have revealed that GFP is
a good candidate as a molecular reporter protein to monitor the alternation of protein localization, gene expression,
and protein trafficking in living cells (Cubitt et al., 1995
).
In this study, we visualized and analyzed the translocation of
-PKC-GFP fusion protein with confocal laser scanning
fluorescence microscopy, using various stimulations, such
as phorbol esters, Ca2+ ionophore, K+ depolarization, and
receptor-mediated stimulus.
Materials and Methods
-phorbol 12, 13-didecanoate (4
-PDD) were purchased from Sigma
Chemical Co. (St. Louis, MO). (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), 2-amino-8-phosphonopentanoic acid (AP-5),
and (RS)-
-methyl-4-carboxyphenyl-glycine ([RS]-MCPG) were from
Tocris (Bristol, UK). Nicardipine and thapsigargin were from Wako Pure Chemical Ltd. (Osaka, Japan).
-Conotoxin GVIA was from Peninsula Laboratories Inc. (Belmont, CA). Cytochalasin D and colchicine were from
nacalai tesque (Kyoto, Japan). All other chemicals were of analytical grade.
) were a
gift from Dr. Nakanishi (Department of Biological Science, Kyoto University Faculty of Medicine, Kyoto, Japan). COS-7 cells were cultured
in DME containing 25 mM glucose, which was buffered with 44 mM
NaHCO3 and supplemented with 10% FBS, in a humidified atmosphere
containing 5% CO2 at 37°C. NG 108-15 cells were cultured in the same
conditions as for COS-7 cells. NIH3T3 cells were cultured in DME supplemented with 10% calf serum instead of FBS. CHO/mGluR1 cells were
cultured as described (Tanabe et al., 1992
). All media were supplemented
with penicillin (100 U/ml) and streptomycin (100 µg/ml), and the FBS
used was not heat-inactivated.
-PKC-GFP
Fusion Protein
). A cDNA
fragment encoding GFP with a HindIII site in the 5
-terminal and an
EcoRI site in the 3
-terminal end was obtained by PCR using pGFP 10.1 as a template. The sense and antisense primers used were 5
-TTAAGCTTATGGTGAGCAAGGGCCAGGAG-3
and 5
-CCGAATTCTTACTTGTACAGCTCGTCCAT-3
, respectively. A rat
-PKC cDNA was obtained from its cDNA clone of
CKR
1 (Ono et al., 1988a
). After
digestion with EcoRI, an insert fragment encoding rat
-PKC was subcloned into an expression plasmid for mammalian cells, pTB 701 (Ono et
al., 1988a
) (designated as BS 55) (Fig. 1). A cDNA fragment of
-PKC
with an EcoRI site in the 5
terminus and a HindIII site in 3
terminus was
also produced by PCR using BS 55 as a template. The sense and antisense primers used were 5
-TTGAATTCATGGCGGGTCTGGGTCCTGG-3
and 5
-TTAAGCTTATGGCGGGTCTGGGTCCTGG-3
, respectively.
PCR products for both GFP and
-PKC were together subcloned into the
EcoRI site in pTB701 (BS 186) (Fig. 1).
Fig. 1.
Constructs of -PKC-GFP fusion protein and its mutants. BS 186 was a
-PKC-GFP fusion protein in which
-PKC
(BS 55) and GFP were bound at the COOH terminus of
-PKC.
In the C1 region of
-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
-PKC, the Ca2+ binding domain, was deleted for the production
of BS 239 (C2 deletion) as described in the text.
[View Larger Version of this Image (33K GIF file)]
-PKC-GFP, whose
zinc finger-like motif in the C1 region was mutated. We used pTB966, a
plasmid containing a cDNA for mutated C1 region, as a template for PCR
(Ono et al., 1989
). The protein derived from pTB966 was reported to have
no binding activity for TPA (Ono et al., 1989
). We also produced a mutant
-PKC-GFP construct whose C2 region was deleted. For this, pTB971
was used as a template for PCR (Ono et al., 1989
). The protein derived
from pTB971 no longer had binding activity for Ca2+ (Ono et al., 1989
).
PCR was performed to obtain a cDNA fragment containing C1 and C2 regions with a sense primer (5
-TTGAATTCATGGCGGGTCTGGGTCCTGG-3
) and an antisense primer (5
-TTGGATCCTCTGCGCTCTGCCAG-3
) using pTB966 or pTB971 as templates. These PCR products
were digested with EcoRI and BamHI and then subcloned into BS 186, whose EcoRI/ BamHI fragment in
-PKC was removed. These constructs
were designated as BS 238 (C1 mutant) and BS 239 (C2 deletion), respectively. All PCR products were verified by sequencing. The structures of three
-PKC-GFP proteins (BS 186, BS 238, and BS 239) are summarized in Fig. 1.
-PKC-GFP Protein in Cultured Cells
-PKC-GFP or
-PKC were
transfected into 6 × 106 cells using a Gene Pulser (960 µF, 220 V; Bio-Rad
Labs, Hercules, CA). Transfections into NG-108, NIH3T3, and CHO/
mGluR1 cells were carried out by lipofection using Tfx-TM-50 (Promega
Corp., Madison, WI) according to the manufacturer's standard protocol.
After the transfection, cells were cultured at 30°C to obtain the optimal
fluorescence of GFP. The fluorescence of
-PKC-GFP was detected 2 or 3 d
after the transfection. Experiments were performed 3-5 d after the transfection.
-PKC-GFP in
COS-7 Cells
1 and NMDAR
1 subunits, kindly donated by Dr. Mishina (Department of Pharmacology, Tokyo University, Faculty of Medicine, Tokyo, Japan) (Ikeda et al., 1992
; Meguro et al.,
1992
; Yamazaki et al., 1992
), were subcloned into an expression plasmid,
pTB 701. The same amount of
-PKC-GFP, NMDAR
1, and NMDAR
1 plasmids (total 32 µg) was transfected into COS-7 cells by electroporation as described above. To prevent NMDA receptor-mediated cell death, transfected cells were cultured in the presence of 100 µM AP-5, an antagonist of NMDA receptor, until the experiment was performed.
-PKC-GFP and
-PKC
-PKC-GFP (BS 186) or
-PKC (BS 55) cDNAs were transiently transfected into 6 × 106 COS-7 cells by electroporation. The same number of
transfected cells were divided into two culture dishes 8 cm in diameter and cultured at 30°C for 3 d. After treatment with 5 µM TPA for 90 min at
room temperature, cells were harvested with PBS(
) and centrifuged.
The cell pellet/dish was resuspended in 200 µl homogenate buffer (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 50 mM Tris/HCl, 200 µg/ml
leupeptin, 1 mM PMSF, pH 7.4). After the sonication (UD-210 TOMY
SEIKO Co. Ltd., Tokyo, Japan; output 3, duty 50%, 10 times, at 4°C),
samples were centrifuged at 19,000 g for 30 min at 4°C, and supernatant
was collected as the cytosol fraction. The pellet was resuspended with 200 µl of homogenate buffer containing 0.5% Triton-X and used as particulate fraction after the sonication as described above.
monoclonal antibody
(diluted 1:2,000) (Hashimoto et al., 1988
) or anti-GFP polyclonal antibody
(diluted 1: 2,000) (CLONTECH Laboratories, Inc., Palo Alto, CA) for 30 min at 25°C. After washing with 0.01 M PBS containing 0.03% Triton X- 100, the filters were incubated with goat anti-mouse IgG (for
-PKC antibody)
or anti-rabbit IgG (for GFP antibody) for 15 min and then incubated for
15 min with rabbit peroxidase antiperoxidase complex. After three rinses, the immunoreactive bands were visualized with a chemiluminescence detection kit (ECL; Amersham, Buckinghamshire, UK).
-PKC,
-PKC-GFP, and its mutants expressed in
COS-7 cells were performed as described previously (Kikkawa et al., 1983
).
The kinase activity in 10 µl of each fraction was assayed by measuring the
incorporation of 32Pi into calf thymus H1 histone from [
-32P]ATP in the
presence of 8 µg/ml phosphatidylserine (PS), 0.8 µg/ml diolein (DO), and
0.5 mM Ca2+. Basal activity was measured in the presence of 0.5 mM
EGTA instead of PS, DO, and Ca2+.
-PKC-GFP and
-PKC, transfected cells
in a dish 8 cm in diameter were harvested with 1 ml of homogenate buffer
containing 1% Triton X-100 and were homogenized by pipetting. After a
centrifuge at 19,000 g for 5 min at 4°C, the supernatant was rotated with
anti-
-PKC monoclonal antibody for 30 min at 4°C, then with protein
A-Sepharose for an additional 30 min. Samples were centrifuged at 2,000 g
for 5 min at 4°C, and pellets were washed three times with PBS(
). Finally, 10 µl of suspended pellet with 50 µl PBS(
) was used for kinase assay as described above.
-PKC-GFP Translocation
-PKC-GFP-transfected cells were spread onto the glass bottom culture
dishes (MatTek Corp., Ashland, MA) and cultured for at least 16 h before
the observation. For COS-7 cells and CHO/mGluR1 cells, the culture medium was replaced with normal Hepes buffer composed of: 135 mM NaCl,
5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM Hepes, 10 mM glucose,
pH 7.3. When necessary, CaCl2 or MgCl2 were eliminated. In the case of
NG-108 cells, the buffer used was composed of: 165 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM Hepes, 10 mM glucose, pH 7.4.
-PKC-GFP was monitored with a confocal laser
scanning fluorescent microscope (model LSM 410 invert; Carl Zeiss, Jena,
Germany) at 488-nm argon excitation using a 515-nm-long pass barrier filter. Translocation of
-PKC-GFP was triggered by a direct application of
various stimulants at high concentration into the Hepes buffer to obtain
the appropriate final concentration. To observe NMDA-induced translocation, NMDA was applied into the dish in the presence of 10 µM glycine
and absence of MgCl2. To induce K+ depolarization in NG-108 cells, the
buffer was exchanged to a high K+-containing buffer composed of: 52 mM
NaCl, 100 mM KCl, 1 mM MgCl2, 10 mM CaCl2, 5 mM Hepes, 10 mM glucose, pH 7.4. All experiments were performed at room temperature.
-PKC-GFP-transfected Cells by
Anti-
-PKC Antibody
-PKC-GFP-transfected COS-7 cells, cultured in glass bottom dishes,
were stimulated by TPA or A23187 and it was confirmed that fluorescence was completely translocated. Then cells were fixed with 4%
paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4, for 30 min. After two washes with 0.1 M PBS, pH 7.4, cells were treated
with PBS containing 0.3% Triton X-100 and 5% normal goat serum for 10 min. Cells were sequentially incubated with anti-PKC-
monoclonal antibody (Hashimoto et al., 1988
) (diluted 1:1,000) for 40 min in PBS with 0.03%
Triton X-100 (PBS-T) and 5% normal goat serum and then with Cy5-labeled
goat anti-mouse IgG for 30 min at room temperature. The fluorescence of
-PKC-like immunoreactivity was observed with a confocal laser scanning
fluorescent microscope at 633-nm argon excitation and a 665-nm red glass
filter.
Results
-PKC-GFP Fusion Protein
-PKC-GFP had the characteristics
of a phospholipid-dependent/calcium-activated protein kinase (PKC), we carried out immunoblotting and kinase assay of
-PKC-GFP and
-PKC expressed in COS-7 cells. As
shown in Fig. 2 A,
-PKC and
-PKC-GFP were recognized as specific bands with the reasonable molecular masses of 80 and 110 kD, respectively.
-PKC-GFP was
also detected as a single 110-kD band by anti-GFP polyclonal antibody (data not shown). The amount of membrane-associated
-PKC-GFP was increased after the treatment with TPA as seen in the case of
-PKC (Fig. 2 A).
The degradative products of
-PKC-GFP and
-PKC could
not be detected by the antibodies against
-PKC and GFP.
Furthermore, kinase activities of
-PKC-GFP and of
-PKC
were translocated from cytosol to membrane fractions
(Fig. 2 B). The kinase assays revealed that both immunoprecipitated
-PKC and
-PKC-GFP were dependent on
PS/DO and Ca2+ (Fig. 2 C). These results suggested that
-PKC-GFP had similar enzymological properties to the
native
-PKC.
Fig. 2.
Enzymological property of -PKC and
-PKC-GFP
transiently expressed in COS-7 cells. (A) Immunoblotting analysis
by anti-
-PKC antibody revealed that expressed
-PKC (BS 55)
and
-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
-PKC and
-PKC-GFP associated with the particulate fraction. p, pellet (particulate fraction);
s, supernatant (cytosol fraction). (B) Kinase activity of expressed
-PKC and
-PKC-GFP in cytosol and particulate fractions. Kinase activities of both
-PKC and
-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
-PKC and
-PKC-GFP immunoprecipitated by anti-
-PKC antibody. Kinase activities of expressed
-PKC and
-PKC-GFP, which were
immunoprecipitated by anti-
-PKC antibody, were measured in
the presence or absence of activators of
-PKC. The kinase activity of
-PKC-GFP maximized in the presence of PS, DO, and Ca2+, as did that of
-PKC. The enzymological properties of
-PKC and
-PKC-GFP were very similar.
[View Larger Version of this Image (43K GIF file)]
-PKC-GFP Induced by
TPA and A23187
-PKC-GFP was observed in the
perikarya of the transfected COS-7 cells, and faint fluorescence was seen in the nuclei (Fig. 3, A and B). The activation of
-PKC-GFP by 5 µM TPA induced the obvious
translocation of the fluorescence from the cytoplasm to the
membrane. Translocation began at 10 min and was completed by 60 min after the treatment with TPA (Fig. 3 A).
The fluorescence remained on the plasma membrane for
at least 90 min after TPA treatment and did not return to
the cytoplasm in the cells tested. The
-PKC-GFP in the
nuclei did not appear to be translocated. The TPA-induced
translocation of
-PKC-GFP occurred more rapidly when
the experiments were performed at 37°C (Fig. 3 B, upper
trace). The translocation completed 10 min after the treatment with lower concentration of TPA (200 nM). In contrast, an inactive phorbol ester, 4
-PDD, at 500 nM failed
to induce the translocation within 30 min (Fig. 3 B, lower
trace).
Fig. 3.
Phorbol ester-
induced translocation of
-PKC-GFP in COS-7 cells.
(A) Change in the fluorescence of
-PKC-GFP expressed in COS-7 cells by 5 µM TPA at room temperature.
-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
-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
-PKC-GFP expressed in
COS-7 cells by 200 nM TPA
and 500 nM 4
-PDD at 37°C.
Lower concentration of TPA
(200 nM) induced the translocation of
-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, 4
-PDD, an inactive
phorbol ester, at 500 nM
failed to induce the translocation of
-PKC-GFP even at
37°C. Bars, 10 µm.
[View Larger Version of this Image (79K GIF file)]
-PKC-GFP translocation, the time course of which was
significantly different from the TPA-induced translocation.
The Ca2+ ionophore-induced translocation was rapid and
reversible (Fig. 4 A). The fluorescence of
-PKC-GFP
translocated transiently at only 30 s after the stimulation.
The first phase of the translocation was quickly reversed.
The
-PKC-GFP was retranslocated to cytoplasm at 90 s
after the stimulation. The second and third phase of the translocation was observed 20 and 45 min after the stimulation, and finally
-PKC-GFP was accumulated as patchy
dots at the plasma membrane 60 min after A23187 treatment (Fig. 4 A, upper and middle traces). In the lower
trace of Fig. 4 A, the profiles of the GFP intensity on the
same line across the cell at various time points were
shown. The translocation of
-PKC-GFP to the membrane was detected as the increase in the intensity at the
fringe of the cell at 30 s and 45 min. Very similar profiles
of the GFP intensity were obtained at 0 and 5 min, and the
fading of the fluorescence is negligible between the two
different time points.
Fig. 4.
Ca2+ ionophore-
induced translocation of
-PKC-GFP in COS-7 cells.
(A) Change in the fluorescence of
-PKC-GFP expressed in COS-7 cells by
80 µM A23187, Ca2+ ionophore. A23187 also translocated the fluorescence of
-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
-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.
-PKC-GFP eventually accumulated as patchy dots
on the plasma membrane
and in neighboring cytoplasm. Bars, 10 µm.
[View Larger Version of this Image (83K GIF file)]
-PKC-GFP translocation by A23187 was not always reversible (Fig. 4 B, left cells). The unidirected translocation (Fig. 4 B, left cells) was more common than the
reversible translocation (Fig. 4 B, right cells). However,
-PKC-GFP was always accumulated on plasma membrane as patchy dots (Fig. 4 B). A23187 at lower concentration (10 µM) also showed similar effects (data not shown).
-PKC-GFP
-PKC-GFP translocation, we studied the effects of thapsigargin, which inhibits endoplasmic
reticulum Ca2+-ATPase and increases the concentration
of cytosolic Ca2+, on translocation of
-PKC-GFP. Application of 5 µM thapsigargin induced a rapid translocation
of fluorescence, which began within 1 min after the stimulation (Fig. 5). Finally, fluorescence was accumulated as
patchy dots on the plasma membrane as seen in A23187-induced translocation. Accumulation of
-PKC-GFP also
occurred in the perikaryon.
Fig. 5.
Thapsigargin-
induced translocation of
-PKC-GFP. 5 µM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-
ATPase, also induced rapid
translocation of
-PKC-GFP.
Fluorescence of
-PKC-GFP
accumulated as patchy dots
as in A23187-induced translocation. Bar, 10 µm.
[View Larger Version of this Image (71K GIF file)]
-PKC-GFP Translocation
-PKC-GFP partially returned to the
cytoplasm (Fig. 6, lower trace). TPA-induced translocation
was not inhibited by either pre- or post-treatment with
Ca2+ chelators (data not shown).
Fig. 6.
Effects of Ca2+ chelators on A23187-induced
translocation of -PKC-GFP.
Pretreatment with 2.5 mM
EGTA and 15 µM BAPTA-AM completely blocked
A23187 (50 µM)-induced
-PKC-GFP translocation
(upper trace). Treatment with
2.5 mM EGTA and 15 µM
BAPTA-AM retranslocated
-PKC-GFP from membrane
to cytosol, even after A23187-
induced translocation had
occurred (lower trace). Bars,
10 µm.
[View Larger Version of this Image (60K GIF file)]
-PKC-GFP
), suggesting that the fluorescence of
translocated
-PKC-GFP did not exactly reveal the localization of
-PKC. Therefore, we immunostained the transfected cells using a monoclonal anti-
-PKC antibody that
recognizes the regulatory domain of
-PKC after the translocation was completed. As shown in Fig. 7,
-PKC-like Cy5
fluorescence was colocalized with the GFP fluorescence
even after the TPA- or A23187-induced translocation was
completed. These results suggested that fluorescence of
GFP showed the localization of
-PKC itself.
Fig. 7.
Comparison of the fluorescence of -PKC-GFP with
the
-PKC-like immunoreactivity. Immunostaining of
-PKC-
GFP-transfected COS-7 cells by anti-
-PKC antibody showed
that GFP fluorescence and
-PKC-like immunoreactivity had very
similar localizations, even after the TPA- or A23187-induced translocation was completed. Bar, 10 µm.
[View Larger Version of this Image (66K GIF file)]
-PKC-GFP Fusion Protein
-PKC in the
-PKC-GFP translocation, we constructed
mutant
-PKC-GFP, BS 238 (C1 mutant), and BS 239 (C2
deletion), as described above (Fig. 1). BS 238 is a mutant
of the C1 region that binds TPA; it therefore was not expected to be activated by TPA. Indeed, TPA did not induce the translocation of BS 238 (C1 mutant), while A23187 did (Fig. 8 A). The Ca2+ ionophore-induced translocation of BS 238 was insufficient compared with that of
wild-type
-PKC-GFP (BS 186) (Fig. 8 A and 4 A). BS 239 (C2 deletion) is a deletion mutant of the C2 region that binds calcium ion. In contrast to BS 238, BS 239 was translocated by TPA but not by A23187 (Fig. 8 B).
Fig. 8.
Translocation of
mutant -PKC-GFPs (BS
238 and BS 239) expressed in
COS-7 cells. (A) Translocation of BS 238 (C1 mutant
-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
-PKC-GFP). (B)
Translocation of BS 239 (C2
deletion
-PKC-GFP). In
contrast to BS 238, BS 239 was translocated by 5 µM
TPA but not by 50 µM A23187. Bars, 10 µm.
[View Larger Version of this Image (65K GIF file)]
-PKC-GFP in Transfected NG 108-15 Cells
-PKC-GFP protein was further examined under physiological conditions. To investigate
whether depolarization and subsequent activation of the
voltage-gated Ca2+ channel induce
-PKC translocation,
we expressed
-PKC-GFP fusion protein in NG 108-15 cells,
which have voltage-gated Ca2+ channels (Atlas and Adler,
1981
). After the extracellular K+ was elevated from 5 to
100 mM, the fluorescence of
-PKC-GFP was rapidly translocated from cytosol to membrane (Fig. 9). After the
translocation was completed, patchy dotlike fluorescence
accumulated in membrane and cytosol as seen in A23187-
or thapsigargin-induced translocation (Fig. 9). Reversible
translocation, as seen in Fig. 3 B, also occurred in the other
cells tested (data not shown). K+ depolarization-induced
translocation was abolished by 10 µM nicardipine or 10 µM
-conotoxin GVIA, blockers of L- and N-type Ca2+ channels, respectively, but not by 2 µM tetrodotoxin, a Na+
channel blocker (data not shown). These findings suggested that translocation was triggered by Ca2+ influx
through a certain type of voltage-gated Ca2+ channel expressed in NG 108-15 cells.
Fig. 9.
K+ depolarization-
induced translocation of
-PKC-GFP expressed in
NG 108-15 cells. Replacing
the external solution with a high K+-containing one rapidly induced translocation of
-PKC-GFP. The fluorescence of
-PKC-GFP accumulated as patchy dots on
the plasma membrane and in
neighboring cytoplasm, as
in Ca2+ ionophore-induced
translocation. Bar, 10 µm.
[View Larger Version of this Image (34K GIF file)]
-PKC-GFP
-PKC-GFP by receptor-mediated
stimulation was further examined. In COS-7 cells coexpressing NMDA receptor channels (
1 and
1 subunits),
the treatment with 1 mM NMDA plus 10 µM glycine induced faint but significant translocation of
-PKC-GFP (Fig. 10). Simultaneous application of 500 µM AP-5, an
antagonist of NMDA receptor, blocked NMDA-induced
translocation (Fig. 10).
Fig. 10.
NMDA-induced
translocation of -PKC-
GFP in COS-7 cells coexpressing NMDA receptors.
NMDAR
1 and NMDAR
1
subunits were cotransfected with
-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
-PKC-GFP.
Simultaneous application of
100 µM AP-5 with 1 mM
NMDA blocked NMDA-
induced
-PKC-GFP translocation. Bars, 10 µm.
[View Larger Version of this Image (94K GIF file)]
-PKC-GFP
-PKC-GFP into the CHO cells stably expressing mGluR1 to investigate G-protein-coupled receptor-mediated translocation of
-PKC. Activation of mGluR1
receptor by 1 mM trans-ACPD, an agonist of mGluR1, induced a rapid translocation of
-PKC-GFP with re-translocation to the cytoplasm within 20 min (Fig. 11). Simultaneous application of 2.5 mM RS-MCPG, an antagonist of
mGluR1, completely blocked mGluR1-mediated translocation of
-PKC-GFP (Fig. 11).
Fig. 11.
mGluR1-mediated translocation of -PKC-GFP.
-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
-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
-PKC-GFP. Bar, 10 µm.
[View Larger Version of this Image (66K GIF file)]
-PKC-GFP Translocation
-PKC
translocation, we studied the effects of cytochalasin D, an
inhibitor of actin polymerization, on the translocation of
-PKC-GFP. Pretreatment with 10 µM cytochalasin D for
25-40 min altered the shapes of cells; however, it did not
inhibit the translocation of
-PKC-GFP induced by either
TPA or A23187 (Fig. 12 A). We also examined the effects
of colchicine, an inhibitor of microtubule polymerization, to investigate the relationship between
-PKC translocation and microtubules. Colchicine, like cytochalasin D, did
not affect the TPA- or A23187-induced translocation of
-PKC-GFP (Fig. 12 B). To assess whether the pretreatment with cytochalasin D or colchicine described above
actually acted on cytoskeleton, we stained filamentous actin with rhodamine-phalloidin or microtubules with anti-
-tubulin antibody. Both 10 µM cytochalasin and 100 µM
colchicine disrupted actin fibers and microtubules, respectively (data not shown).
Fig. 12.
Involvement of
-PKC-GFP translocation in
cytoskeleton. (A) Effects of
10 µM cytochalasin D on
-PKC-GFP translocation.
Treatment with 10 µM
-PKC-GFP affected neither
TPA- nor A23187-induced translocation of
-PKC-GFP.
(B) Effects of 100 µM colchicine on
-PKC-GFP translocation. Treatment with 100 µM colchicine did not affect
TPA- or A23187-induced
translocation. In these experiments,
-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.
[View Larger Version of this Image (50K GIF file)]
Discussion
-PKC-
GFP protein by measuring kinase activity with or without
activators of PKC. As shown in Fig. 2 C,
-PKC-GFP protein expressed in COS-7 cells had similar enzymological
character to the native
-PKC. In addition, immunoblotting
analysis revealed that
-PKC-GFP of reasonable molecular size, but not the degradation product of
-PKC-GFP,
was present as a donor of GFP fluorescence, even after the
translocation from cytosol to membrane by the stimulation with TPA (Fig. 2 A). Kinase activity of
-PKC-GFP was
also translocated from cytosol to membrane (Fig. 2 B).
These results suggest that
-PKC-GFP protein did not
lose its enzymological character as a
-PKC, even though a
GFP, a protein with 238 amino acids, was added to the
COOH terminus of
-PKC. When the GFP was added to the NH2 terminus of
-PKC (GFP-
-PKC), the distribution of GFP-
-PKC within a cell was similar to the present
observation of
-PKC-GFP (data not shown). The NH2-terminal methionine of cPKC, however, is known to be
posttranslationally cleaved and replaced with an acetyl group
(Tsutakawa et al., 1995
). Therefore, it is suggested that the
fusion protein
-PKC-GFP, rather than GFP-
-PKC, is
better to monitor the exact localization of
-PKC itself.
). If
-PKC-GFP was proteolysed during its
translocation, GFP fluorescence did not reveal the exact
localization of
-PKC. Therefore, we also carried out the
immunostaining of
-PKC-GFP with the antibody that
recognizes the regulatory domain of
-PKC when the translocation was completed. As shown in Fig. 7, the immunoreactivity of
-PKC-GFP coincided with the fluorescence of GFP. Furthermore, the fact that the line intensity
profiles across the cell were similar before and after a transient translocation to the membrane (Fig. 4 A) suggests
that the fluorescence of
-PKC-GFP exactly revealed the
localization of
-PKC-GFP itself, and the time-dependent
movement of GFP fluorescence showed the translocation
events of
-PKC in real time.
), although
-PKC-GFP was
translocated more slowly and a high dose of TPA was necessary for the translocation at room temperature. The discrepancy was due to the temperature since 200 nM TPA
was enough to translocate
-PKC-GFP to the membrane, and the translocation was completed within 10 min when
the experiment was performed at 37°C (Fig. 3 B).
-PKC-GFP at 37°C was
still slower than that induced by Ca2+ ionophore. Ca2+
chelators blocked the translocation induced by Ca2+ ionophore, suggesting that Ca2+-induced translocation depended
on the intracellular Ca2+ concentration ([Ca2+]i). In this
regard, the wavelike translocation by A23187 may reflect
the alternation of [Ca2+]i . To prove this, we observed the
A23187-induced change in [Ca2+]i by loading cells with
calcium green-1-AM, a fluorescent Ca2+ indicator, using a
confocal laser fluorescent microscope. A transient elevation of fluorescence was observed; however, no wavelike
phenomenon of the [Ca2+]i could be detected (data not
shown). In addition, A23187 commonly induced the unidirected translocation of
-PKC-GFP, and the typical reversible translocation as shown in Fig. 3 was infrequent.
The reason why Ca2+ ionophore induced the wavelike translocation of
-PKC-GFP is unclear at present; however,
possible explanations can be proposed. First, a wavelike
alternation of [Ca2+]i is induced by A23187 under certain
conditions only, and the experiments using calcium green-1-AM were not performed under such conditions or the
calcium green-1-AM was not a suitable drug with which to
fine-tune change in [Ca2+]i becuase of its effect as a Ca2+
chelator. Alternatively, when the A23187-induced elevation
of [Ca2+]i was insufficient, the
-PKC-GFP translocation
was incomplete and transient, but subsequent production
of phospholipids by various Ca2+-activated phospholipases induced a second or third translocation of
-PKC-
GFP. The results of the kinase assay of
-PKC-GFP that both phospholipids and Ca2+ were needed for the full activation of
-PKC-GFP support this idea. Thapsigargin also
induced rapid translocation of
-PKC-GFP, indicating
that Ca2+ influx from intracellular Ca2+ stores could translocate
-PKC-GFP.
-PKC in TPA-induced translocation was different from in
the Ca2+-induced one. Fluorescence of
-PKC-GFP was accumulated in plasma membrane in TPA-induced translocation, whereas it was accumulated in plasma membrane
and submembrane cytoplasm as patchy dots in Ca2+-induced
translocation. These findings suggest that TPA-induced and Ca2+-induced translocation are mediated by different
pathways and that the final localization of
-PKC is distinct after each stimulation.
).
-PKC was also reported to be translocated to Golgi organelle by TPA in NIH3T3 cells stably overexpressing
-PKC
(Goodnight et al., 1995
). In contrast,
-PKC-GFP was
mainly translocated from cytosol to membrane by TPA in
our study. As far as we could determine in various cells
(COS-7, CHO, NG-108, etc.),
-PKC was not translocated to the Golgi complex. Transiently expressed
-PKC may
have different properties of translocation from native or
stably expressed
-PKC. Otherwise, an addition of GFP
may alter the translocation nature of
-PKC.
-PKC-GFP was translocated by
various physiological stimuli. The findings showed that
-PKC is activated and translocated in living cells. In addition, receptor mediated-translocation of
-PKC-GFP
were rapid and reversible. In particular, mGluR1-mediated translocation was transient, indicating that
-PKC
translocation induced by receptor-mediated breakdown
of phosphatidylinositol was not sustained in living cells. As
mGluR1-mediated elevation of Ca2+ was found to be transient by measuring fluorescence of intracellular-loaded Ca2+ green-1-AM (data not shown), mGluR1-mediated
translocation of
-PKC-GFP may depend on intracellular
Ca2+.
-PKC is associated with the activation mechanism, we examined the
translocation of mutant
-PKC-GFPs. TPA induced translocation of BS 239 (C2 mutant) but not BS 238 (C1 mutant). In contrast, Ca2+ ionophore translocated BS 238 but
not BS 239. The kinase activity of BS 238 was dependent
on Ca2+, in parallel with the results of BS 238 translocation,
while the kinase activity of BS 239 depended on neither
Ca2+ nor TPA but was blocked by the staurosporine, a potent kinase inhibitor, suggesting that BS 239 may become a
constitutively active form by a mutation. TPA-induced
translocation of
-PKC may not be coupled with its kinase
activity, as the staurosporine did not block TPA-induced
translocation (data not shown). As shown in Fig. 8 A,
Ca2+ ionophore-induced translocation of BS 238 (C1 mutant) was insufficient in all cells tested. Based on the results of the kinase assay (Fig. 2 C), Ca2+ alone appears not
to induce the complete translocation of
-PKC. As Ca2+
has been reported to activate the production of phospholipids, including DG, both calcium and phospholipids were
needed to induce complete translocation of
-PKC.
; Jaken et al., 1989
; Papadopoulos and Hall, 1989
; Kiley and Jaken, 1990
; Mochly-Rosen et al., 1990
). In
II-PKC, which was reported to be translocated to actin fiber
(Goodnight et al., 1995
), it was proposed that receptors for
activated C-kinase (RACK), present in the detergent-
insoluble fraction and bound activated
II-PKC, play a
role in the mechanism of PKC translocation (Mochly-Rosen et al., 1991
; Ron and Mochly-Rosen, 1995). To elucidate whether cytoskeleton proteins such as actin or microtubule are involved in PKC translocation, the effects of
cytochalasin D, an inhibitor of actin polymerization, and
colchicine, an inhibitor of microtubule polymerization, on
the translocation of
-PKC-GFP were examined. As shown
in Fig. 12, pretreatment with neither cytochalasin D nor
colchicine affected
-PKC-GFP translocation. Furthermore,
-PKC-GFP translocation occurred even when the
glucose in the external solution was eliminated (data not
shown). These results indicated that PKC translocation
did not need the cytoskeleton and glucose-dependent motor protein that are essential for some types of protein
trafficking, such as an axonal flow or vesicle transport
(Bloom, 1992
; Cheney et al., 1993
). In addition, based on
the findings that the C2 deletion mutant of
-PKC (BS
239) could be translocated by TPA, phorbol ester-induced
translocation of
-PKC from cytosol to plasma membrane
is probably not necessary for the association of RACK because RACK was reported to bind the C2 region of PKC
(Ron and Mochly-Rosen, 1995).
-PKC is a useful tool for investigating the mechanism and significance
of
-PKC translocation in living cells.
Received for publication 4 August 1997 and in revised form 30 September 1997.
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.jpWe thank Dr. Ushio Kikkawa for many useful discussions.
This work was supported by grants from the Ministry of Education, Science, Sports and Culture in Japan, the Yamanouchi Foundation for Research on Metabolic Disorders, and the Kato Memorial Bioscience Foundation.
4-PDD, 4
-phorbol 12, 13-didecanoate;
DG, diacylglycerol;
DO, diolein;
GFP, green fluorescent protein;
NMDA, N-methyl-D-aspartate;
MCPG,
-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.
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