Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
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Abstract |
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Effects of fatty acids on translocation of the
- and
-subspecies of protein kinase C (PKC) in living
cells were investigated using their proteins fused with
green fluorescent protein (GFP).
-PKC-GFP and
-PKC-GFP predominated in the cytoplasm, but only a
small amount of
-PKC-GFP was found in the nucleus.
Except at a high concentration of linoleic acid, all the
fatty acids examined induced the translocation of
-PKC-GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on
-PKC-GFP in the nucleus. Arachidonic and linoleic acids induced slow
translocation of
-PKC-GFP from the cytoplasm to the
perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower
translocation of
-PKC-GFP by arachidonic acid was
identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied
among the 11 fatty acids tested. In general, a higher
concentration was required to induce the translocation
of
-PKC-GFP than that of
-PKC-GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of
-PKC-GFP. Simultaneous application of arachidonic acid (25 µM) and DiC8 (10 µM)
elicited a slow, irreversible translocation of
-PKC-
GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application
of arachidonic acid or DiC8 at the same concentration
induced no translocation.
These findings confirm the involvement of fatty acids
in the translocation of - and
-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that
a combination of intracellular activators alters the target site of PKCs.
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Introduction |
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PROTEIN kinase C (PKC)1 is a family of serine/threonine protein kinases that is activated in the presence of phospholipid and Ca2+ ions (Nishizuka,
1984, 1988
; Parker and Dekker, 1997
; Srinivasan and Blundell, 1997
). To date, at least 12 subspecies have been identified, and these are divided into three groups based on
their structures: conventional, novel, and atypical PKC
(Nishizuka, 1988
, 1992
). The PKC family comprises the
regulatory domain in the amino terminus and a catalytic
domain in the carboxyl terminus. The conventional PKCs
(cPKC), including
-,
I-,
II-, and
-PKC, have two common regions (C1 and C2) in the regulatory domain. The
C1 region has two cysteine-rich loops (zinc finger-like motifs) that are the binding site for diacylglycerol (DAG) and
phorbol ester (Nishizuka, 1988
; Ono et al., 1989
). The C2
region binds to calcium (Ono et al., 1989
). The novel PKCs
(nPKC),
-,
-,
-, and
-PKC, lack the C2 region (Ono et al.,
1988b
; Hug and Sarre, 1993
; Osada et al., 1992
). The atypical PKCs (aPKC),
- and
/
-PKC, lack the C2 region and
have only one cysteine-rich loop in the C1 region (Ono et al.,
1989
; Selbie et al., 1993
; Akimoto et al., 1994
). Calcium,
phosphatidylserine, and DAG are required for the activation of cPKCs, whereas calcium is not required for the activation of nPKCs. aPKCs are insensitive to both DAG and calcium (Newton, 1997
).
cPKCs and nPKCs are translocated from the soluble to
the particulate fraction when activated by several stimuli
(Kraft et al., 1982; Mochly-Rosen et al., 1990
; Jaken, 1996
;
Ohno, 1997
). The molecular mechanism of this translocation, however, has yet to be clarified. A system for monitoring the translocation of
-PKC in living cells has recently been developed that uses protein fused with green
fluorescent protein (GFP) (Sakai et al., 1997
; Oancea et al.,
1998
). This experimental breakthrough enabled us to investigate the subspecies-specific function of PKC by analyzing the different targeting mechanisms of each subspecies.
Several saturated and unsaturated fatty acids also are
reported to potentiate the activity of PKC (McPhail et al.,
1984; Murakami et al., 1986
; Shinomura et al., 1991
;
Asaoka et al., 1992
; Nishizuka, 1995
). For example, saturated fatty acids that have carbon chain lengths of C13
to C18 activate
- and
-PKC in vitro (Kasahara and
Kikkawa, 1995
), and unsaturated fatty acids, such as
arachidonic and oleic acid, enhance the kinase activity of
several PKC subspecies (Sekiguchi et al., 1988
; Lester et al.,
1991
; Shinomura et al., 1991
; Chen and Murakami, 1992
).
These fatty acid-induced activations of PKCs are enhanced by the presence of diacylglycerol. Furthermore, arachidonic acid-induced translocation of
-PKC and oleic
acid-induced translocation of
-,
II-, and
-PKC have
been shown by immunoblot analysis (Khan et al., 1993
;
Huang et al., 1997
). Less information, however, is available on the underlying mechanism of the effects of fatty
acids on PKC translocation. To clarify the physiological involvement of fatty acids in PKC signaling pathways, we
studied the effects of 11 fatty acids on the translocation of
- and
-PKC in living cells that expressed PKC subspecies
fused with GFP.
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Materials and Methods |
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Materials
Tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoic acid were purchased from Doosan Serdary Research Laboratories (Englewood Cliffs, NJ). 1,2-dioctanoylglycerol (DiC8) and BAPTA-AM were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA) and Research Biochemicals International (Natick, MA), respectively. All the other chemicals used were of analytical grade.
Cell Culture
COS-7 cells were purchased from the Riken cell bank (Tsukuba, Japan). The CHO-K1 cell strain was a gift from Dr. Nishijima (National Institute of Health, Tokyo, Japan). COS-7 cells were cultured in DME, and CHO-K1 cells in Ham's F12 medium (GIBCO BRL, Grand Island, NY) at 37°C in a humidified atmosphere containing 5% CO2. Both media contained 25 mM glucose, and both were buffered with 44 mM NaHCO3 and supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). The FBS used was not heat inactivated.
Construct of Plasmids Encoding the - and
-PKC-GFP Fusion Proteins
The plasmid bearing humanized GFP-S65A/Y145F cDNA was donated by Dr. Umesono (Kyoto University, Kyoto, Japan). A cDNA fragment encoding GFP with a HindIII site in the 5'-terminal end and an EcoRI site in the 3'-terminal end were obtained by a PCR with pCMX-SAH/Y145F as the template. The sense primer was 5'-AAGCTTATGGTGAGCAAGGGCGAGGAG-3', and the antisense primer was 5'-TTGAATTCCTAGCTAGCTGGCCAGGATCC-3'.
Rat -PKC cDNA was obtained from the cDNA clone of
CKR
1
(Ono et al., 1988a
). After digestion with EcoRI, an insert fragment encoding rat
-PKC was subcloned to an expression plasmid for mammalian
cells, pTB 701 (Ono et al., 1988b
) (designated BS 55). A cDNA fragment
of
-PKC with an EcoRI site in the 5' terminus and a HindIII site in the 3'
terminus also was produced by a PCR with BS 55 as the template. The
sense primer was 5'-TTGAATTCATGGCGGGTCTGGGTCCTGG-3', and the antisense primer was 5'-TTAAGCTTATGGCGGGTCTGGGTCCTGG-3'. The PCR products of both GFP and
-PKC were digested
with EcoRI and HindIII and then subcloned together into the EcoRI site
in pTB701 (BS 336).
A cDNA fragment encoding GFP with MunI-EcoRI-BglII sites in the
5' terminus and a MunI site in the 3' terminus was obtained by PCR with
pCMX-SAH/Y145F as the template. The sense primer was 5'-TTTCAATTGAATTCAGATCTATGGTGAGCAAGGGCGAGGAG - 3' , and the antisense primer was 5'-GGCAATTGCTAGCTAGCTGGCCAGGATCC-3'. The PCR product was subcloned into pTB 701 (BS
340). The plasmid bearing rat -PKC cDNA in pTB 701 was a gift from
Dr. Ono (Kobe University) (BS 254). A cDNA fragment of
-PKC with a
BglII site in the 5' and 3' termini was produced by a PCR with BS 254 as
the template. The sense primer was 5'-TTAGATCTACCATGGTAGTGTTCAATGG-3', and the antisense primer was 5'-TTAGATCTGGGCATCAGGTCTTCACCAAA-3'. The PCR product for
-PKC was digested with BglII and subcloned to the BglII site in BS 340 (BS 394).
Kinase Assay of Native -PKC and
-PKC-GFP
COS-7 cells were transiently transfected by electroporation with plasmids
encoding -PKC and
-PKC-GFP and then cultured.
-PKC and
-PKC-
GFP were immunoprecipitated with anti-
-PKC monoclonal antibody
(Transduction Laboratories, Lexington, KY), and their kinase activities
were assayed as described elsewhere (Sakai et al., 1997
). In brief, the immunoprecipitate was suspended in 120 µl of Dulbecco's PBS (
), and 10 µl
of the suspended pellet was used for the kinase assay. Kinase activity measurements of the immunoprecipitated
-PKC and
-PKC-GFP were based
on the incorporation of 32P into a fragment of myelin basic protein (Sigma
Chemical Co., St. Louis, MO) from [
-32P]ATP in the presence of 8 µg/ml
phosphatidylserine (PS) and 0.8 µg/ml diolein (DO). Basal activity was
measured in the presence of 0.5 mM EGTA instead of PS or DO.
Transfection of PKCs and Fusion Proteins to Cultured CHO-K1 Cells
Plasmids (~5.5 µg) encoding - or
-PKC-GFP (BS 336 and BS 394) were
transfected to 5 × 106 CHO-K1 cells by lipofection using TransITTM-LT2
(Mirus Co., Madison, WI) according to the manufacturer's standard protocol. The transfected cells were cultured at 37°C to obtain the optimal
GFP fluorescence and then used for immunoblotting, immunostaining,
and the observation of translocation.
Immunostaining of CHO-K1 Cells Expressing -PKC
and Its Fusion Protein
Before and after translocation was induced by 1 µM TPA, cells expressing
-PKC or
-PKC-GFP were fixed with 4% paraformaldehyde and 0.2%
picric acid in 0.1 M PBS for 30 min. After three washes with 0.1 M PBS,
the cells were treated with PBS containing 0.3% Triton X and 10% normal goat serum (NGS) for 20 min. They then were incubated sequentially,
first with anti-
-PKC monoclonal antibody (diluted 1:1,000) for 40 min in
PBS containing 0.03% Triton X (PBS-T) and then with Cy3-labeled goat
anti-mouse IgG (Amersham Corp., Arlington Heights, IL) for 30 min at
room temperature. Fluorescence of
-PKC-like immunoreactivity was observed under a confocal laser scanning fluorescent microscope (model
LSM 410 invert; Carl Zeiss, Jena, Germany) at 588-nm argon excitation with a 590-nm-long pass barrier filter. GFP fluorescence was observed at
488-nm argon excitation with a 510-525-nm-band pass barrier filter.
Observation of - and
-PKC-GFP Translocation
CHO-K1 cells expressing - and
-PKC-GFP were spread on glass-bottomed culture dishes (MatTek Corp., Ashland, MA) and incubated for
16-60 h before observation. 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, and 10 mM glucose, pH 7.3. In the experiment with Ca2+ chelators, the cells were washed with Hepes buffer without
CaCl2 (Ca2+-free Hepes buffer) and then incubated for 30 min with 2.5 mM EGTA and 30 µM BAPTA-AM in Ca2+-free Hepes buffer. Translocation
of the fusion protein was triggered by the addition of the various stimulants to the Hepes buffer to obtain the appropriate final concentration.
All experiments were done at 37°C.
The fluorescence of GFP was monitored under a confocal laser scanning fluorescent microscope at 488-nm argon excitation with a 515-nm-long pass barrier filter. Lastly, the changes in fluorescence were quantified using confocal software.
Codetection of the Golgi Network and -PKC-GFP
Translocated by the Stimulation of Arachidonic Acid
Texas red-conjugated wheat germ agglutinin was used to monitor the
Golgi network. After induction of the translocation of -PKC-GFP by
100 µM arachidonic acid, the cells were fixed and treated with 0.3% Triton X and 10% NGS as described above, after which the cells were incubated with 0.5 µg/ml Texas red-conjugated wheat germ agglutinin (Molecular Probes, Leiden, Netherlands) in PBS-T for 30 min. Finally, the
fluorescence of Texas red and GFP were observed under a confocal laser
scanning fluorescent microscope, the former at 588-nm argon excitation
using a 590-nm-long pass barrier filter and the latter at 488-nm argon excitation using a 510-525-nm-band pass barrier filter.
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Results |
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Properties of -PKC and Its Fusion Protein with GFP
Enzymological and immunochemical properties of -PKC-
GFP and native
-PKC were examined. Fig. 1 A shows
that both the immunoprecipitated
-PKC and
-PKC-GFP
had basal kinase activity and that both were activated at
~3.3-fold the basal level in the presence of PS and DO.
These findings indicate that
-PKC-GFP also is dependent
on PS and DO and that its enzymological property is very
similar to that of the native
-PKC.
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Immunostaining with anti--PKC antibodies showed
that both
-PKC and
-PKC-GFP were expressed in the
cytoplasm of CHO-K1 cells before stimulation (Fig. 1 B, a
and b) and intense immunoreactivities on the plasma
membrane after treatment with 1 µM TPA (Fig. 1 B, d and
e). Within the same cells, localization of the GFP fluorescence of
-PKC-GFP (Fig. 1 B, b) corresponded to that of
the
-PKC-like immunoreactivity (Fig. 1 B, c). Colocalization of GFP and
-PKC-like immunoreactivity also was
observed after treatment with TPA (Fig. 1 B, e and f).
These results indicate that in response to TPA, both native
-PKC and
-PKC-GFP were translocated from the cytoplasm to the plasma membrane without proteolysis. This
was confirmed by immunoblotting using anti-
-PKC antibody (data not shown).
Effects of Fatty Acids on the Translocation of
- and
-PKC-GFP
The effect of a single application of a saturated fatty acid
(such as tridecanoic acid, myristic acid, pentadecanoic
acid, palmitic acid, heptadecanoic acid, or stearic acid) on
the subcellular localization of -PKC-GFP was investigated. All the saturated fatty acids examined induced the
translocation of
-PKC-GFP at 200 µM. Fig. 2 shows sequential pictures of the typical translocation of
-PKC-
GFP induced by saturated fatty acids. When
-PKC-GFP was expressed in CHO-K1 cells, intense fluorescence was
present throughout the cytoplasm, and faint fluorescence
was present in the nucleus. The presence of
-PKC-GFP
in the nucleus was obvious because the nucleolus appeared as a small black circle. Stimulation with 200 µM
tridecanoic acid, pentadecanoic acid, or palmitic acid
caused translocation of the
-PKC-GFP in the cytoplasm
to the plasma membrane at 20 s, but there was a return to
the cytoplasm within 2 min. These fatty acids, however,
had no effect on the
-PKC-GFP in the nucleus. These
fluorescence changes were confirmed by the profiles of
GFP intensity shown in Fig. 2. Myristic acid, heptadecanoic acid, and stearic acid also induced translocation of
-PKC-GFP similar to that shown in Fig. 2 (data not
shown).
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Unsaturated fatty acids also caused the translocation of
-PKC-GFP. Oleic and arachidonic acid at 200 µM induced a very rapid, transient translocation of
-PKC-GFP
from the cytoplasm to the plasma membrane similar to
that of saturated fatty acids (Fig. 3). Linolenic and docosahexaenoic acids induced
-PKC-GFP translocation similar to that of oleic and arachidonic acids (data not shown). Linoleic acid at 200 µM, however, occasionally translocated
-PKC-GFP to different components, but at a lower
concentration (100 µM), it induced a translocation from
the cytoplasm to the membrane similar to that of oleic and
arachidonic acids (Fig. 3). After rapid translocation to the
membrane caused by treatment with 200 µM linoleic acid,
the
-PKC-GFP on the plasma membrane faded slightly, and at 1 min it appeared as an accumulation of dots
throughout the cytoplasm. Finally,
-PKC-GFP again appeared as an accumulation of patchy dots on the plasma
membrane and on the nuclear membrane. It is noteworthy
that the target site for
-PKC-GFP on stimulation with linoleic acid depends on the concentration.
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Translocation of -PKC-GFP was also examined in response to various concentrations of the fatty acids (Table
I). Of the 11 fatty acids tested,
-PKC-GFP was most sensitive to pentadecanoic acid; ~38% of the cells showed
marked translocation in response to 50 µM pentadecanoic
acid. Tridecanoic acid, palmitic acid, heptadecanoic acid,
linoleic acid, and docosahexaenoic acid at 100 µM caused
translocation in more than 40% of the cells, whereas the
other fatty acids required more than 200 µM.
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The sensitivity of -PKC-GFP to fatty acids differed
from that of
-PKC-GFP (Table I). Except for tridecanoic, linoleic, and arachidonic acids, the fatty acids in
general had little effect on the translocation of
-PKC-
GFP. As shown in Table I, only five fatty acids (tridecanoic, pentadecanoic, linoleic, arachidonic, and docosahexaenoic acids) induced translocation when used alone at
200 µM. The other fatty acids required more than 300 µM
for activity or failed to translocate
-PKC-GFP. Tridecanoic acid was the most effective for the
-PKC-GFP
translocation, being more effective for
-PKC-GFP than
-PKC-GFP (Table I).
-PKC-GFP fluorescence was detected in the cytoplasm. Occasionally, fairly intense fluorescence was present
in the perinuclear region (Fig. 4 A, second row), but no
-PKC-GFP was present within the nucleus before stimulation. Except for palmitic acid, the saturated fatty acids
induced rapid, transient translocation of
-PKC-GFP from
the cytoplasm to the membrane (Fig. 4 A). An addition of
200 µM tridecanoic and pentadecanoic acid caused rapid
movement of
-PKC-GFP to the membrane and a return
to the cytoplasm at 2 min. Of the five unsaturated fatty acids tested, the translocation of
-PKC-GFP was reproducibly induced by linoleic, arachidonic, or docosahexaenoic
acid at 200 µM. The translocation induced by docosahexaenoic acid was similar to that induced by the saturated fatty acids in Fig. 4 A (data not shown). Linoleic and
arachidonic acids, however, induced a unique translocation of
-PKC-GFP (Fig. 4 B). Neither linoleic nor arachidonic acid at 200 µM induced rapid, reversible translocation of
-PKC-GFP to the plasma membrane, but both
induced slow, irreversible accumulation of
-PKC-GFP to
perinuclear regions. The accumulation of
-PKC-GFP in
the perinuclear region was evident 3 min after the stimulations with linoleic and arachidonic acids and was still
detectable 15 min after stimulation (data not shown).
Arachidonic acid-induced translocation occurred in the
presence of 10 µM of indomethacine, which inhibits the generation of eicosanoids from arachidonic acid (data not
shown) (Flower et al., 1985
).
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Immunostaining with anti--PKC and -
-PKC antibodies confirmed that these fatty acid-induced translocations
occurred in CHO-K1 cells that expressed native
- and
-PKC (data not shown).
Target Site of -PKC-GFP on Stimulation with
Arachidonic Acid
To identify the compartment in which -PKC-GFP accumulated in response to arachidonic acid, the Golgi network was made visible with Texas red-conjugated wheat
germ agglutinin after arachidonic acid induced the translocation of
-PKC-GFP.
GFP fluorescence was present throughout the cytoplasm, but not in the nucleus, and was most intense in the
perinuclear region (Fig. 5, left). The fluorescence of the
two Texas red-stained cells shown in a micrograph (Fig. 5,
center) indicates that one was the cell detected by GFP
fluorescence and the other was a cell that expressed no
-PKC-GFP. Intense Texas red fluorescence was present
around the nucleus. An overlapping image shows that the
Texas red fluorescence and GFP colocalized in the perinuclear region (Fig. 5, right).
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Effect of Ca2+ Chelators on Fatty Acid-induced
Translocation of - and
-PKC-GFP
To clarify whether the fatty acid-induced translocations of
- and
-PKC-GFP require Ca2+ ions, we examined the effects of Ca2+ chelators on the translocations. Pretreatment
with 2.5 mM EGTA and 30 µM BAPTA-AM blocked the
translocation of
-PKC-GFP induced by docosahexaenoic
acid, but not that of
-PKC-GFP (Fig. 6). Similarly, tridecanoic acid-induced translocation of
-PKC was inhibited by Ca2+ chelators, whereas translocation of
-PKC occurred in the presence of Ca2+ chelators (data not shown).
These findings suggest that the fatty acid-induced translocation of
-PKC-GFP is independent of Ca2+, whereas
Ca2+ is necessary for the fatty acid-induced translocation
of
-PKC.
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Synergistic Effect of Arachidonic Acid
and Diacylglycerol on the Translocation of
- and
-PKC-GFP
As shown in Fig. 7, DiC8 at 10 µM caused very rapid
translocation of -PKC-GFP to the plasma membrane at
15 s, but the translocated
-PKC-GFP returned to the initial state within 3 min (Fig. 7 A), and thereafter no translocation occurred. The application of 1 µM DiC8 alone, however, failed to induce the
-PKC-GFP translocation (Fig.
7 B). Similarly, use of arachidonic acid alone at 200 µM, but
not at 25 µM, caused translocation of
-PKC-GFP (Table
I). Simultaneous applications of 1 µM DiC8 and 25 µM
arachidonic acid induced remarkable translocation of
-PKC-GFP. 15 s after the coapplication, there was very
faint translocation to the plasma membrane, similar to that
induced by a single application of a low concentration of
DiC8 or arachidonic acid. Subsequently, about 3 min after
stimulation, slow translocation of
-PKC-GFP to the plasma membrane became evident. This late phase translocation was unidirected and irreversible (Fig. 7 C). This
synergistic action occurred in the presence of 10 µM indomethacine, indicating that arachidonic acid itself contributed to the unidirected and irreversible translocation
of
-PKC-GFP (data not shown). In contrast, the translocation of
-PKC-GFP was neither markedly enhanced nor altered by the simultaneous application of arachidonic
acid and DiC8.
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Discussion |
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Using - and
-PKC proteins fused with GFP, we showed
the fatty acid-induced translocation of PKCs in living cells
in real time. In order for the proteins' movement to be visible by monitoring the fluorescence of GFP fused to the
protein, the fusion proteins must have the same properties
as native PKCs. Furthermore, because PKCs are reported
to be proteolysed by proteases such as calpain (Kishimoto
et al., 1983
), we examined the enzymological and immunochemical properties of
- and
-PKC-GFP. Because
-PKC-GFP is reported to have the same properties as
native
-PKC (Sakai et al., 1997
), we analyzed the properties of
-PKC-GFP by a kinase assay, immunostaining, and Western blotting and found that (a) the activity of
-PKC-GFP was dependent on PS and DO (Fig. 1 A); (b)
the
-PKC-GFP images immunostained with anti-
-PKC
antibody coincided with the fluorescences of GFP before
and after TPA stimulation (Fig. 1 B); and (c) no
-PKC-
GFP-degraded product was found in the immunoblot
analysis, even after TPA treatment (data not shown).
These findings indicate that the GFP fluorescence fused to
-PKC can be used as a marker for native
-PKC.
We investigated the effects of saturated fatty acids with
carbon chain lengths of C13 to C18 and unsaturated fatty
acids on the translocation of - and
-PKC-GFP because
these fatty acids are known to potentiate the activity of
PKC subspecies (Kasahara and Kikkawa, 1995
). Of the 11 fatty acids examined, the translocation of
-PKC was induced more effectively by the saturated than the unsaturated fatty acids, whereas the kinase activity was enhanced more effectively by the unsaturated fatty acids (Shinomura et al., 1991
). Saturated fatty acids enhanced the kinase activity of both
- and
-PKC by the synergistic action of diacylglycerol, and
-PKC was more sensitive to
saturated fatty acids than
-PKC (Shinomura et al., 1991
;
Kasahara and Kikkawa, 1995
). The translocation of
-PKC,
however, was less sensitive to the saturated fatty acids than was that of
-PKC and was not induced synergistically in the presence of arachidonic acid and DiC8. These
discrepancies may be due to the different methods used to
detect the effects of the fatty acids; the translocation of
PKC was monitored after the extracellular fatty acid application, whereas PKC activity was assayed by applying the
fatty acids directly to the PKCs in vitro. Taking into account that translocation was induced by phorbol ester,
even in the presence of a PKC inhibitor (Sakai et al., 1997
), an increase in the kinase activity of PKC does not
always correspond to the translocation of PKC.
Except for linoleic acid at a high concentration, all the
fatty acids induced similar translocations of -PKC: rapid,
reversible translocation from the cytoplasm to the plasma
membrane. In contrast, both arachidonic and linoleic acids
generated the slow translocation of
-PKC from the cytoplasm to the perinuclear area, and saturated fatty acids induced a translocation of
-PKC similar to that of
-PKC.
The target site of the arachidonic acid-induced translocation of
-PKC was identified as the Golgi network. This is
consistent with previous reports showing that
-PKC was
localized to the Golgi via its zinc-finger domain (Lehel et al.,
1995
) and that arachidonic acid stimulated
-PKC redistribution in heart cells (Huang et al., 1997
). These findings
indicate that the target site of
-PKC differs in response to
the fatty acid used and strongly suggest that the mechanism of translocation induced by arachidonic acid differs
for
- and
-PKC. These interpretations are supported by
the different effects of fatty acids on the kinase activities of PKC subspecies in vitro (Shinomura et al., 1991
; Koide
et al., 1992
; Kasahara and Kikkawa, 1995
). Saturated fatty
acids with carbon chain lengths of C12 to C14 activated
-,
-,
-, and
-PKC, but not
-PKC. Phorbol ester synergically enhanced the activity of
-,
-, and
- PKCs when simultaneously treated with fatty acids, whereas it suppressed the activity of
-PKC.
Some fatty acids mobilize intracellular Ca2+ (Gamberucci et al., 1997; Schaloske et al., 1998). We therefore
clarified whether fatty acid translocation depends on an
increase in the intracellular Ca2+ concentration. Docosahexaenoic and tridecanoic acids induced translocation of
-PKC in the presence of Ca2+ chelators, indicating that
the fatty acid-induced translocation of
-PKC is independent of the increase in the intracellular Ca2+ concentration, although none of the fatty acids were tested in the
presence of Ca2+ chelators. In contrast, neither docosahexaenoic nor tridecanoic acid caused the translocation of
-PKC in the presence of Ca2+ chelators. This suggests
that fatty acids cause the translocation of
-PKC through
an increase in the intracellular Ca2+ concentration, but it
does not exclude the possibility that they directly act on
-
and
-PKC and induce their translocation. Intracellular Ca2+ appears to be indispensable for
-PKC translocation
because the DiC8-induced translocation of
-PKC was
blocked by Ca2+ chelators (data not shown).
Furthermore, arachidonic acid increases the activities of
-PKC and
-PKC by the synergistic action of DiC8
(Kasahara and Kikkawa, 1995
). As shown in Fig. 7, arachidonic acid also increased translocation sensitivity to DiC8
and then induced the additional, unidirectional translocation of
-PKC. In contrast, synergistic translocation of
-PKC could not be induced by arachidonic acid and
DiC8. Oancea et al. (1998)
also detected no synergistic effect of arachidonic acid on DiC8-induced translocation of
the first cystein-rich region (Cys-1) of
-PKC, but they did
show that arachidonic acid inhibited the DiC8-induced
translocation of the Cys-1 of PKC to the plasma membrane. These findings suggest that the synergistic effect of
arachidonic acid on
-PKC activity occurs through an unknown mechanism that differs from the targeting mechanism by arachidonic acid of Cys-1 (or
-PKC) to the Golgi
complex.
The anchoring proteins of PKC, RACKs, have recently
been identified (Mochly-Rosen et al., 1991). RACKs bind
activated PKC (Jaken, 1997
) and are thought to be involved in PKC translocation. To clarify the mechanism of
PKC translocation, it is necessary to examine the effects of
these anchoring proteins on PKC translocation using
GFP-labeled PKC in living cells.
In conclusion, each PKC subspecies has a different targeting mechanism that depends on the extracellular stimuli used, i.e., the fatty acid. Moreover, the synergistic actions of intracellular activators have an important role in targeting, thereby contributing to the subspecies-specific function.
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Footnotes |
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Received for publication 22 May 1998 and in revised form 10 August 1998.
Y. Shirai and K. Kashiwagi equally contributed to the performance of all
the experiments and writing of the manuscript.
Address all correspondence to Naoaki Saito, Laboratory of Molecular
Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. Tel.: 81-78-803-1251. Fax: 81-78-803-0993. E-mail: naosaito{at}kobe-u.ac.jp
We thank Dr. Yasutomi Nishizuka for his helpful discussions of our work.
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Yamanouchi Foundation for Research on Metabolic Disorders, and the Kato Memorial Bioscience Foundation.
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Abbreviations used in this paper |
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cPKC, nPKC, and aPKC, conventional, novel, and atypical PKCs; DAG, diacylglycerol; DiC8, 1,2-dioctanoylgrecerol; DO, diolein; GFP, green fluorescent protein; PKC, protein kinase C; PS, phosphatidylserine; TPA, 12-o-tetradecanoylphorbol 13-acetate.
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