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Molecular and Cellular Biology, March 2001, p. 1769-1783, Vol. 21, No. 5
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1769-1783.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Subtype-Specific Translocation of the
Subtype
of Protein Kinase C and Its Activation by Tyrosine Phosphorylation
Induced by Ceramide in HeLa Cells
Taketoshi
Kajimoto,
Shiho
Ohmori,
Yasuhito
Shirai,
Norio
Sakai, and
Naoaki
Saito*
Laboratory of Molecular Pharmacology,
Biosignal Research Center, Kobe University, Nada-ku, Kobe 657-8501, Japan
Received 19 July 2000/Returned for modification 30 August
2000/Accepted 7 December 2000
 |
ABSTRACT |
We investigated the functional roles of ceramide, an intracellular
lipid mediator, in cell signaling pathways by monitoring the
intracellular movement of protein kinase C (PKC) subtypes fused to
green fluorescent protein (GFP) in HeLa living cells. C2-ceramide but not C2-dihydroceramide induced
translocation of
PKC-GFP to the Golgi complex, while
PKC- and
PKC-GFP did not respond to ceramide. The Golgi-associated
PKC-GFP
induced by ceramide was further translocated to the plasma membrane by
phorbol ester treatment. Ceramide itself accumulated to the Golgi
complex where
PKC was translocated by ceramide. Gamma interferon
also induced the
PKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and
Mg2+-dependent neutral sphingomyelinase. Photobleaching
studies showed that ceramide does not evoke tight binding of
PKC-GFP
to the Golgi complex but induces the continuous association and
dissociation of
PKC with the Golgi complex. Ceramide inhibited the
kinase activity of
PKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of
PKC-GFP
immunoprecipitated from ceramide-treated cells was increased. The
immunoprecipitated
PKC-GFP was tyrosine phosphorylated after
ceramide treatment. Tyrosine kinase inhibitor abolished the
ceramide-induced activation and tyrosine phosphorylation of
PKC-GFP.
These results suggested that gamma interferon stimulation followed by
ceramide generation through Mg2+-dependent sphingomyelinase
induced
PKC-specific translocation to the Golgi complex and that
translocation results in
PKC activation through tyrosine
phosphorylation of the enzyme.
 |
INTRODUCTION |
Protein kinase C (PKC) is a family
of phospholipid-dependent serine/threonine protein kinases consisting
of at least 10 subspecies that can be classified into three subgroups,
classical, novel, and atypical PKC (43, 44, 52, 54). The
classical PKC members (
,
I,
II, and
), each of which has a
Ca2+ binding region (C2 region) and two cysteine-rich
regions, are activated by Ca2+, phosphatidylserine (PS),
and diacylglycerol (DG) or phorbol esters. The novel PKC members (
,
,
, and
), lacking the C2 region, are activated by PS and DG
or phorbol esters without Ca2+. The atypical PKC members
(
and
/
), which lack the C2 region and have only one
cysteine-rich region, are dependent on PS but are not affected by DG,
phorbol esters, or Ca2+ (52). Although a
considerable number of studies have demonstrated the involvement of PKC
in various cellular functions (2, 6, 42, 45, 62), the
individual roles of each PKC subtype in cellular functions remain
unclear. Recent studies in living cells using green fluorescent protein
(GFP)-tagged PKC have shown that each PKC subtype has a spatially
and temporally different targeting mechanism that is dependent on the
extracellular signals contributing to the subspecies-specific functions
of PKC (46, 49, 56, 59). Based on these findings, it was
proposed that the PKC targeting mechanism is a determinant for the
specific function of each PKC subtype in response to various stimuli in
various cell types.
Ceramide has recently emerged as an intracellular lipid mediator
implicated in various cellular responses, such as programmed cell
death, cell differentiation, growth inhibition, and long-term depression of synaptic transmission (5, 18, 19, 24, 47, 51, 64,
65). Ceramide is generated by transient hydrolysis of
sphingomyelin, and many reports have indicated that ceramide is
produced via receptor-mediated stimulation by various extracellular ligands, including vitamin D3 (50), gamma
interferon (IFN-
) (25), tumor necrosis factor alpha
(TNF-
) (11, 25), interleukin-1 (36), and
nerve growth factor (5, 10). Recently, the regulation of
PKC activity by ceramide has been reported, but the results are still
controversial; ceramide has been shown to activate
PKC or inhibit
PKC autophosphorylation in renal mesangial cells in vitro
(22). Furthermore, it is also reported that ceramide
induces the translocation of
PKC and
PKC from the membrane to the
cytosol in human myelogenous leukemia HL-60 cells (57) or
of
PKC from the cytosol to the membrane in renal mesangial cells and
in smooth muscle cells (22, 23). These apparently
contradictory results may have been due to differences not only in
methods but also in time points and cell types examined, suggesting the
necessity to observe the localization of each PKC subtype after
ceramide treatment continuously in living cells.
In the present study, we investigated the intracellular movement
of GFP-tagged PKC subtypes in living cells after treatment with various
stimuli, such as ceramide and IFN-
. We also examined the effect of
ceramide on the kinase activity of PKC subtypes. We demonstrated here
the
PKC-specific translocation to the Golgi complex by ceramide and
the activation of
PKC through tyrosine phosphorylation of the enzyme.
 |
MATERIALS AND METHODS |
Materials.
D-Erythro-C2-ceramide and
D-erythrodihydro-C2-ceramide were purchased
from Biomol Research Laboratories (Plymouth Meeting, Pa.).
D-Erythro-C6-ceramide and
6-{[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]
hexanoyl} sphingosine (C6-NBD-ceramide) were purchased from Molecular Probes (Eugene, Oreg.).
12-O-Tetradecanoylphorbol-13-acetate (TPA) was purchased
from Sigma Chemical Co. (St. Louis, Mo.). D609 was purchased from
Cayman Chemical (Ann Arbor, Mich.). IFN-
was a kind gift from
Shionogi Research Laboratory and Co., Ltd. (Osaka, Japan). TNF-
was
purchased from Gibco BRL (Grand Island, N.Y.). Scyphostatin was a kind
gift from Takeshi Ogita (Biomedical Research Laboratories, Sankyo Co.,
Ltd., Tokyo, Japan). Glutathione (GSH) was purchased from Nacalai
Tesque Inc. (Kyoto, Japan). Tyrphostin AG490 and genistein were
purchased from Calbiochem-Novabiochem Co. (La Jolla, Calif.).
Anti-
PKC,
PKC, and
PKC monoclonal antibodies were purchased
from Transduction Laboratories (Lexington, Ky.). Anti-
PKC and
PKC
polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc.
(Santa Cruz, Calif.). Anti-
PKC polyclonal antibody was purchased
from Upstate Biotechnology (Lake Placid, N.Y.) and that used for
immunoblotting was produced as described previously (53).
Anti-phosphotyrosine antibody, clone 4G10, was purchased from Upstate
Biotechnology. Peroxidase-conjugated goat anti-mouse or anti-rabbit
immunoglobulin G (IgG) was purchased from Amersham Corp. (Arlington
Heights, Ill.). Cy3-labeled goat anti-mouse or anti-rabbit IgG was
purchased from Amersham Corp. Calf thymus H1 histone was purchased from
Boehringer GmbH (Mannheim, Germany). Myelin basic protein (MBP) was
purchased from Sigma Chemical Co. Sodium fluoride (NaF) was purchased
from Nacalai Tesque Inc. Sodium orthovanadate was purchased from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan). All the other chemicals used were of analytical grade.
Cell culture.
HeLa cells and HEK293 cells were purchased
from Riken Cell Bank (Tsukuba, Japan). The CHO-K1 cell strain was a
gift from Masahiro Nishijima (National Institute of Health, Tokyo,
Japan). HeLa cells were cultured in minimum essential medium (Gibco
BRL) which was buffered with 44 mM NaHCO3 and supplemented
with 10% fetal bovine serum (FBS) in 5% CO2 at 37°C in
a humidified incubator. HEK293 cells were maintained in minimum
essential medium supplemented with 44 mM NaHCO3 and 10%
horse serum in a humidified atmosphere containing 5% CO2
at 37°C. CHO-K1 cells were cultured as described previously
(49, 59). All media were supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml), and the FBS used was not heat inactivated.
Preparation of
PKC-GFP adenovirus.
The adenoviral
plasmids (pAdEasy-1 and pAdEasy-2) and the shuttle vectors (pShuttle,
pShuttle-CMV, pAdTrack, and pAdTrack-CMV) were gifts from Bert
Vogelstein (Johns Hopkins University Oncology Center, Baltimore, Md.).
Generation of an adenoviral vector including
PKC-GFP cDNA was
performed according to the methods described on the web site of Johns
Hopkins University Oncology Center
(http://www.coloncancer.org/adeasy.html). Briefly, the plasmid
encoding GFP (BS 348) was digested by BgIII and
HindIII. The plasmid encoding
PKC (BS390) was
digested with XhoI and BamHI, the digested
products were subcloned into the XhoI and
HindIII sites of the expression vector pShuttle-CMV, and
the new plasmid was designated pBsAd010.
For the production of recombinant virus (
PKC-GFP in pAdEasy-1),
pBsAd010 linearized with PmeI was cotransformed with
supercoiled circular pAdEasy-1 into competent Escherichia
coli BJ5183 cells by electroporation. Transformation yielded
approximately 10 kanamycin-resistant clones, of which about two-thirds
contained recombinants, based on the sizes of undigested miniprep
plasmid DNA. Candidate clones were digested with several restriction
endonucleases to verify proper recombination. HEK293 cells at 50 to
70% confluency were prepared in 25-cm2 flasks for
transfection of adenoviral vector. Recombinant adenoviral vector DNA
(~4 µg) including
PKC-GFP was digested with PacI and transfected into HEK293 cells by lipofection using TransIT-LT2 (Mirus,
Madison, Wis.) according to the manufacturer's standard protocol.
Transfected cells were monitored for GFP expression and were collected
7 to 10 days after transfection and then resuspended in 2 ml of
phosphate-buffered saline without Ca2+ or Mg2+
[PBS(
)]. After three cycles of freezing in a methanol-dry ice bath
and rapid thawing at 37°C, 500 µl of viral lysate was used to
infect 106 cells in 25-cm2 flasks. Three or
four days later, viruses were harvested as described above. To generate
higher titer viral stocks, this process was repeated three times. In
the final round, a total of 108 cells in 24 75-cm2 flasks were used to obtain a multiplicity of
infection of 5. Three to five days after the final infection, the
resultant viruses were purified by CsCl banding, and final yields were
measured as described previously (21). Final yields were
generally 1011 to 1012 PFU.
Expression of
PKC-,
PKC-, and
PKC-GFP or
PKC protein
in HeLa cells.
GFP was fused to the C terminus of each PKC
subtype. For lipofection, plasmids (~5.5 µg) including
PKC-,
PKC-, and
PKC-GFP or
PKC cDNA were transfected into 5 × 106 HeLa cells using TransIT-LT2 (Mirus) according to the
manufacturer's standard protocol. For adenoviral infection, HeLa cells
(5 × 106) were incubated in serum-free medium for
2 h with recombinant
PKC-GFP adenovirus with a multiplicity of
infection of 10 in a humidified atmosphere containing 5%
CO2 at 37°C. After infection, the viral supernatant was
removed, and the cells were cultured in normal serum-containing medium.
Adenoviral infection was performed in the immunoblotting experiments
and kinase assays. The fluorescence of
PKC-,
PKC-, and
PKC-GFP
became detectable 16 h after transfection. All experiments were
performed 2 days after transfection.
Observation of
PKC-,
PKC-, and
-PKC-GFP
translocation.
HeLa cells expressing
PKC-,
PKC-, and
PKC-GFP were spread onto glass-bottomed culture dishes (MatTek,
Ashland, Mass.) and cultured for at least 16 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. The
fluorescence of GFP was monitored under a confocal laser scanning
fluorescence microscope (LSM 410 or 510; Carl Zeiss, Jena, Germany) at
a 488-nm excitation wavelength with a 515-nm-long pass or 510- to
525-nm band pass barrier filter. Translocation of the fusion protein
was triggered by addition of various stimulators at high concentrations
into the HEPES buffer to obtain the appropriate final concentrations.
All experiments were performed at 37°C.
Immunostaining of endogenous
PKC,
PKC, and
PKC or
expressed
PKC.
Before and after treatment with 10 µM
C2-ceramide or C6-NBD-ceramide, HeLa cells were
fixed with a fixative containing 4% paraformaldehyde and 0.2% picric
acid in 0.01 M PBS (pH 7.4) for 30 min. After washing twice with PBS,
the cells were treated with PBS containing 0.3% Triton X-100 and 5%
normal goat serum (NGS) for 10 min. The cells were then incubated with
the anti-
PKC and -
PKC monoclonal antibodies (diluted 1:200) or
the anti-
PKC polyclonal antibody (Upstate Biotechnology) (diluted
1:200) in PBS with 0.03% Triton X-100 (PBS-T) and 5% NGS for 1 h
at 25°C. After washing in PBS-T, the cells were incubated with
Cy3-labeled goat anti-mouse or anti-rabbit IgG (diluted 1:1,000) for 30 min. After three washes with PBS-T for 10 min each time, the
fluorescence of Cy3 was observed under a confocal laser scanning
fluorescent microscope with excitation at 588 nm using a 590-nm-long
pass barrier filter.
Codetection of the Golgi complex and
PKC-GFP translocated by
ceramide.
Texas red-conjugated wheat germ agglutinin was used to
monitor the Golgi complex. After translocation of
PKC-GFP by 10 µM C2-ceramide, the cells were fixed with a fixative
containing 4% paraformaldehyde and 0.2% picric acid in 0.01 M PBS (pH
7.4) for 30 min. After washing twice with PBS, the cells were treated
with PBS containing 0.3% Triton X-100 and 5% NGS for 10 min. The
cells were then incubated with 1 µg of Texas red-conjugated wheat
germ agglutinin (Molecular Probes, Leiden, The Netherlands) per ml for
40 min in PBS-T and 5% NGS. After three washes with PBS-T for 10 min
each time, the fluorescence of Texas red and GFP was observed under a
confocal laser scanning fluorescent microscope, with excitation at 488 nm using a 510- to 525-nm band pass barrier filter for the former and
with excitation at 588 nm using a 590-nm-long pass barrier filter for
the latter.
Cell fractionation and immunoprecipitation.
For detection of
endogenous PKC isozymes in HeLa cells, cultured HeLa cells were
harvested with 1 ml of homogenate buffer (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 20 mM Tris-HCl, 200 µg of leupeptin per ml, 1 mM
phenylmethylsulfonyl fluoride, pH 7.4) and centrifuged at
2,000 × g. For immunoblot analysis of tyrosine phosphorylation, we also used the homogenate buffer containing 10 mM
NaF and 1 mM Na3VO4. The cells were resuspended
with 300 µl of homogenate buffer containing 1% Triton X-100 and
sonicated (UD-210; Tomy Seiko Co. Ltd., Tokyo, Japan) (output, 5; duty, 50%) 10 times at 4°C, and the supernatant was used after
centrifugation at 19,000 × g for 15 min.
For subcellular fractionation, the HeLa cells transfected with the
adenoviral vector containing

PKC-GFP were treated with
10 µM
C
2-ceramide for 20 min at 37°C and then harvested with 1
ml of homogenate buffer and centrifuged at 2,000 ×
g.
The cells
were resuspended with 300 µl of homogenate buffer and
sonicated
as described above. The samples were centrifuged at
55,000 ×
g for 30 min at 4°C, and the supernatant
was collected as the cytosol
fraction. The pellet was sonicated with
300 µl of homogenate buffer
containing 1% Triton X-100 and
centrifuged at 19,000 ×
g for 15
min, and then the
supernatant was collected as the particulate
fraction.
For immunoprecipitation of endogenous

PKC,

PKC, or

PKC or
expressed

PKC-GFP, the cytosol, particulate, or total fraction
was
rotated with the subtype-specific antibodies (anti-

PKC,

PKC,
or

PKC) or anti-GFP polyclonal antibody (Molecular Probes) (diluted
1:50) for 2 h at 4°C and then with protein G-Sepharose for an
additional 2 h. Samples were centrifuged at 2,000 ×
g for 5 min
at 4°C, and pellets were washed three times with
PBS(

).
Finally, the pellet was suspended in 50 µl of PBS(

) and used for
phosphorylation or immunoblotting studies as described
below.
Immunoblotting analysis.
The same amounts of samples from
each fraction were subjected to sodium dodecyl sulfate-7.5%
polyacrylamide gel electrophoresis according to the method of Laemmli
(28), and the separated proteins were electorophoretically
transferred onto polyvinylidene difluoride (PVDF) filters (Millipore
Co., Bedford, Mass.). Nonspecific binding sites on the PVDF filters
were blocked by incubation with 5% nonfat milk or 2% bovine serum
albumin in PBS-T for 18 h. The PVDF filters were then incubated
with the anti-PKC monoclonal antibodies (
PKC,
PKC, or
PKC)
(diluted 1:1,000), the anti-PKC polyclonal antibodies (
PKC,
PKC,
or
PKC) (diluted 1:1,000), the anti-GFP polyclonal antibody
(Clontech, Palo Alto, Calif.) (diluted 1:1,000), or
anti-phosphotyrosine antibody (diluted 1:1,000) for 1 h at 25°C.
After washing in PBS-T, the filters were incubated with
peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (diluted
1:10,000) for 30 min. After three rinses, the immunoreactive bands were
visualized using an enhanced chemiluminescence detection kit
(Amersham). After immunoblot using anti-phosphotyrosine antibody, the
PVDF filter was washed according to the manufacturer's protocol for
reprobing with anti-GFP polyclonal antibody.
Fluorescence photobleaching.
Photobleaching experiments were
performed as follows. After translocation of
PKC-GFP by 10 µM
C2-ceramide, a circular region in the Golgi complex or a
square region of perikarya within a plane of the cell was photobleached
by scanning for 15 s with an argon laser of the highest power. Recovery
and fading of fluorescence in the selected regions were then analyzed
by imaging the entire cell by confocal fluorescent microscopy with low
laser power at the indicated times after photobleaching. For all of the
images, the noise levels were reduced by line scan averaging.
In vitro and in vivo kinase assay of
PKC.
The
immunoprecipitated samples (10 µl of suspended pellet) were used for
both in vitro and in vivo kinase assays. In vitro kinase assays of
PKC-GFP expressed in HeLa cells were performed as described
previously (49). Briefly, the kinase activity in 10 µl
of each sample was assayed by measuring the incorporation of
32Pi into H1 histone from
[
-32P]ATP in the presence of PS (8 µg/ml), diolein
(DO) (0.8 µg/ml), or C2-ceramide at various
concentrations. For the in vivo kinase assay, endogenous
PKC,
PKC, or
PKC or exogenous
PKC-GFP was immunoprecipitated from
HeLa cells at various time points after stimulation with 10 µM
C2-ceramide, and then the kinase activities were measured
with H1 histone (
PKC and
PKC) or MBP (
PKC) as the substrate
without any PKC activators such as PS, DO, or C2-ceramide.
 |
RESULTS |
Expression of PKC isozymes in HeLa cells.
The expression of
endogenous PKC subtypes in HeLa cells was examined by immunoblotting
using subtype-specific antibodies. In HeLa cell lysates, each antibody
against
PKC,
PKC, or
PKC detected an immunoreactive band of
reasonable molecular weight, while
and
subtypes of PKC were not
detected, indicating that the
,
, and
subtypes of PKC are
expressed in HeLa cells (Fig. 1). In this
study, therefore, we focused on the localization of
PKC,
PKC, and
PKC but not of
PKC or
PKC.

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FIG. 1.
Immunoblotting analysis of endogenous PKC subtypes in
HeLa cells. Total cell lysates (25 µg) extracted from HeLa cells
(lanes 1) were separated by sodium dodecyl sulfate-7.5%
polyacrylamide gel electrophoresis, transferred onto nitrocellulose
membranes, and stained with antibodies against each PKC subtype. Rat
brain homogenate was used as a positive control for PKC, PKC,
PKC, and PKC, and recombinant PKC expressed in CHO-K1 cells
was used as a positive control for PKC (lanes 2). The , , and
subtypes of PKC were detected with reasonable molecular masses in
HeLa cells. The results shown are representative of two independent
experiments.
|
|
Translocation of
PKC,
PKC, and
PKC induced by
C2-ceramide and phorbol ester.
When
PKC- and
PKC-GFP were expressed in HeLa cells, intense and homogeneous
fluorescence of
PKC- and
PKC-GFP was observed throughout the
cytoplasm, with no signals detected in the nucleus (Fig. 2A and
C). In contrast, faint but significant
fluorescence of
PKC-GFP was seen in the nucleus in addition to
intense fluorescence in the cytoplasm, and occasionally,
PKC-GFP was
found more densely in the perinuclear region than in the surrounding
cytoplasm (Fig. 2A and C). Localization of the fluorescence did not
change for at least 60 min when observed under a confocal laser
scanning fluorescence microscope without stimulation.

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FIG. 2.
Ceramide- or TPA-induced translocation of PKC subtypes
in HeLa cells. (A) C2-ceramide (C2-Cer)-induced
translocation of PKC-, PKC-, and PKC-GFP overexpressed in HeLa
cells. PKC-, PKC-, and PKC-GFP were seen throughout the
cytoplasm of HeLa cells, and faint signals for PKC-GFP were also
seen in the nucleus. The addition of 10 µM C2-ceramide
induced translocation of PKC-GFP but not of PKC- or PKC-GFP
from the cytoplasm to the perinuclear region. (B) Immunocytochemical
localization of endogenous PKC, PKC, and PKC before and after
C2-ceramide treatment in HeLa cells. Endogenous PKC,
PKC, and PKC were visualized by immunostaining with anti- PKC,
PKC, or PKC antibodies and Cy3-labeled secondary antibodies. The
addition of 10 µM C2-ceramide induced the accumulation of
endogenous PKC but not of PKC or PKC to the perinuclear
region. (C) TPA-induced translocation of PKC-, PKC-, and
PKC-GFP overexpressed in HeLa cells. TPA at 1 µM induced
translocation of PKC-GFP from the cytoplasm to the plasma membrane.
TPA at 1 µM induced translocation of PKC-GFP from the cytoplasm
and nucleoplasm to the plasma membrane and nuclear membrane,
respectively. Application of 1 µM TPA failed to induce translocation
of PKC-GFP. The results shown are representative of three
independent experiments. Bars, 10 µm.
|
|
The effects of C
2-ceramide, a membrane-permeable analogue
of ceramide, on the cellular localization of

PKC-,

PKC, and

PKC-GFP
were investigated in HeLa cells. The

PKC-GFP accumulated
significantly
in the perinuclear region after treatment with
C
2-ceramide at
10 µM. The intensity of fluorescence in
the perinuclear region
reached the maximum level at 20 min after
treatment. The

PKC-GFP
in the nucleoplasm was not altered by
C
2-ceramide. The fluorescence
remained in the perinuclear
region for at least 60 min after C
2-ceramide
treatment and
did not return to the cytoplasm. On the other hand,
application of 10 µM C
2-ceramide failed to induce any translocation
of

PKC- or

PKC-GFP (Fig.
2A). To verify the

PKC-specific
translocation
by ceramide, we further examined the effect of ceramide
on endogenous

PKC,

PKC, and

PKC in HeLa cells by
immunocytochemistry. As
shown in Fig.
2B, endogenous

PKC but not

PKC or

PKC was significantly
accumulated in the perinuclear
region after the treatment, as
seen in the case of

PKC-GFP. TPA at 1 µM induced translocation
of both

PKC- and

PKC-GFP but not of

PKC-GFP from the cytoplasm
to the plasma membrane within 15 min.
Translocation from the nucleoplasm
to the nuclear membrane was also
observed only in the case of

PKC-GFP (Fig.
2C). The fluorescence of

PKC- and

PKC-GFP remained
on the plasma membrane or on the
nuclear membrane for at least
60 min after TPA
treatment.
C
2-dihydroceramide, a derivative of C
2-ceramide
lacking the C
4-C
5 double bond of the sphingoid
backbone (
4), did not induce
significant translocation of

PKC-GFP (Fig.
3A).
C
2-ceramide is
known to be converted to
C
2-sphingomyelin by sphingomyelin synthase
(SMS) (
35,
55). To determine whether C
2-ceramide but not
C
2-sphingomyelin
translocates

PKC-GFP, we studied the
effects of D609, an inhibitor
of SMS (
35,
41,
58), on the
C
2-ceramide-induced translocation
of

PKC-GFP.
Pretreatment with 200 µg of D609/ml for 30 min failed
to inhibit the
C
2-ceramide-induced translocation of

PKC-GFP (Fig.
3B).
After C
2-ceramide-induced translocation of

PKC-GFP to
the
perinuclear region, TPA treatment (1 µM, 3 min) induced
translocation
of both the perinuclear and cytosolic

PKC-GFP to the
plasma membrane
(Fig.
3C). The

PKC-GFP in the nucleoplasm was
translocated to
the nuclear membrane 10 min after TPA treatment (data
not shown).

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FIG. 3.
Characterization of the ceramide-induced translocation
of PKC-GFP. (A) C2-dihydroceramide
(DH-C2-Cer) (10 µM), an inactive ceramide, failed to
induce translocation of PKC-GFP. (B) Preincubation with D609 (200 µg/ml) for 30 min did not affect the C2-ceramide
(C2-Cer)-induced translocation of PKC-GFP. (C) After
translocation of PKC-GFP to the perinuclear region by treatment with
10 µM C2-ceramide for 20 min, 1 µM TPA irreversibly
induced translocation of all the PKC-GFP to the plasma membrane
within 3 min. The results shown are representative of three independent
experiments. Bars, 10 µm.
|
|
Translocation of

PKC-GFP by ceramide was further examined by
immunoblotting analysis. To determine whether

PKC-GFP was
translocated
from the cytosol to the particulate fraction by
C
2-ceramide, immunoblotting
analysis was performed.

PKC-GFP was immunoprecipitated from the
transfected HeLa cells using
anti-N terminus of

PKC monoclonal
antibody and stained with anti-C
terminus of

PKC polyclonal antibody
as described in Materials and
Methods. As shown in Fig.
4 (left
panel),

PKC-GFP was predominantly present in the cytosolic fraction
before
C
2-ceramide treatment, and C
2-ceramide (10 µM, 20 min)
caused translocation of

PKC-GFP from the cytosol to
the particulate
fraction. In addition, we obtained the same results
using anti-GFP
antibody instead of anti-N terminus of

PKC antibody
for immunoprecipitation
(data not shown). These results suggested that
no degradation
of

PKC-GFP occurred in the nontreated cells or even
in the cells
treated with C
2-ceramide. We further examined
whether the total
amounts of

PKC-GFP were altered during
C
2-ceramide treatment
by immunoblot analysis using
anti-

PKC monoclonal antibody. The
amount of

PKC-GFP in the total
homogenate of transfected HeLa
cells was not altered by
C
2-ceramide treatment (Fig.
4, right
panel).

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FIG. 4.
Ceramide-induced translocation of PKC-GFP assessed by
immunoblotting analysis. With immunoblotting analysis, PKC-GFP
was detected as a 110-kDa band that was more abundant in the cytosolic
fraction (c). C2-ceramide treatment (10 µM, 20 min)
induced translocation of PKC-GFP from the cytosolic fraction to the
particulate fraction (p). No degradation products were detected before
or after treatment with C2-ceramide (left panel). The level
of PKC-GFP in the total homogenate was not changed after ceramide
treatment for 20 min (right panel). The results shown are
representative of three independent experiments.
|
|
Intracellular movement of ceramide.
To compare the time course
of
PKC-GFP translocation with permeation of ceramide into cells, we
monitored the movement of ceramide in HeLa cells using a fluorescent
analogue of ceramide, C6-NBD-ceramide. After application of
10 µM C6-NBD-ceramide to HeLa cells, the fluorescence of
C6-NBD-ceramide was first detected on the plasma membrane
at 1 min, and weak signals were in the perinuclear region, and then the
intensity of the fluorescence on the plasma membrane gradually
increased until 10 min. Obvious accumulation of the fluorescence was
seen in the perinuclear region at 3 min, and the intensity of
fluorescence was markedly increased at 10 min (Fig. 5A, top
row). The C6-NBD-ceramide
accumulated at the perinuclear region was not altered by TPA treatment
(1 µM, 15 min) (data not shown). C6-ceramide also induced
the translocation of
PKC-GFP similarly to the effect of
C2-ceramide. The accumulation of
PKC-GFP was first seen
at 1 min and was apparent 3 min after treatment with
C6-ceramide, and the intensity of GFP fluorescence in the
perinuclear region increased until 10 min (Fig. 5A, bottom row) and
reached a maximum at 20 min (data not shown).

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FIG. 5.
Localization of fluorescent ceramide
(C6-NBD-ceramide) in HeLa cells. (A)
C6-NBD-ceramide (NBD-C6-Cer) at 10 µM rapidly
accumulated on the plasma membrane and perinuclear region of HeLa cells
1 min after the treatment, and the fluorescence in the perinuclear
region increased significantly until 10 min (top row).
C6-ceramide (C6-Cer)-induced translocation of
PKC-GFP showed a similar time course to that of
C6-NBD-ceramide (bottom row). (B) HeLa cells transfected
with PKC were fixed after treatment with 10 µM
C6-NBD-ceramide for 20 min. Cells were immunostained with
anti- PKC monoclonal antibody and with Cy3-labeled IgG as secondary
antibody to make the expressed PKC visible. The localization of
C6-NBD-ceramide (NBD) is shown in green (left) and of
PKC is shown in red (center). On the merged image, the overlapped
signals of C6-NBD-ceramide and Cy3 appear in yellow
(right). The results shown are representative of three independent
experiments. Bars, 10 µm (A, top row) and 20 µm (A, bottom row, and
B).
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To identify whether C
6-ceramide translocates

PKC-GFP to
the same intracellular compartment that C
6-NBD-ceramide
accumulates
in, the

PKC was visualized with anti-

PKC monoclonal
antibody
in HeLa cells overexpressing

PKC after
C
6-NBD-ceramide treatment.
As shown in Fig.
5B, intense NBD
fluorescence was present in the
perinuclear region.

PKC
immunoreactivity also accumulated in
the perinuclear region. Merged
images showed that the fluorescence
of NBD and

PKC immunoreactivity
were colocalized in the perinuclear
region, indicating that
C
6-NBD-ceramide and

PKC are targeted
to the same
perinuclear
compartment.
Translocation of
PKC-GFP induced by IFN-
.
The effect of
IFN-
, which hydrolyzes sphingomyelin to generate ceramide, on the
translocation of
PKC-GFP was investigated in HeLa cells, since these
cells are known to express IFN-
receptors (33). IFN-
at 100 U/ml induced significant
PKC-GFP translocation from the
cytoplasm to the perinuclear region within 5 min, and the intensity of
fluorescence increased in the perinuclear region until 30 min (Fig. 6A,
top row). We examined the influence of serum deprivation on the translocation of
PKC-GFP induced by IFN-
. When the culture medium was replaced with serum-free medium, IFN-
induced the same translocation of
PKC-GFP as seen in the presence of FBS (Fig. 6A, bottom row). Serum deprivation did not alter
the localization of
PKC-GFP until at least 60 min after treatment
(data not shown). The application of 100 U of IFN-
per ml failed to
induce
PKC- or
PKC-GFP translocation for at least 60 min (Fig. 6B
and C).

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FIG. 6.
Effects of IFN- on PKC subtype translocation in HeLa
cells. (A) Treatment with IFN- (100 U/ml) induced translocation of
PKC-GFP from the cytoplasm to the perinuclear region within 5 min
after treatment of HeLa cells in culture medium containing FBS (top
row). The translocation of PKC-GFP was not altered by eliminating
FBS from the culture medium (bottom row). (B) IFN- (100 U/ml) did
not affect the localization of PKC-GFP expressed in HeLa cells. (C)
IFN- (100 U/ml) did not affect the localization of PKC-GFP
expressed in HeLa cells. The results shown are representative of three
independent experiments. Bars, 10 µm.
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|
We further examined the downstream signaling pathway after activation
of the IFN-

receptor, which contributes to the translocation
of

PKC. IFN-

has been known to cause the activation of
sphingomyelinase
and then produce ceramide from sphingomyelin
(
25). To determine
whether the IFN-

-induced
translocation of

PKC-GFP is mediated
by the activation of
sphingomyelinase, we first examined the effect
of the Mg
2+
chelator EDTA, which inhibits Mg
2+-dependent neutral
sphingomyelinase, a subtype of sphingomyelinase
(
5,
14).
As shown in Fig.
7A, pretreatment with
Mg
2+-free HEPES buffer containing 0.5 mM EDTA for 30 min
entirely
blocked the IFN-

-induced translocation of

PKC-GFP (Fig.
7A,
top row), while in normal HEPES buffer IFN-

induced
translocation
of

PKC-GFP from the cytoplasm to the perinuclear
region (Fig.
7A, bottom row). The effects of other inhibitors of
Mg
2+-dependent neutral sphingomyelinase (scyphostatin and
GSH) (
3,
34,
63) were further examined. Pretreatment with
50 µM scyphostatin
for 15 min effectively blocked the translocation
of

PKC-GFP induced
by IFN-

, and the further application of 10 µM C
2-ceramide also
rescued the perinuclear translocation
of

PKC-GFP (Fig.
7B, top
row). Similarly, pretreatment with 5 mM GSH
for 30 min effectively
inhibited the translocation of

PKC-GFP
induced by IFN-

, and
the further application of 10 µM
C
2-ceramide rescued the perinuclear
translocation of

PKC-GFP fluorescence (Fig.
7B, bottom row).
Tyrosine kinases such as
JAK1 and JAK2 are involved in the downstream
stages of IFN-

signaling pathways (
40). To clarify whether
the
perinuclear translocation of

PKC-GFP induced by IFN-

is
mediated
by the activation of JAK1 and JAK2 in HeLa cells, we
investigated the
effects of genistein or tyrphostin AG490 on the
IFN-

-induced
translocation of

PKC-GFP. Pretreatment with 100
µM genistein, a
nonspecific tyrosine kinase inhibitor, for 30
min effectively blocked
the translocation of

PKC-GFP induced
by IFN-

, and further
application of 10 µM C
2-ceramide rescued
the perinuclear
translocation of

PKC-GFP (Fig.
8, top
row). Pretreatment
with 100 µM
tyrphostin AG490, a specific inhibitor of JAK2 tyrosine
kinase
(
1,
37,
66), for 30 min also blocked the translocation
of

PKC-GFP induced by IFN-

, and the further application of 10
µM
C
2-ceramide rescued the perinuclear translocation of

PKC-GFP
fluorescence as seen in the case of treatment with
sphingomyelinase
inhibitors (Fig.
8, bottom row).

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FIG. 7.
Effects of sphingomyelinase inhibitors on
IFN- -induced translocation of PKC-GFP. (A) Treatment with
Mg2+-free HEPES [Mg( ) HEPES] buffer containing 0.5 mM
EDTA for 30 min blocked the IFN- (100 U/ml)-induced translocation of
PKC-GFP (top row). IFN- induced translocation of PKC-GFP in
normal HEPES buffer containing 1 mM Mg2+ (bottom row). (B)
IFN- -induced translocation of PKC-GFP was inhibited by
pretreatment with 50 µM scyphostatin (Scypho.) for 15 min. However,
scyphostatin did not inhibit the 10 µM C2-ceramide
(C2-Cer)-induced translocation of PKC-GFP (top row). GSH
treatment (5 mM, 30 min) also abolished IFN- - but not
C2-ceramide-induced translocation of PKC-GFP (bottom
row). The results shown are representative of three independent
experiments. Bars, 10 µm.
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FIG. 8.
Effects of tyrosine kinase inhibitors on IFN- -induced
translocation of PKC-GFP. IFN- -induced translocation of
PKC-GFP was inhibited by pretreatment with 100 µM genistein for 30 min. However, genistein did not alter 10-µM ceramide
(C2-Cer)-induced translocation of PKC-GFP (top row).
Similarly, preincubation with tyrphostin AG490 (100 µM) for 30 min
abolished IFN- -but not C2-ceramide-induced translocation
of PKC-GFP (bottom row). The results shown are representative of
three independent experiments. Bars, 10 µm.
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|
Ceramide is also generated by the activation of TNF-

receptors,
which are expressed in HeLa cells (
11,
13,
25,
38).
We
studied the effects of TNF-

on the translocation of

PKC-GFP.
TNF-

at 100 U/ml induced apparent

PKC-GFP translocation from
the
cytoplasm to the perinuclear region within 20 min, and the
intensity of
the fluorescence increased slowly in the perinuclear
region until 60 min (data not
shown).
Target site of
PKC-GFP in response to ceramide.
To identify
the intracellular compartment where
PKC-GFP accumulated in response
to C2-ceramide, the Golgi complex was visualized with Texas
red-conjugated wheat germ agglutinin in HeLa cells expressing
PKC-GFP after ceramide treatment. As shown in Fig. 9, intense GFP fluorescence was present
in the perinuclear region in addition to moderate fluorescence
throughout the cytoplasm. Texas red fluorescence accumulated in the
perinuclear region and was also seen on the nuclear membrane. Merged
images showed that the fluorescence of GFP and that of Texas red were
colocalized in the perinuclear region, indicating that
PKC-GFP is
targeted to the Golgi complex in response to ceramide. Colocalization
of
PKC-GFP and Texas red-conjugated wheat germ agglutinin was also seen after stimulation with C6-ceramide or IFN-
(data
not shown).

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FIG. 9.
Colocalization of PKC-GFP and wheat germ agglutinin
binding sites in PKC-GFP-expressing HeLa cells treated with
ceramide. HeLa cells transfected with PKC-GFP were fixed after
treatment with 10 µM C2-ceramide for 20 min. Cells were
treated with Texas red-conjugated wheat germ agglutinin (WGA) to make
the Golgi complex visible. The localization of PKC-GFP is shown in
green (left). The Golgi complex is shown in red (center). On the merged
image, overlapping GFP and Texas red signals appear yellow (right). The
results are representative of four independent experiments. Bar, 10 µm.
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FRAP of
PKC-GFP translocated by ceramide.
We investigated
the interaction of
PKC-GFP with the Golgi complex by fluorescence
recovery after photobleaching (FRAP). We measured the fluorescence
recovery of the
PKC-GFP in the bleached area and also the
fluorescence fading in the unbleached area after photobleaching with an
argon laser at 488 nm. As shown in Fig. 10, after treatment with 10 µM
C2-ceramide for 30 min, photobleaching of a circular area
in the Golgi complex abolished the fluorescence of
PKC-GFP in the
circle. The GFP fluorescence in the circle recovered within 40 s
to a level similar to that in the unbleached Golgi complex. The
recovery of fluorescence was significantly faster than the
translocation of
PKC-GFP induced by ceramide (Fig. 5A). In contrast,
the fluorescence in the unbleached perikarya faded gradually.
Photobleaching was also applied to a square area in perikarya (Fig.
10). GFP fluorescence of the bleached area (perikarya) rapidly
recovered (within 30 s), and the fluorescence in the Golgi complex
rapidly faded.

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FIG. 10.
FRAP of PKC-GFP after translocation induced by
ceramide. (A) Fluorescence recovery of PKC-GFP after photobleaching
of the Golgi complex (a to c) or of the cytoplasm (d to f). The images
were obtained before (a and d) and 0 s (b and e), 132 s (c),
or 38 s (f) after photobleaching. The bleached areas are shown in
red circles (a to c) and orange squares (d to f). The blue squares (a
to c) and green circles (d to f) show the areas where fluorescence
fading was measured. (B and C) Measurement of fluorescence recovery of
PKC-GFP after photobleaching of the Golgi complex (B) or of the
cytoplasm (C). Time-dependent recovery (red circle and orange square)
of fluorescence in the bleached areas and fading (blue square and green
circle) of the fluorescence in the unbleached areas are shown as
percentages of the fluorescence before bleaching. Arrowheads (a to f)
indicate the time points of the pictures in panel A. The results shown
are representative of three independent experiments.
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Changes in kinase activity of
PKC by C2-ceramide, in
vitro and in vivo.
The effects of C2-ceramide on the
kinase activity of
PKC-GFP were examined by an in vitro kinase
assay. As shown in Fig. 11A,
C2-ceramide at 10 µM failed to activate
PKC-GFP in
vitro. In the presence of PS and DO, the kinase activity of
PKC-GFP was increased 2.9-fold, and C2-ceramide inhibited the
activation of
PKC-GFP by PS and DO. The activity of
PKC-GFP in
the presence of the cofactors was dose-dependently inhibited by
C2-ceramide, and the maximal level was seen at 10 µM
(31% inhibition).

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FIG. 11.
Effects of ceramide on kinase activity of PKC
subspecies, in vitro and in vivo. (A) Effects of
C2-ceramide on kinase activity of PKC-GFP assessed by in
vitro kinase assay. Kinase activities of the immunoprecipitated
PKC-GFP were measured in the presence of various concentrations of
C2-ceramide (C2-Cer) or activators of PKC
such as PS and DO. Data are expressed as percentages of the control
level. Statistical significance: *, P < 0.05 versus
kinase activity of PS and DO. (B) Changes in kinase activity of
PKC-GFP in HeLa cells after C2-ceramide treatment
assessed by in vivo kinase assay. PKC-GFP was immunoprecipitated
from HeLa cells overexpressing PKC-GFP at various time points after
ceramide treatment. The kinase activity of PKC-GFP was assayed with
H1 histone as the substrate without any activators such as PS or DO.
Data are expressed as percentages of the control level (the kinase
activity before stimulation). (C) Effects of C2-ceramide on
kinase activity of endogenous PKC subtypes assessed by in vivo kinase
assay. Endogenous PKC, PKC, and PKC were immunoprecipitated
from HeLa cells before and after C2-ceramide
(C2-Cer) treatment. The kinase activity of PKC, PKC,
and PKC was assayed with H1 histone ( PKC and PKC) or MBP
( PKC) as the substrate without any activators such as PS or DO. Data
are expressed as percentages of the control level (the kinase activity
of each PKC subtype immunoprecipitated from untreated cells).
Statistical significance: *, P < 0.01 versus kinase
activity of control. All results represent the means and standard
errors of more than three determinations.
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In contrast, the in vivo kinase assay indicated that the kinase
activity of the immunoprecipitated

PKC-GFP was increased
in HeLa
cells treated with C
2-ceramide. Treatment with 10 µM
C
2-ceramide
increased the kinase activity of the
immunoprecipitated

PKC-GFP
in a time-dependent manner, and at 20 min
after treatment with
C
2-ceramide, the kinase activity was
increased 1.7-fold (Fig.
11B). To examine whether endogenous

PKC is
also activated by ceramide,
we performed the in vivo kinase assay of
endogenous

PKC,

PKC,
and

PKC in untransfected HeLa cells.
After the treatment with
10 µM C
2-ceramide for 20 min,
the kinase activity of

PKC but
not of

PKC or

PKC was
significantly increased 1.6-fold (Fig.
11C).
Tyrosine phosphorylation of
PKC-GFP after treatment with
C2-ceramide in HeLa cells.
It has been reported that
PKC is activated by tyrosine phosphorylation after various
stimulations (8, 9, 17, 27, 30, 31, 60). To elucidate
whether or not the ceramide-induced activation of
PKC-GFP is through
tyrosine phosphorylation, we investigated the effect of tyrosine kinase
inhibitor on the C2-ceramide-induced activation of
PKC-GFP. Treatment with 10 µM C2-ceramide induced significant activation of
PKC-GFP, while treatment with 10 µM C2-dihydroceramide for 20 min did not induce significant
activation of
PKC-GFP (Fig. 12A).
The activation of
PKC-GFP by C2-ceramide was abolished
by the pretreatment with 200 µM genistein, a nonspecific tyrosine
kinase inhibitor (Fig. 12A). To determine whether or not
PKC-GFP was
tyrosine phosphorylated after the treatment with C2-ceramide, tyrosine phosphorylation of
PKC-GFP was
analyzed by immunoblotting using an anti-phosphotyrosine antibody.
Although C2-dihydroceramide did not cause any tyrosine
phosphorylation of
PKC-GFP,
PKC-GFP was significantly tyrosine
phosphorylated by treatment with C2-ceramide (Fig. 12B). In
addition, pretreatment with 200 µM genistein effectively blocked the
tyrosine phosphorylation of
PKC-GFP induced by
C2-ceramide. Immunoblotting with the anti-GFP antibody
revealed that similar amounts of
PKC-GFP were immunoprecipitated in
all samples.

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FIG. 12.
Effects of a tyrosine kinase inhibitor on kinase
activity and tyrosine phosphorylation of PKC-GFP after
C2-ceramide treatment. (A) Effect of genistein on the
ceramide-induced activation of PKC-GFP assessed by in vivo kinase
assay. After pretreatment with genistein (200 µM), the PKC-GFP was
immunoprecipitated from ceramide (C2-Cer)- or
C2-dihydroceramide (DH-C2-Cer)-treated cells.
The kinase activities of the immunoprecipitated PKC-GFP were assayed
with H1 histone as the substrate without any activators such as PS or
DO. Data are expressed as percentages of the control level (the kinase
activity of PKC-GFP from untreated cells). Statistical significance:
*, P < 0.01 versus kinase activity of control. (B)
Effect of genistein on tyrosine phosphorylation of PKC-GFP in
transfected HeLa cells treated with C2-ceramide. The
PKC-GFP was prepared as described for panel A, and tyrosine
phosphorylation of PKC-GFP was analyzed by immunoblotting using
anti-phosphotyrosine (anti-p-Tyr) antibody (top row). Immunoblotting of
the same membrane was performed using anti-GFP polyclonal antibody as
described in Materials and Methods (bottom row). All results represent
the means and standard errors of more than three determinations.
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 |
DISCUSSION |
We have studied the targeting mechanism of PKC subtypes in living
cells using GFP fusion proteins to elucidate the individual function of
each PKC subtype. In CHO-K1 cells overexpressing
PKC-,
PKC-, and
PKC-GFP, spatial and temporal targeting varied depending on PKC
subtype and extracellular stimulus (49, 56, 59). We
further demonstrated that PKC translocation to the specific intracellular compartment (PKC targeting) is necessary for the recognition and phosphorylation of the substrates in the compartment (48). This subtype-and stimulus-specific targeting
strongly suggested that the targeting mechanisms of PKC subtypes
determine their individual roles in cell signaling pathways. In the
present study, we examined the effects of ceramide on PKC translocation to clarify how ceramide is involved in various cell responses, especially in PKC-mediated signaling pathways.
Since HeLa cells express IFN-
receptors coupled to the
sphingomyelin-ceramide pathway, we used HeLa cells that endogenously express
PKC,
PKC, and
PKC for the present study. Among these three endogenously expressed PKC subtypes, only
PKC was translocated to the Golgi complex by a permeable ceramide analogue. Immunoblotting analysis indicated that ceramide induced translocation of
PKC from
the cytosol to the particulate fraction, suggesting that
PKC was
associated with the Golgi membrane after ceramide treatment. Immunocytochemical studies also revealed that ceramide translocated endogenous
PKC but not
PKC or
PKC to the Golgi complex. The results of the present study in living cells suggested that only
PKC, at least in HeLa cells, is responsible for the ceramide-induced cellular responses, although it is possible that other PKC subtypes expressed at undetectable levels in HeLa cells are also involved in the
responses or that ceramide acts on PKC without translocation of PKC to
specific subcellular compartments. Shirai et al. demonstrated that
among
PKC,
PKC, and
PKC, only
PKC was insensitive to various fatty acids, including arachidonic acid, which induced translocation of
PKC to the Golgi complex (59).
Ceramide also translocated
PKC but not
PKC to the Golgi complex
in the present study (data not shown). Since ceramide translocates both
PKC and
PKC to the Golgi complex and arachidonic acid
translocates only
PKC but not
PKC, it is suggested that
arachidonic acid-induced translocation of
PKC to the Golgi complex
may occur by a mechanism different from that involved in
ceramide-induced translocation of
PKC and
PKC to the Golgi
complex. Previous biochemical studies, however, showed that
PKC and
PKC have ceramide-binding abilities and that treatment with ceramide
translocated
PKC as well as
PKC from the membrane to the cytosol
fraction (57). It was also reported that ceramide induced
translocation of
PKC from the cytosol to the membrane fraction
(22) and that
PKC was translocated to the perinuclear
region by ceramide (16). In the present study using living
HeLa cells, neither
PKC nor
PKC responded to ceramide 60 min
after treatment. The precise reason for this discrepancy is not
clear, but it may have been due to differences in the cell types or
experimental conditions used.
Many studies have shown that ceramide is produced through
sphingomyelin hydrolysis after exposure to various extracellular stimuli, including IFN-
(25) and TNF-
(11,
25). As shown in Fig. 6, physiological receptor stimulation by
IFN-
evoked translocation of only
PKC, but not
PKC or
PKC,
from the cytoplasm to the Golgi complex as seen when treated with
ceramide. Since Mg2+-dependent neutral sphingomyelinase
inhibitors such as scyphostatin and GSH inhibited IFN-
- but not
ceramide-induced translocation of
PKC, it is likely that the
translocation of
PKC occurred downstream of the
Mg2+-dependent neutral sphingomyelinase pathway.
Furthermore, the chelation of extracellular Mg2+ completely
blocked the translocation of
PKC, demonstrating that the
Mg2+-dependent neutral sphingomyelinase is activated
outside the plasma membrane. Since D609, an inhibitor of SMS, did not
alter the ceramide-induced translocation of
PKC or that induced by
IFN-
, ceramide generated by hydrolysis of sphingomyelin, but not
sphingomyelin generated from ceramide, induced PKC translocation.
Although serum deprivation has been reported to induce sphingomyelin
hydrolysis and generation of ceramide within 10 h after treatment
(24), the serum deprivation did not alter localization of
PKC, at least within the 60-min observation period in the present
study. Furthermore, because IFN-
induced translocation both in the
presence and absence of serum, translocation of
PKC did not occur
through an unknown effect of serum. The TNF-
receptor is also known
to be expressed in HeLa cells (13, 38), and TNF-
also
induced similar but slower translocation of
PKC (data not shown).
From the present findings that both AG490, a JAK2 inhibitor, and
genistein, a tyrosine kinase inhibitor, completely blocked
IFN-
-induced translocation of
PKC, it is likely that
Mg2+-dependent neutral sphingomyelinase is activated
downstream of the IFN-
receptor-JAK pathway and that ceramide is
subsequently produced, leading to translocation of
PKC to the Golgi
complex, although the detailed pathway between JAK2 and
Mg2+-dependent sphingomyelinase is currently unclear.
Ceramide is widely used as a marker for the Golgi complex, as ceramide
accumulates in this organelle (32). As shown in Fig. 5,
C6-NBD-ceramide accumulated to the perinuclear region with a time course similar to that of C6-ceramide-induced
translocation of
PKC, and finally, ceramide and
PKC accumulated
to the same compartment, the Golgi complex (Fig. 5B). This simultaneous
translocation of
PKC with ceramide to the Golgi complex suggested
that the translocation of
PKC was due to its association with
ceramide accumulating in the Golgi complex. However, NBD-ceramide was
transiently accumulated on the plasma membrane just after application,
but ceramide treatment did not cause translocation of
PKC to the plasma membrane. These observations suggested that ceramide may act on
PKC only at the Golgi complex but not at the plasma membrane. Although it is unclear whether
PKC binds ceramide directly or indirectly, it is possible that other components, such as anchoring protein, are necessary for the association of
PKC with ceramide in
the Golgi complex. While the Golgi-associated
PKC following ceramide
treatment was further translocated to the plasma membrane by TPA, the
Golgi-associated NBD-ceramide was not altered by TPA treatment. This
strongly suggested that the binding of
PKC to the Golgi complex is
reversible and that the association and dissociation of
PKC with the
Golgi complex occurred continuously. Rapid recovery of fluorescence
into the bleached areas and fading of the fluorescence in the
unbleached areas (Fig. 10) suggested that
PKC does not bind tightly
to the Golgi complex but continuously moves in both directions between
the Golgi complex and the cytoplasm.
The effects of ceramide on PKC activity are controversial; ceramide has
been reported to inhibit
PKC activity by an indirect mechanism
(29) and to have no direct effect on PKC activity (20), while Huwiler et al. reported an inhibitory effect
of ceramide on
PKC activity in the presence of PKC activators such as DG and PS (22) in vitro. We found that ceramide did not
affect the basal activity of
PKC but dose-dependently inhibited the kinase activity in the presence of PS and DO in vitro. It is
noteworthy, however, that the activity of the immunoprecipitated
PKC
was increased after ceramide treatment in vivo (Fig. 11B). An increase in kinase activity of immunoprecipitated
PKC was also seen after treatment with IFN-
(1.6-fold increase) but not with dihydroceramide (data not shown). These results suggested that
PKC is not activated by a direct interaction with ceramide but is activated by unknown factors that are modulated by ceramide in the Golgi complex. Tyrosine phosphorylation is a candidate to explain the unknown factor activating
PKC in the Golgi complex. There is increasing evidence that
PKC is activated by its tyrosine phosphorylation after various stimulations of the cells (8, 9, 17, 27, 30, 31, 60). To elucidate the
involvement of tyrosine phosphorylation in ceramide-induced activation
of
PKC, we examined the effect of a tyrosine kinase inhibitor on
ceramide-induced activation of
PKC and also on the ceramide-induced
tyrosine phosphorylation of
PKC. As shown in Fig. 12, ceramide
induced tyrosine phosphorylation of
PKC as well as the activation of
PKC, and genistein, a tyrosine kinase inibitor, abolished both
tyrosine phosphorylation and activation of
PKC. Considering that
genistein did not block the ceramide-induced translocation of the
PKC (Fig. 8), tyrosine phosphorylation of
PKC is not necessary
for the translocation of
PKC to the Golgi complex. Currently, the
tyrosine kinase which phosphorylates
PKC in response to ceramide
remains unclear. These results strongly suggest that ceramide induces
the
PKC-specific translocation to the Golgi complex and also induces
the
PKC-specific activation by tyrosine phosphorylation in the Golgi complex.
Ceramide, one of the most important second messengers, has been shown
to regulate various biological processes (19, 24, 47, 51, 64,
65). Among these multiple functions, ceramide has attracted
attention as an intracellular mediator of apoptosis. C2-ceramide as well as ceramide generation after treatment
with TNF-
caused DNA fragmentation (15, 47).
Furthermore, Chin et al. reported that IFN-
also induced apoptosis
through the STAT signaling pathway (7). The involvement of
PKC in apoptosis in various types of cells was demonstrated after
exposure to various extracellular stimuli (12, 26, 39,
61). These reports strongly suggested that ceramide-induced
translocation of
PKC to the Golgi complex is an important step for
apoptosis. However, Obeid et al. showed that PKC activation by TPA
inhibited ceramide-induced DNA fragmentation (47) and
suggested the involvement of two signaling pathways, ceramide- and
PKC-associated pathways, in the regulation of apoptosis. The present
study showed that TPA evokes translocation of
PKC to the plasma
membrane even after ceramide translocates
PKC to the Golgi complex,
suggesting that the inhibitory effect of TPA on DNA fragmentation is
due to the different targeting of PKC but not to the activation of PKC
in the same intracellular compartment to which PKC was translocated by ceramide.
In conclusion, IFN-
stimulation followed by ceramide generation
through Mg2+-dependent neutral sphingomyelinase induced
PKC-specific translocation to the Golgi complex, and this
translocation resulted in
PKC activation through tyrosine
phosphorylation of the enzyme.
 |
ACKNOWLEDGMENT |
This work was supported by grants from the Ministry of Education,
Science, Sports, and Culture in Japan (09NP0601, 11780448, 12680754, 12210107), the Sankyo Foundation of Life Science, the Uehara Memorial
Foundation, and the Hyogo Science and Technology Association.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. Phone: 81-78-803-5961. Fax: 81-78-803-5971. E-mail: naosaito{at}kobe-u.ac.jp.
 |
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Molecular and Cellular Biology, March 2001, p. 1769-1783, Vol. 21, No. 5
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1769-1783.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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