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INTRODUCTION |
Protein kinase C (PKC)1
comprises a family of phospholipid-dependent
serine-threonine kinases that play important roles in signal
transduction of various physiological stimuli, including growth
factors, hormones, and neurotransmitters (1-3). Activation of PKC
leads to the phosphorylation of proteins that are involved in the
regulation of cell growth, differentiation, and apoptosis (4-7). PKC
consists of at least 11 isoforms showing diversity in their structures,
cellular distributions, and biological functions (8). The members of
the classical PKCs 
, 
1, 2, and 
bind phorbol esters and are
Ca2+-dependent. The novel PKCs 
, 
, 
,
and 
do not depend on Ca2+ but bind phorbol esters. The
third subfamily includes the atypical PKCs (PKC
and PKC
/
),
which do not bind either Ca2+ or phorbol esters, and
PKCµ, which exhibits unique characteristics (9). All PKC isoforms can
be divided into an N-terminal regulatory domain and a C-terminal
catalytic domain with serine-threonine kinase activity (10, 11). Both
domains contain conserved (C) regions of extended sequence homology and
variable (V) regions. In the classical PKC isoforms the regulatory
domain contains a Ca2+-binding domain, and in both the
classical and novel PKC isoforms it contains a pair of highly conserved
zinc fingers (C1 domains) that bind phorbol esters and a
pseudosubstrate region (12-14). PKC chimeras have been used to study
the role of the regulatory and catalytic domains of different PKC isoforms.
PKC is a widely expressed member of the novel PKCs (15). This
isoform has been associated with the proliferation of various cells in
a cell type-specific manner. For example, PKC inhibited the
proliferation of smooth muscle cells (16) and glial cells (17) and
caused cell arrest at the G2/M phase of the cell cycle in
Chinese hamster ovary cells (18). In contrast, in breast cancer cells
PKC has been shown to increase tumorigenicity (19). PKC has also
been reported to play a role in cell differentiation. Thus, PKC has
been shown to undergo translocation and activation during
differentiation of keratinocytes (20) and overexpression of this
isoform induced squamous (21) and myeloid cell differentiation (22).
Recent studies suggest that PKC associates with different tyrosine
kinases and that this association can induce the tyrosine phosphorylation of PKC itself and can affect the activity of both
the tyrosine kinases and PKC (15). PKC has been shown to be tyrosine
phosphorylated in response to various stimuli such as PMA, EGF, PDGF
(23-25), ligands for the IgE receptor (26, 27), ATP, and
H2O2 (28). The phosphorylation site(s) and the role of tyrosine phosphorylation of PKC in its activity and in its
function are just beginning to be understood. Two specific sites in the
regulatory domain, tyrosines 187 and 52, have been reported so far to
be phosphorylated in response to PDGF and FcRI, respectively (29),
and tyrosine 311 has been shown to be phosphorylated by Src (30). In
contrast, tyrosine residues in the catalytic domain of PKC have been
reported to be phosphorylated in response to
H2O2 (28).
In a recent study (23), we found that in C6 glioma cells tyrosine
phosphorylation of PKC in the regulatory domain mediated the
inhibitory effect of this isoform on the expression of the astrocytic
marker, glutamine synthetase (GS). In the present study, we found that
tyrosine phosphorylation of PKC also plays a role in the inhibitory
effect of PKC on cell proliferation and have identified different
tyrosine residues that are involved in the selective effects of PKC
on cell proliferation and GS expression.
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EXPERIMENTAL PROCEDURES |
Materials--
PDGF and an affinity-purified polyclonal
anti-PKC antibody against a polypeptide corresponding to amino acids
726-737 of PKC were purchased from Life Technologies, Inc.
Monoclonal anti-PKC and anti-GS antibodies were obtained from
Transduction Laboratories (Lexington, KY). Polyclonal anti-PKC
antibodies and anti-Src, -Fyn, and -Lyn antibodies were from
Santa Cruz (Santa Cruz, CA). PMA was from Alexis Co. (San Diego, CA).
Leupeptin, aprotinin, phenylmethylsulfonyl fluoride, and sodium
vanadate were obtained from Sigma.
Generation of PKC Chimeras--
The PKC chimeras were generated
by exchanging the regulatory and catalytic domains of PKC, -, and
- as described by Acs et al. (31). PKC/ refers to
the chimera with the PKC regulatory domain and the PKC catalytic
domain, and PKC/ refers to the reciprocal chimera. The PKC
cDNAs were subcloned into the metallothionein promoter-driven
eukaryotic expression vector (MTH). The vector sequence encodes a
C-terminal PKC-derived 12-amino acid tag (MTH) that is added to
the expressed proteins (44). The expression of these chimeras
and their activities in C6 cells were described recently (23).
Site-directed Mutagenesis of PKC--
Mouse PKC was cloned
into the pGEM-T vector (Promega, Madison, WI) as described previously
(23). This plasmid served as our "master" vector for the
site-directed mutagenesis, using the Transformer Site-Directed
Mutagenesis Kit from CLONTECH (Palo Alto,
CA). Conversion of tyrosine residues at sites 52, 64, 155, 187, and 565 into phenylalanine was performed as described previously (23).
PKC and the PKC mutants were subcloned into the metallothionein promoter-driven eukaryotic expression vector (MTH).
Construction of PKC-GFP Fusion Protein--
cDNAs
encoding the murine PKC and the various PKC mutants were fused
into the N-terminal-enhanced GFP vector pEGFP-N1
(CLONTECH, Palo Alto, CA). The original pEGFP-N1
vector was modified by the insertion of an MluI site in the
plasmid polylinker. The restriction site was created by ligating a
phosphorylated linker containing the MluI site into pEGFP-N1
digested with SmaI. The construct was verified by
sequencing. The clones containing the GFP-PKC- or GFP-fused
to the different PKC mutants were constructed by the excision of
PKC or the specific mutants from MTH-PKC plasmids by digestion with
XhoI and MluI. The inserts were then ligated into
the modified GFP vector using the same restriction sites. DNA
sequencing of the GFP-PKC constructs confirmed the intended reading frame.
C6 Glial Cultures and Cell Transfection--
C6 cells of late
passages (50), that exhibit an astrocytic phenotype, were used in
this study. Cells of these passages showed somewhat smaller response to
overexpression of PKC as compared with the C6 cells of passage 30, which exhibit progenitor properties (17, 23). For the current studies
we chose cells of late passages, because we wanted to focus on the
effects of PKC on the expression of GS and not on general aspects of
cell differentiation. Cells (1 × 105 cells/ml) were
seeded on tissue culture dishes (10 cm) and were grown in medium
consisting of Dulbecco's modified Eagle's medium containing
10% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin (50 units/ml), and streptomycin (0.05 mg/ml). The cells were
transfected either with the empty vectors, the different PKC
expression vectors, or a Fyn dominant negative mutant in pSG5 (kindly
provided by Alan P. Saltiel) using LipofectAMINE (Life Technologies,
Inc.) as described previously (17). Experiments were routinely
carried out on a clone of the transfected cells, but all the results
were confirmed on one pool and two additional individual clones.
For overexpression of the GFP-PKC fusion proteins, C6 cells were
seeded onto 40-mm round glass coverslips at a density of 5 × 104 cells/coverslip. Twenty-four hours later, cells were
transfected with the different GFP-PKC constructs using
LipofectAMINE Plus reagent according to the manufacturer's
instructions. All experiments were performed 48 h
post-transfection.
Preparation of Cell Homogenates--
Cells were washed and
resuspended in serum-free medium. The plates were placed on ice,
scraped with a rubber policeman, and centrifuged at 1,400 rpm for 10 min. The supernatants were aspirated, and the cell pellets were
resuspended in 100 µl of lysis buffer (25 mM Tris-HCl, pH
7.4, 50 mM NaCl, 0.5% sodium deoxycholate, 2% Nonidet
P-40, 0.2% SDS, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 50 µM leupeptin, 0.5 mM
Na3VO4) on ice for 15 min. The cell lysates
were centrifuged for 15 min at 14,000 rpm in an Eppendorf
microcentrifuge, supernatants were removed, and 2× sample buffer was added.
Immunoblot Analysis--
Lysates (20 µg of protein) were
resolved by SDS-PAGE (10%) and were transferred to nitrocellulose
membranes. The membranes were blocked with 5% dry milk in
phosphate-buffered saline and subsequently stained with the primary
antibody. Specific reactive bands were detected using a goat
anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase
(Bio-Rad), and the immunoreactive bands were visualized by the ECL
Western blotting detection kit (Amersham Pharmacia Biotech).
Immunoprecipitation--
Immunoprecipitation was performed as
described previously (23). Briefly, C6 cells overexpressing
PKC or the PKC mutants were serum-starved overnight and treated
for different periods of time with PMA (10 nM) or PDGF (100 ng/ml). The samples were preabsorbed with 25 µl of protein
A/G-Sepharose (50%) for 10 min, and immunoprecipitation was performed
using 4 µg/ml antibody for 1 h at 4 °C and then incubated
with 30 µl of A/G-Sepharose for an additional hour. Following washes,
the pellets were resuspended in 25 µl of SDS sample buffer and boiled
for 5 min. The entire supernatants were subjected to Western blotting.
Membranes were incubated with horseradish peroxidase-conjugated
secondary antibodies for 1 h at room temperature. The membranes
were washed and visualized by the ECL system.
PKC Kinase Assay--
PKC activity was assayed by measuring the
incorporation of 32P from [-32P]ATP into
substrate in the presence of 100 µg/ml phosphatidylserine and 1 µM PMA as described previously (23).
Cell Proliferation Assay--
Cells overexpressing the wild-type
PKC or the PKC mutants were seeded in triplicate and incubated in
the absence or presence of ZnCl2 (20 µM) for
24 h followed by treatment with PMA (30 nM) for an
additional 48 h. Cells were pulsed with 0.5 µCi of
[3H]thymidine for the last 6 h and then harvested.
The incorporation of [3H]thymidine was determined in a
Beckman Scintillation counter.
Confocal Microscopy--
Confocal fluorescent images were
collected with a Bio-Rad MRC 1024 confocal scan head (Bio-Rad) mounted
on a Nikon microscope with a 60× planapochromat lens. Excitation at
488 nm was generated by a krypton-argon gas laser with a 522/32
emission filter for green fluorescence. For kinetics of GFP-PKC
translocation in living cells, cells plated on a 40-mm-round coverslip
were enclosed in a Bioptechs Focht Chamber System (Bioptechs, Butler,
PA). The chamber was inverted and attached to the microscope stage with a custom stage adapter; a temperature controller set at 37 °C was
connected, and medium was perfused through the chamber with a Lambda
microperfusion pump. Sequential images of the same cell were collected
at various time points using LaserSharp Software.
Statistical Analysis--
The results are presented as the mean
values ± S.E. All data were analyzed using a paired Student's
t test to determine the level of difference between the treatments.
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RESULTS |
The Regulatory Domain of PKC and Its Tyrosine Phosphorylation
Mediate the Decrease in Cell Proliferation Induced by PMA--
In a
recent study we demonstrated that the regulatory domain of PKC
mediated its inhibitory effect on the expression of the astrocytic
marker GS (23). To characterize the effect of PKC on C6 cell
proliferation we first examined the relative contributions of the
regulatory and catalytic domains of this isoform. For these studies, we
used chimeras between the regulatory and catalytic domains of PKC,
-, and -, combined at the highly conserved hinge region. The
expression and activity of C6 cells overexpressing the different
chimeras were already described previously (23).
Cells were pretreated for 24 h with ZnCl2, followed by
PMA treatment (20 nM) for an additional 48 h. This
concentration of PMA induced prolonged activation of PKC without
marked down-regulation. Cells overexpressing PKC/ exhibited a
lower rate of cell proliferation than control cells. In contrast, cells
overexpressing PKC/ and / exhibited a higher rate of cell
proliferation than controls both in the presence and absence of PMA.
Similar to cells expressing PKC/, cells overexpressing the
chimeras containing the regulatory domain of PKC, namely / and
/, also showed a reduced level of cell proliferation, whereas
cells expressing chimeras containing the catalytic domain of PKC
together with the regulatory domain of PKC or - exhibited an
increased level of proliferation similar to that observed with cells
expressing PKC/ or PKC/ (Fig. 1A). Untreated cells
overexpressing PKC/ or chimeras containing the regulatory domain
of PKC also showed decreased cell proliferation as compared with
control vector cells, but the magnitude of this decrease was much lower
than that observed in PMA-treated cells (Fig. 1A).
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Fig. 1.
Proliferation of C6 cells overexpressing
different PKC chimeras and the PKC5
mutant. C6 cells overexpressing different PKC chimeras
(A) or the PKC5 mutant (B) were plated in
24-well plates in the absence and presence of PMA (20 nM)
for 48 h. [3H]Thymidine was added to the cells for
the last 6 h, and the assay was performed as described under
"Experimental Procedures." The results are expressed as the percent
of the control untreated cells and represent the mean ± S.E. of
three separate experiments. *, p < 0.05; **,
p < 0.002, as compared with control untreated
cells.
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We reported that the expression of the PKC5 mutant (in which
tyrosines 52, 64, 155, 187, and 565 were mutated to phenylalanine) in
C6 cells abolished the decrease in the expression of the astrocytic marker GS induced by PMA or PDGF. Expression of the PKC5 mutant also
resulted in a lower tyrosine phosphorylation of PKC in response to
these treatments (23). Using cells expressing PKC5 we found that in
response to PMA (20 nM) these cells displayed enhanced proliferation as compared with vector control cells, contrasting with
cells overexpressing PKC WT which exhibited a significantly lower
proliferative response (Fig. 1B). Similar results were
obtained when the proliferation of C6 cells expressing PKC and
PKC5 was followed over the course of 5 days (data not shown).
Overexpression of PKC Mutants--
To identify the specific
tyrosines that are involved in the inhibitory effect of PKC on cell
proliferation and GS expression, we examined the role of tyrosines 52, 155,and 187, which were already reported to be phosphorylated in other
systems (25, 29). C6 cells were stably transfected with PKC mutants
in which one of these three tyrosines was mutated to phenylalanine.
Fig. 2A illustrates a
representative Western blot of C6 cells overexpressing PKC, PKC5,
PKCY155F, PKCY187F, and PKCY52F mutants, and the vector
control. Using the previously described tagging system (44), we
were able to detect specifically the transfected PKC isoforms using an
antibody against the PKC epitope tag on the constructs. The levels
of the overexpressed PKC WT and the PKC mutants in the
transfected cells we used were 8-10-fold higher than the endogenous
PKC as determined using an anti-PKC specific antibody (data not
shown). Cells overexpressing PKC or the PKC mutants expressed
similar levels of PKC, -, -, -, -, -, -, and -µ
(data not shown), thus excluding the possibility that the different
effects of PKC, and the PKC mutants were indirectly mediated by
changes in the expression of the other PKC isoforms. To establish that
the overexpressed PKC mutants were functionally active, we measured
kinase activity on cell lysates. All PKC mutants expressed higher
kinase activity as compared with the control vector cells (Fig.
2B).
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Fig. 2.
Expression of the PKC
mutants. Stable transfectants of C6 cells overexpressing
PKC WT, the various PKC mutants, or the empty vector
(M) were harvested and subjected to SDS-PAGE and Western
blot analysis. The membranes were probed with anti- antibody, which
recognized the -tag (A). Cell lysates of C6 clones
overexpressing PKC WT or the different PKC mutants were analyzed for
kinase activity by measuring 32P incorporation into
substrate in the presence of 1 µM PMA and 100 µg/ml
phosphatidylserine. The values for the PKC and the PKC mutants
are expressed as percent of control (empty vector cells)
(B). The results represent one of three separate experiments
which gave similar results.
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Tyrosine Phosphorylation of the PKC Mutants by PMA and
PDGF--
The degree of tyrosine phosphorylation of the various PKC
mutants was examined in response to PMA and PDGF. As illustrated in
Fig. 3, a small basal level of tyrosine
phosphorylation was observed in untreated cells. PMA and PDGF induced
marked tyrosine phosphorylation of PKC WT but, as already described,
no significant degree of enhanced tyrosine phosphorylation of PKC5
(p > 0.07, n = 5). The degree of
tyrosine phosphorylation of PKCY155F and PKCY187F in response to
PMA was intermediate between that of PKC WT and that of the PKC5
mutant, suggesting that both tyrosines 155 and 187 are being
phosphorylated in response to PMA. In contrast, cells overexpressing
PKCY52F exhibited a level of tyrosine phosphorylation similar to
that of PKC WT, suggesting that this tyrosine may not be
phosphorylated in response to PMA. As already described, PDGF also
induced tyrosine phosphorylation of PKC and this effect was
abolished in the PKC5 cells. We found that treatment of cells overexpressing PKCY187F with PDGF resulted in a very low tyrosine phosphorylation of PKC. In contrast, PDGF-stimulated cells
overexpressing PKCY155F or PKCY52F exhibited similar levels of
tyrosine phosphorylation to those observed in cells overexpressing
PKC WT.
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Fig. 3.
Tyrosine phosphorylation of
PKC mutants in response to PMA and PDGF.
C6 cells transfected with PKC WT or the PKC mutants were treated
with either PMA (20 nM) or PDGF (100 ng/ml) for 20 min.
Cells were then harvested, and immunoprecipitation of PKC was
performed as described under "Experimental Procedures." Following
SDS-PAGE, membranes were stained with anti-phosphotyrosine antibody
(anti-Tyr(P)) or with anti-PKC antibody. The results
represent one of three separate experiments which gave similar
results.
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Tyrosine 155 Mediates the Inhibitory Effect of PKC on Cell
Proliferation, whereas Tyrosine 187 Mediates the Inhibitory Effect of
PKC on GS Expression--
We then examined the degree of cell
proliferation and GS expression in cells overexpressing the different
PKC mutants. We found that, in response to PMA, cells overexpressing
PKCY155F displayed an increased cell proliferation over control
vector expressing cells and significantly increased proliferation
compared with cells overexpressing PKC WT (Fig.
4A). Interestingly, the degree
of cell proliferation in the PKCY155F cells was similar to that
obtained with the PKC5 overexpressing cells. In contrast, cells
overexpressing the PKCY187F or PKCY52F mutant exhibited a reduced
degree of cell proliferation as compared with vector control cells and
were similar to cells overexpressing PKC WT.
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Fig. 4.
Proliferation and GS expression of C6 cells
overexpressing the PKC mutants. C6
cells overexpressing PKC or the PKC mutants were plated in
24-well plates in the absence and presence of PMA (20 nM)
for 48 h. [3H]Thymidine was added to the cells for
the last 6 h, and the assay was performed as described under
"Experimental Procedures" (A). The results are expressed
as the percent of the control untreated cells and represent the
mean ± S.E. of five separate experiments. For GS expression,
stable transfectants of PKC WT or the PKC mutants were treated
with PDGF (100 ng/ml) for 48 h. Cells were harvested, and the
level of GS was determined using Western blot analysis (B).
The results are from one representative experiment out of four separate
experiments. *, p < 0.02; **, p < 0.001, as compared with control untreated cells.
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The effects of the PKC mutants were also examined on the expression
of GS in response to PDGF. Cells were pretreated for 24 h with
ZnCl2 and then with PDGF (100 ng/ml) for an additional 48 h. As described previously, cells overexpressing PKC WT
exhibited lower levels of GS expression upon PDGF treatments, whereas
cells overexpressing PKC5 exhibited increased levels of GS compared with vector control cells. Cells overexpressing PKCY155F or
PKCY52F exhibited lower GS expression in PDGF-treated cells similar
to the level obtained in cells overexpressing PKC WT. Opposite
results were obtained in cells overexpressing PKCY187F. These cells
exhibited an increased expression of GS in response to stimulation with PDGF, similar to the results obtained in cells overexpressing PKC5
(Fig. 4B). Similar results with the mutants were obtained in
cells treated with 20 nM PMA (data not shown).
Translocation and Degradation of PKC and the PKC
Mutants--
One possible explanation for the differential effect of
the mutants on cell proliferation and GS expression is translocation to
different cellular compartments following stimulation. We therefore examined the translocation of the different PKC mutants in response to PMA and PDGF. For these experiments we used GFP-tagged PKC WT or
the different PKC mutants. Cells were transiently transfected with
the specific GFP-PKC mutants, and the response of the cells to PMA
or PDGF was monitored over a period of 30 min. Stimulation of the cells
with 100 nM PMA induced initial translocation of PKC to
the plasma membrane followed by some translocation of PKC to the
perinuclear membrane (Fig. 5). PDGF also
induced translocation of PKC, but the magnitude of this
translocation was much lower than that induced by PMA. PDGF induced
some membranal translocation of PKC and distribution of PKC
around the perinuclear membrane (Fig. 5). A similar pattern of
translocation was observed for PKC and the PKC mutants in
response to PMA (Fig. 6) and PDGF (data
not shown). Thus, PMA induced translocation of the PKC mutants to
both the plasma membrane and the perinuclear membrane (Fig. 6). The
kinetics of the translocation as well as the response to lower
concentrations of PMA were likewise similar in all of the mutants (data
not shown). PMA or PDGF did not induce any changes in cells
overexpressing GFP protein alone (data not shown).
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Fig. 5.
Kinetics of PKC
translocation in response to PMA and PDGF. Cells were
transiently transfected with GFP-PKC. Following 48 h, cells
were treated with either PMA (100 nM) (A) or
PDGF (100 ng/ml) (B), and sequential confocal images were
taken every 30 s for a period of 30 min. The figures present
images taken at time 0, 10, and 30 min after treatment. Cells shown are
representative of four independent experiments.
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Fig. 6.
Cellular localization of
PKC and the PKC
mutants in PMA-treated C6 cells. Cells were transiently
transfected with GFP-PKC or the different GFP-PKC mutants.
Following 48 h, cells were treated with PMA (100 nM),
and sequential confocal images were taken every 30 s for a period
of 30 min. The figures present images taken at time 0 ( PMA) and 30 min (+PMA) after treatment. Cells shown are representative of four
independent experiments.
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Recent studies suggested that tyrosine phosphorylation of PKC may
play a role in its degradation (30). Since the inhibitory effects of
PMA on GS expression and cell proliferation require a prolonged
exposure of the cells to PMA, we examined whether the various PKC
mutants exhibit different degrees of degradation as compared with
PKC WT. For these experiments, cells overexpressing PKC WT or the
PKC mutants were treated for 24 h with a range of
concentrations of PMA, and the expression was examined by Western blot
analysis using the anti- tag antibody. PMA induced a
dose-dependent decrease in the expression of the exogenous
PKC WT. Similar results were obtained for PKC5 or the different
PKC mutants (data not shown).
Phosphorylation of PKC by PDGF Is Inhibited by PP1 and
PP2--
To examine the role of Src-related kinases in the tyrosine
phosphorylation of PKC, we employed the Src kinase inhibitors PP1
and PP2. Pretreatment of the cells with either PP1 or PP2 abolished the
tyrosine phosphorylation of PKC in response to PDGF (Fig.
7A). PP1 and PP2 also
abrogated the inhibitory effect of PKC on GS expression (Fig.
7B), providing further support that the
tyrosine-phosphorylated form of PKC is involved in the inhibition of
GS expression. Since PP1 has also been reported to inhibit the kinase
activity of the PDGFR at a similar concentration range (30),
our results suggest that either Src-related kinases or the PDGFR are
involved in the phosphorylation of PKC in response to PDGF.
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Fig. 7.
PP1 and PP2 inhibit the PDGF-induced tyrosine
phosphorylation of PKC and the decrease in GS
expression. C6 cells were treated with PP1 or PP2 for 1 h
following by stimulation with PDGF for an additional 15 min
(A). Cells were then harvested and immunoprecipitation of
PKC was performed as described under "Experimental Procedures."
Following SDS-PAGE, membranes were stained with anti-phosphotyrosine
antibody (anti-Tyr(P)) or with anti-PKC antibody. C6 cells were
pretreated with PP1 or PP2 for 1 h and then with PDGF for 48 h (B). GS expression was determined using Western blot
analysis. The results represent one of three separate experiments that
gave similar results.
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Association of PKC with Src-related Kinases--
To examine the
association of PKC with Src-related kinases we performed
co-immunoprecipitation of PKC with Src, Fyn, and Lyn.
We found that in unstimulated C6 cells PKC was constitutively associated with p60Src (Fig.
8A). Stimulation of the cells
with either PMA or PDGF for 1-60 min did not induce further
association of PKC with Src (data not shown). In contrast,
PDGF induced association of Fyn with PKC and, to a lesser extent,
association of Lyn (Fig. 8A). To further characterize the
association of Fyn with PKC, we performed a kinetic study and found
that PDGF induced association of Fyn following 1 min of treatment and
that this association was decreased after 30 min (Fig.
8B).
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Fig. 8.
Association of Src-related kinases with
PKC. C6 cells were treated with PDGF for
15 min, immunoprecipitation of PKC was performed as described under
"Experimental Procedures," and the membranes were stained with
anti-Src, anti-Fyn, or anti-Lyn (A). Cells overexpressing
PKCWT or PKCY187F were treated with PDGF for different periods of
time. PKC or PKCY187F was immunoprecipitated using anti-PKC
antibody, and the association of Fyn with PKC was measured
(B). The results represent one of three separate experiments
that gave similar results.
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Since the decrease in GS expression by PDGF appeared to involve
phosphorylation of tyrosine 187 in PKC, we examined the role of this
tyrosine residue in the association of Fyn and PKC. Stimulation of
cells overexpressing PKCY187F with PDGF (Fig. 8B) or PMA
(data not shown) did not lead to association of the mutated PKC with Fyn as determined by co-immunoprecipitation, indicating the tyrosine 187 is essential for the association. In contrast, the mutant PKCY155F associated with Fyn similarly to PKC WT (data not
shown), suggesting the tyrosine 155 does not play a role in the
association of PKC and Fyn.
Fyn Is Involved in the Inhibitory Effect of PKC on GS
Expression--
Since Fyn associates via tyrosine 187 with PKC in
response to PDGF treatment, we wanted to examine the role of Fyn in the tyrosine phosphorylation of PKC and in the inhibitory effect of
PKC on GS expression. We stably transfected a Fyn dominant negative
mutant in C6 cells and examined their response to PDGF. Cells
overexpressing empty vector exhibited an increase in the tyrosine
phosphorylation of PKC following 10 min of PDGF treatment. In
contrast, a decrease in the degree of the tyrosine phosphorylation of
PKC was observed in cells overexpressing the Fyn dominant negative
mutant (Fig. 9A). We also
examined the expression of GS in the different cells. PDGF decreased
the expression of GS in cells overexpressing control vector. In
contrast, there was no significant decrease in the expression of GS in
response to PDGF in cells overexpressing the Fyn dominant negative
mutant (Fig. 9B). Interestingly, the inhibitory effect of
the Fyn dominant negative on the decrease in GS expression induced by
PDGF was more marked than the decrease in tyrosine phosphorylation of
PKC, suggesting that Fyn is not the only tyrosine kinase
phosphorylating PKC in response to PDGF. Similar results with the
Fyn dominant negative mutant were obtained in cells treated with 20 nM PMA (data not shown). In contrast, Fyn dominant negative
did not abolish the decrease in cell proliferation induced by PMA,
suggesting that the effect of Fyn dominant negative was specific to
GS expression (data not shown).
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Fig. 9.
Effects of the Fyn dominant negative mutant
on tyrosine phosphorylation of PKC and GS
expression by PDGF. For tyrosine phosphorylation of PKC, cells
overexpressing Fyn dominant negative mutant or the control vector
(Control) were treated with PDGF (100 ng/ml) for 10 min,
immunoprecipitation of PKC was performed as described under
"Experimental Procedures," and the membranes were stained with
anti-phosphotyrosine (A). C6 cells expressing a Fyn dominant
negative mutant or the control vector (Control) were treated
with PDGF for 24 h, and the expression of GS was determined using
Western blot analysis (B). The results represent one of five
separate experiments, which gave similar results.
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DISCUSSION |
In this study we explored the mechanisms involved in the
inhibitory effects of PKC on C6 cell proliferation and GS
expression. We found that the regulatory domain of PKC is
responsible for the effects of this isoform on C6 cell proliferation.
These results are similar to those we recently described regarding the
regulation of GS expression by PKC (23). Chimeras have been
used to delineate the contributions of individual PKC domains to the
specific functions of different PKC isoforms in a number of systems.
Both the catalytic and the regulatory domains of PKC may determine
isoform-specific functions depending on the specific system. For
example, the catalytic domain of PKC was found to confer
isoform-specific function in the differentiation of erythroleukemia
cells (14), and the catalytic domain of PKC in reciprocal - and
-chimeras mediated PMA-induced macrophage differentiation of mouse
promyelocytes (32). In contrast, the regulatory domain of PKC
enhanced cell growth and induced colonies in soft agar in NIH 3T3 cells
(33). Recently, it has been reported that the regulatory domain of
PKC overexpressed by itself inhibited mammary tumor cell metastases
(34).
PKC has been shown to become tyrosine-phosphorylated in the
regulatory domain in response to PMA and PDGF in C6 cells (23). Tyrosine phosphorylation of PKC has been reported in response to EGF
stimulation in keratinocytes (24), in response to carbachol, substance
P and PMA stimulation in parotid acinar cells (35), in response to
H2O2 in CHO-K1 cells (28), and in response to activation of the IgE receptor in RBL-2H3 cells (26, 27). Constitutive
tyrosine phosphorylation was reported in Ras-transformed mouse
keratinocytes (36). Our results suggest that Src-related kinases are
involved in the tyrosine phosphorylation of PKC in response to PDGF,
since the Src kinase inhibitors PP1 and PP2 reduced significantly the
phosphorylation induced by PDGF. Our results using cells overexpressing
a Fyn dominant negative mutant further suggest that Fyn contributes to
PKC tyrosine phosphorylation in response to PDGF. Since PP1 has also
been reported to inhibit the kinase activity of PDGFR (3), we cannot
exclude at this point that this receptor directly phosphorylates PKC
upon PDGF binding. Indeed, Li et al. (22) reported that the
PDGF receptor phosphorylated PKC in vitro.
The effect of tyrosine phosphorylation on the activity of PKC or on
its function differs, depending on the specific system. Thus, tyrosine
phosphorylation of PKC has been reported to reduce its activity in
Ras-transformed cells and in response to activation of the EGF receptor
(24, 36). In contrast, tyrosine phosphorylation of PKC by Fyn
increased the kinase activity (37). Recently it was suggested that the
tyrosine phosphorylation of PKC in response to engagement of the IgE
receptor leads to altered substrate specificity (26). A previous report
has shown that mutation of PKC at tyrosine 187 did not change kinase
activity (25). In this study we did not further explore the kinase
activity of the different PKC mutants using different substrates, since
the nature of the endogenous substrates involved in the effects of PKC on cell proliferation and GS expression have not yet been identified.
Our results using the PKC5 mutant suggest that tyrosine
phosphorylation of PKC plays a role in the inhibitory effect exerted by this isoform on cell proliferation. These results are similar to our
recent findings, which showed a role for tyrosine phosphorylation of
PKC in the inhibitory effect of this isoform on the expression of
the astrocytic marker GS (23). Thus, the PKC5 mutant appears to act
in an opposite way to PKC in the effect of this isoform on both GS
expression and cell proliferation. Since tyrosine phosphorylation of
PKC in response to PMA occurs only in the regulatory domain of this
isoform in the C6 cells (23), our results are consistent with the
importance of the regulatory domain of PKC in the inhibitory effect
of this isoform on different cellular functions.
Although tyrosine phosphorylation in the regulatory domain of PKC
mediates the inhibitory effects of this isoform on both GS expression
and cell proliferation, it appears that different tyrosine residues are
involved in the different effects. Thus, tyrosine 155 is implicated as
a phosphorylation site that is involved in the inhibitory effect of
PKC on cell proliferation but not on GS expression, whereas tyrosine
187 appears to be involved in the inhibitory effect of PKC on GS
expression. There have been a number of reports regarding the
phosphorylation of specific tyrosines on PKC. For example, tyrosine
phosphorylation in the catalytic domain of PKC was described in
response to treatment with H2O2 (28). Tyrosine
311 was reported to be phosphorylated in response to Src (30), and
engagement of the FcR1 resulted in tyrosine phosphorylation of
tyrosine 52 in the regulatory domain (27, 29). Interestingly, tyrosine
52 did not appear to be phosphorylated in response to either PMA or
PDGF in the C6 cells. Our results of PDGF-induced phosphorylation of
tyrosine 187 in PKC are consistent with the results of Li et
al. (25), who reported that PMA and PDGF induced phosphorylation
of PKC on tyrosine 187 in NIH 3T3 cells. The authors concluded that
this phosphorylation site was not important for the monocytic
differentiation of 32D cells by PMA. Thus, the monocytic
differentiation of 32D cells resembles C6 cell proliferation and
contrasts with GS expression in its control by tyrosine 187 phosphorylation of PKC.
One of the factors that could explain the different effects of the
PKC mutants is a distinct pattern of translocation. Translocation of
PKC to specific cellular compartments could lead to different effects
due to the phosphorylation of different substrates and to the
association of PKC with specific proteins present in these locations. One of the important factors that can determine the localization of PKC following its activation is association with RACKs
(receptors for activated protein kinase Cs) (38). It is currently not
clear to what extent tyrosine kinases can act as RACKs and affect the
translocation of PKC isoforms. In a recent study, Ron et al.
(39) suggested that Fyn, which is associated with PKC, might act as
a RACK of this PKC isoform. We found that PMA induced initial
translocation of PKC to the plasma membrane followed by
translocation to the perinuclear membrane. This pattern of
translocation is similar to the translocation of PKC reported in
CHO-K1 cells (40). PDGF also induced membranal translocation of PKC,
although to a lesser extent, with some accumulation around the
perinuclear membrane. Stimulation of cells overexpressing PKC WT or
the different PKC mutants with PMA or PDGF resulted in a similar
pattern of translocation. Thus, the differential effects of the
different PKC mutants on cell proliferation and GS expression are
probably not due to their different translocation following activation.
At this point, however, we cannot exclude the possibility that the
PKC mutants undergo translocation to different membranal subdomains,
causing their association with distinct signaling molecules.
PKC has been reported to associate with different tyrosine kinases
such as Src (27, 30, 41, 42), Lyn (27), and c-Abl (43) in either
a phosphorylation-dependent or -independent manner. The
ability of PKC to be tyrosine phosphorylated on more than one
tyrosine suggests that PKC can associate with different tyrosine
kinases. We found that PKC associated with Src in a phosphorylation-independent manner and with Fyn following stimulation of PDGF via tyrosine 187. Specifically, the effect of PKC on cell
proliferation and GS expression in C6 cells may be mediated by either
different downstream PKC substrates or different tyrosine kinases that
are associated with PKC. Indeed, our results suggest that Fyn is
involved in the inhibitory effect of PDGF on GS expression, since
overexpression of the Fyn dominant negative mutant abrogated the
inhibitory effect of PDGF. It has been reported that the differential interaction of PKC with tyrosine kinases may lead to changes in the
activity and substrate recognition of PKC and to changes in the
activity of the associated tyrosine kinases (26, 27). Thus, the
differential phosphorylation of specific tyrosine residues may generate
diversity in the effects of PKC and positions this isoform as an
important component in a complex bi-directional interaction between
serine-threonine and tyrosine kinase signaling. The identities of the
tyrosine kinases that are associated with PKC via tyrosine 155 and
are involved in the inhibitory effect of PKC on cell proliferation
are currently under investigation.
on Distinct
Tyrosine Residues Regulates Specific Cellular Functions*
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Faculty of Life
Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. Tel.: 972-3-531-8266; Fax: 972-3-736-9929; E-mail:
chaya@mail.biu.ac.il.
