From the Department of Biology, University of York, York YO10 5DD, United Kingdom
Received for publication, October 16, 2000, and in revised form, November 22, 2000
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ABSTRACT |
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We have recently demonstrated that in quiescent
fibroblasts protein kinase C (PKC) The PKC1 family of
related phospholipid-dependent serine/threonine kinases are
involved in the control of many cellular processes, including cell
growth and differentiation (1, 2). To date 11 PKC isoforms have been
identified: the conventional PKCs ( Recent work from many groups has highlighted the importance of
phosphorylation in the regulation of PKC activity (4-7). Initially phospholipid-dependent kinase 1 phosphorylates a conserved
site on the lip of the catalytic region that corresponds to
Thr566 in PKC PKC The regulation of how PKC becomes dephosphorylated has not been well
studied. Most work has been carried out using TPA to induce activation
that leads to dephosphorylation and degradation. Our system involving
cell passage allows us to examine the control of the putative
Ser729-specific phosphatase rather than to look at the
complete dephosphorylation of the protein. Our results suggest that
dephosphorylation of PKC In this study we have examined how the dephosphorylation of PKC Materials
Cells were from the European Collection of Animal Cell Cultures
(Porton Down, UK) and the William Dunn Cell Bank (Oxford, UK). Cell
culture plastic was from Nunc (Life Technologies, Inc.). All other cell
culture reagents were from Life Technologies, Inc. except serum, which
was from PAA Laboratories (Linz, Austria). All other chemicals were
from Sigma, and chemical inhibitors were from Calbiochem (Nottingham,
UK) unless otherwise stated. Nitrocellulose membrane (Hybond C) was
from Amersham Pharmacia Biotech. Dried milk powder was Marvel (Premier
Beverages, Stafford, UK). BCA reagents were from Pierce.
The polyclonal PKC Methods
Tissue Culture--
3T3 and 3T6 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum in a humidified incubator at 5% CO2 as described
previously (26). Cells were passaged by rinsing with Tris saline,
followed by release with Tris trypsin. Cells were resuspended in fresh
medium as above, plated into fresh tissue culture flasks, and allowed
to settle for 15 min before harvesting unless otherwise stated.
Inhibitor Studies--
Cells were treated with inhibitors at
concentrations described in the text. Inhibitors were dissolved in
Me2SO unless otherwise stated and effects compared with
Me2SO control treated cells. Solvent concentrations were
never greater than 0.01%. Quiescent cells were treated for 30 min
before passage, and fresh inhibitor was added after passage. PKC Plasmids and Cell Transfection--
Hemagglutinin-tagged pCH3 Western Blotting--
Cells were scraped from flasks into lysis
buffer (50 mM Tris, 0.5 mM EDTA, 2 mM EGTA, pH 7.5, with 0.5% Nonidet P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM
[4-(2-aminoethyl)benzenesulfonylfluoride, 50 mM NaF, 5 mM sodium pyrophosphate, 10 µM sodium
orthovanadate). Protein concentration was determined using the BCA
assay (Pierce). Laemmli loading buffer was added, and equal amounts of
protein (30 µg) were run on SDS-PAGE gels, transferred to
nitrocellulose, and probed for PKC Immunoprecipitation--
Cells were harvested in
immunoprecipitation buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% SDS, 1% Triton
X-100, 1% deoxycholic acid, 0.2 mM
[4-(2-aminoethyl)benzenesulfonylfluoride, 50 mM NaF, 5 mM sodium pyrophosphate, 10 µM sodium
orthovanadate). Samples were precleared with protein A-Sepharose at
4 °C for 1 h on a rotary stirrer. Equal amounts of cell protein
were incubated with anti-PKC [35S]Methionine Incorporation--
Cells were
incubated in methionine-free Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) for 1 h. 3.7 Mbq of [35S]Met/Cys
(Promix; Amersham Pharmacia Biotech) was then added for 2 h. Cells
were then passaged into fresh medium and flasks, and 3.7 Mbq
[35S]Met/Cys was retained in the medium. Cells were
harvested at different time points, and PKC PKC Serum and Readhesion Are Required for the Formation of
PKC
Readhesion is also necessary for the formation of PKC
Because re-entry into the cell cycle from G0 is necessary
but not sufficient for PKC PP1, PP2A, and PP2B Are Not Important in PKC PKC
Transfection of fibroblasts with full-length PKC The Production of PKC TPA Stimulates PKC We have previously shown that when fibroblasts are passaged there
is a change in the phosphorylation status of PKC We have shown that the removal of the phosphate group at
Ser729 from PKC Our inhibitor studies clearly show that the formation of
PKC Interestingly, OA treatment and also treatment with the calpain and
proteasome inhibitor ALLN, increased the level of PKC Our time course results show that PKC Inhibitors of PKC PKC PKC Our working model for PKC95 is
phosphorylated at Ser729, Ser703, and
Thr566 and that upon passage of quiescent cells
phosphorylation at Ser729 is lost, giving rise to
PKC
87. Ser729 may be rephosphorylated later,
suggesting cycling between PKC
87 and
PKC
95. Here we show that the dephosphorylation at
Ser729 is insensitive to okadaic acid, calyculin, ascomycin
C, and cyclosporin A, suggesting that dephosphorylation at this site is
not mediated through protein phosphatases 1, 2A or 2B. We demonstrate
that this dephosphorylation at Ser729 requires serum and
cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine,
and Ro-31-8220. These results suggest that the phosphorylation status
of Ser729 in the hydrophobic domain at Ser729
is regulated independently of the phosphorylation status of other sites
in PKC
, by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser729.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
I,
II,
and
), which are regulated by calcium and diacylglycerol, the
novel PKCs (
,
, and
), which are calcium independent but dependent upon diacylglycerol, and the atypical PKCs (
,
, and
), which are both diacylglycerol- and calcium-independent. Another isoform, PKCµ, is known as protein kinase D (3).
(8, 9). Phosphorylation at this site is
important for the enzymatic activity of PKC (4). Two further
phosphorylation sites have been identified in the C-terminal region of
the enzyme at the turn and hydrophobic motifs (4-6). Phosphorylation
at the turn motif (Ser703 in PKC
) is believed to be
mediated through autophosphorylation (5). There is some debate as to
whether the hydrophobic site (Ser729 in PKC
) becomes
phosphorylated as a result of autophosphorylation or by a separate
kinase. Phosphorylation at this hydrophobic site may be modulated by
PKC
and appears to be sensitive to rapamycin (10, 11). Although
phosphorylation at these two C-terminal sites is not essential for the
catalytic activity of PKC, they seem to regulate the stability of the
enzyme (12-16).
is the only isoform that has oncogenic potential (17, 18) that
may be mediated through its interaction with Raf 1 kinase (19, 20).
PKC
is also unique in having actin and Golgi-binding domains
(21-25). PKC
from fibroblasts migrates on SDS-PAGE as two distinct
forms, with molecular sizes of 95 and 87 kDa (PKC
95 and
PKC
87) that differ in their intracellular localization.
In quiescent cells the PKC
95 form predominates, whereas
after passage PKC
87 becomes the major species. We have
recently reported that these forms differ in their phosphorylation
status at Ser729 (26). Thr566 and
Ser703 are phosphorylated in both these forms of PKC
,
and the protein has complete N and C termini (26). The formation of
PKC
87 upon cell passage is not due to new protein
synthesis. We have therefore suggested that a phosphatase responsible
for dephosphorylation of Ser729 is activated upon cell
passage. Removal of the Ser729 phosphate from the
hydrophobic domain may reduce the stability of the enzyme, rendering it
more accessible to phosphatase attack and potentially to degradation
(27). Alternatively, the change in localization of PKC
on passage
may make it accessible to a Ser729 phosphatase. Therefore
regulation of phosphorylation at Ser729 may prove to be yet
another level of control for PKC.
is a two-stage process. A specific
phosphatase removes the phosphate at Ser729 followed by
either the removal of other phosphate groups or rephosphorylation at
Ser729 and recycling of the enzyme to the 95-kDa form.
at
Ser729 in 3T3 and 3T6 fibroblasts is regulated. We present
evidence that the dephosphorylation of Ser729 is not
mediated by protein phosphatases 1, 2A, and 2B but is dependent
upon serum and cell readhesion, and that MAPK and mTOR (mammalian target of
rapamycin) pathways are involved. PKC is also
important in regulating the phosphorylation status of
Ser729.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PKC
antibodies used for Western blotting
and immunoprecipitations were generated to the C-terminal peptide
sequence by Professor N. Groome (Oxford Brookes University, UK) as
previously described (26). Peroxidase-conjugated secondary antibodies
were from Sigma.
and
PKC
translocation inhibitor and activator peptides coupled to
anttenepaedia carrier protein were generously supplied by Professor D. Mochly-Rosen (Stanford, CA).
RD was generously donated by Professor S. Jaken (University of
Vermont). 3T3 and 3T6 cells were transiently transfected with Superfect
(Qiagen) and were analyzed after 48 h, were quiescent, or were
passaged. Transfection was monitored by Western blotting for the
hemagglutinin tag. PKC
antisense was created through cloning the
full-length PKC
into the pZeo SV vector (Invitrogen). Stable
transfections were selected using Zeocin (Invitrogen), and individual
colonies were expanded and analyzed by Western blotting. In all cases
results were compared with mock transfected cells.
as described previously (26).
antibody overnight (1:250), and this
was recovered with protein A-Sepharose. After washing with
immunoprecipitation buffer, the pellet was resuspended in 10% SDS and
Laemmli loading buffer and resolved by SDS-PAGE.
were immunoprecipitated
as described above. Samples were resolved by SDS-PAGE, and gels were
fixed in isopropanol/acetic acid/H20 (25/20/65, v/v) for
1 h and then incubated for 15 min in Amplify (Amersham Pharmacia
Biotech) before drying and exposing to x-ray film. All data shown are
typical of at least three independent experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
87 Is Formation upon Cell Passage Is Not the
Result of New Protein Synthesis--
In quiescent cells
PKC
95 predominates, whereas 15 min after passage into
serum with readhesion PKC
87 becomes the major form (Fig.
1A). We have recently shown
that PKC
95 and PKC
87 differ in their
phosphorylation at Ser729 and that the formation of
PKC
87 is most probably the result of dephosphorylation
of PKC
95 at Ser729 (26). It is likely that
the apparent increase in total PKC
protein (Fig. 1A) is
the result of increased immunoreactivity of our antibody with
PKC
87 compared with PKC
95 since the
polyclonal PKC
antibody used in these studies is raised against the
C-terminal region of PKC
(-NQEEFKGFSYFGEDLMP), which includes
Ser729. This observation applies to other Western blots.
This is confirmed by [35S]Met/Cys incorporation studies
that reveal that no new PKC
synthesis occurs within 15 min of
passage although synthesis is detected 48-72 h after passage (Fig.
1B). Western blots show that PKC
95 reappears
and becomes the predominant PKC form within 1 h after cell passage
(Fig. 1A). Because there is no synthesis of PKC
over this
time period (Fig. 1B), this suggests that PKC
95 detected within 1 h of passage is derived through
the rephosphorylation of PKC
87.
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Fig. 1.
A, Western blot for PKC from 3T3
cells. Lane 1, quiescent cells; lane 2, 15 min
after passage; lane 3, 1 h after passage; lane
4, 2 h after passage; lane 5, 4 h after
passage; lane 6, 8 h after passage.
PKC
95 and PKC
87 are indicated.
B, fluorogram of PKC
immunoprecipitated from
[35S]Met/Cys-labeled 3T3 cells. Lane 1, 15 min
after passage; lane 2, 30 min after passage; lane
3, 1 h after passage; lane 4, 4 h after
passage; lane 5, 8 h after passage; lane 6,
12 h after passage; lane 7, 24 h after passage;
lane 8, 48 h after passage. PKC
is indicated.
87 upon Cell Passage--
When quiescent cells are
passaged into serum-free medium and allowed to adhere for 15 min, no
formation of PKC
87 is observed (data not shown).
However, when cells are passaged into increasing concentrations of
serum, PKC
87 is formed in increasing amounts (Fig.
2A). This result shows that
some factor(s) in serum is/are necessary for the formation of
PKC
87 upon cell passage. Fibroblasts at 70% confluency
contain both PKC
95 and PKC
87 (Fig.
2B). When these cells are serum-starved for 24 h, to
promote entry into G0 (28), PKC
95 becomes
the predominant form (Fig. 2B), as is observed in cells grown to confluency and quiescence (Fig. 2A). Readdition of
serum to serum-starved cells in G0 does not promote the
formation of PKC
87, although passage of these cells does
(Fig. 2B). This is also the case if fresh serum is added to
quiescent fibroblasts (results not shown). These findings suggest that
serum is necessary but not sufficient for the formation of
PKC
87 upon cell passage.
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Fig. 2.
A, Western blot for PKC from 3T3
cells. Lane 1, quiescent; lane m, marker lane;
lane 2, passaged into medium containing 1% serum;
lane 3, passaged into medium containing 10% serum;
lane 4, passaged into medium containing 20% serum.
PKC
95 and PKC
87 are indicated.
B, Western blot for PKC
from 3T3 cells. Lane
1, 70% confluent; lane 2, serum-starved for 24 h;
lane 3, passaged, after serum starvation for 24 h;
lane 4, cells to which fresh medium has been added for 15 min, after serum starvation for 24 h. PKC
95 and
PKC
87 are indicated. C, Western blot for
PKC
in 3T3 cells. Lane 1, quiescent; lane 2,
passaged; lane 3, passaged into flasks coated with 12 mg/ml
poly-HEME to prevent adherence; lane 4, passaged into flasks
coated with 24 mg/ml poly-HEME to prevent adherence.
PKC
95 and PKC
87 are indicated.
D, Western blot for PKC
from 3T3 cells. Lane
1, quiescent; lane 2, quiescent, treated for 15 min
with 2 µM nocodazole; lane 3, quiescent,
treated with 200 nM cytochalasin D for 15 min.
PKC
95 and PKC
87 are indicated.
87
upon passage. This is shown by the finding that when quiescent
fibroblasts are passaged in serum containing medium onto
poly-HEME-coated plastic, or shaken to prevent adhesion, no formation
of PKC
87 is observed (Fig. 2C). These results
emphasize the need for both serum factors and readhesion in
PKC
87 formation. We have confirmed through
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide analysis
that 3T3 and 3T6 fibroblasts require both serum and readhesion to exit
G0 and proliferate (data not shown). It should be noted
that trypsinization of fibroblasts does not stimulate PKC
87 formation (data not shown).
87 formation (Fig.
2D), it is clear that other factors are also important in
regulating the dephosphorylation of Ser729. One such factor
may be the disruption of cell-substratum interactions and resulting
changes in the cytoskeleton that occur upon cell passage and
readhesion. Indeed, if the actin cytoskeleton is disrupted through
cytochalasin D treatment of quiescent cells, PKC
87 is
formed (Fig. 2D). The microtubule disrupting drug nocodazole does not produce the same effect (Fig. 2D).
87
Formation upon Passage--
Treatment of cells with okadaic acid (OA)
and calyculin (PP1 and PP2A inhibitors (29)) or cyclosporin A and
ascomycin (inhibitors of PP2B (29)) did not inhibit the formation of
PKC
87 upon passage (Fig.
3) (ascomycin C data not shown). This
suggests that these protein phosphatases are not involved in catalyzing the removal of the Ser729 phosphate and that an
OA-insensitive phosphatase is mediating this dephosphorylation. We
observed that OA increased PKC
87 formation upon cell
passage. It is possible then that inhibition of PP1 and/or PP2A
increases the activity of the Ser729 phosphatase.
Alternatively, the inhibitors may be preventing further
dephosphorylation of PKC
87 at Ser703 and
Thr566, thus preserving PKC
87. In fact we
occasionally detect a faster migrating PKC
band (PKC
84); formation of PKC
84 is inhibited
by OA, supporting the latter idea. Hansra et al. (30) have
shown that TPA-induced dephosphorylation of PKC
could be only
partially inhibited by OA and have speculated that another protein
phosphatase, insensitive to OA, may be involved. These workers have
also described the importance of vesicle transport in the
dephosphorylation of PKC
, showing that incubation at 18 °C
inhibited dephosphorylation (31). We find that incubation at 18 °C
to inhibit vesicle transport had no effect on the production of
PKC
87 (Fig. 3). In fact, similarly to OA, it increased
the formation of PKC
87.
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Fig. 3.
Western blot for PKC
from 3T3 cells. Lane 1, passaged; lane
2, passaged, Me2SO solvent control; lane 3, passaged in
the presence of NaF; lane 4, passaged in the presence of 1 nM calyculin; lane 5, passaged in the presence
of 10 nM calyculin; lane 6, passaged in the
presence of 1 µM okadaic acid; lane 7,
passaged in the presence of 10 µM okadaic acid;
lane 8, passaged in the presence of 1 µM
cyclosporin A; lane 9, passaged in the presence of 10 µM cyclosporin A; lane 10, passaged and
maintained at 18 °C. PKC
95 and PKC
87
are indicated.
87 Production upon Passage Is Increased by
Inhibiting Calpain, PI 3-Kinase, and PKC
--
Treatment of
fibroblasts with the calpain and proteasome inhibitor ALLN (32)
increased the level of PKC
87 produced upon cell passage
(Fig. 4A). This may implicate
calpain in the activation of a putative Ser729 phosphatase,
or, probably more likely, this reflects an inhibition of the
degradation of PKC
87. Treatment of cells with the PI
3-kinase inhibitors LY294002 and wortmannin (Refs. 33 and 34;
wortmannin data not shown) also caused an increase in formation of
PKC
87 upon cell passage (Fig. 4B). PI
3-kinase has already been demonstrated to play a role in PKC
phosphorylation through its activation of phospholipid-dependent kinase 1 (7, 8). Our findings also implicate PI 3-kinase in the control of PKC
dephosphorylation and
degradation.
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Fig. 4.
A, Western blot for PKC from 3T3
cells. Lane 1, passaged, Me2SO solvent control;
lane 2, 3T3 cells passaged in the presence of 10 µM ALLN. PKC
95 and PKC
87
are indicated. B, Western blot for PKC
from 3T3
cells. Lane 1, passaged, Me2SO solvent control;
lane 2, passaged in the presence of 10 µM
LY294002. PKC
95 and PKC
87 are indicated.
C, Western blots for PKC
and PKC
from 3T3 cells
transfected with PKC
antisense. Lane 1, mock transfected,
quiescent; lane 2, mock transfected, passaged; lane
3, transfected, quiescent; lane 4, transfected,
passaged. PKC
95 and PKC
87 are indicated.
D, Western blot for PKC
from 3T3 cells transfected with
pCH3RD
. Lane 1, mock transfected, quiescent; lane
2, quiescent, transfected; lane 3, passaged, mock
transfected; lane 4, passaged, transfected.
PKC
95 and PKC
87 are indicated.
E, Western blots for PKC
from 3T3 cells. Lane
1, quiescent; lane 2, quiescent, treated with PKC
translocation inhibitor peptide; lane 3, quiescent, treated
with PKC
translocation inhibitor peptide; lane 4,
quiescent, treated with PKC
translocation agonist peptide;
lane 5, quiescent, treated with PKC
translocation
activator peptide; lane 6, passaged cells untreated control;
lane 7, passaged cells treated with PKC
translocation
inhibitor peptide; lane 8, passaged cells control, treated
with scrambled peptide; lane 9, passaged cells treated with
PKC
translocation inhibitor peptide; lane 10, passaged
cells, control, treated with scrambled peptide; lane 11,
passaged cells treated with PKC
translocation activator peptide;
lane 12, passaged cells treated with PKC
translocation
activator peptide. PKC
95 and PKC
87 are
indicated.
antisense reduced
PKC
expression and increased PKC
87 formation upon
cell passage (Fig. 4C). Expression of the regulatory domain
of PKC
has been shown to be a specific inhibitor of PKC
(35, 36).
Fibroblasts transfected with a construct containing this domain showed
increased formation of PKC
87 upon cell passage (Fig.
4D). PKC
translocation inhibitor and activator peptides
were also used to modulate PKC
in fibroblasts (37, 38). A PKC
translocation inhibitor peptide increased PKC
87
formation upon passage, whereas control and activator peptides had no
effect (Fig. 4E). Peptide modulators of PKC
had no effect on the formation of PKC
87 upon cell passage.
Conformation of the specificity and activity of these peptides was
confirmed through analysis of PKC isoform localization to membrane and
cytosol fractions (data not shown).
87 upon Passage Involves MAPK,
mTOR, and PKC--
The production of PKC
87 upon passage
is inhibited passaging cells in the presence of PD98059, a MEK
inhibitor (Ref. 39 and Fig.
5A), rapamycin, an inhibitor
of mTOR (Ref. 40 and Fig. 5B), and by chelerythrine or
Ro-31-8220, PKC inhibitors (Refs. 41 and 42; Fig. 5B;
Ro-31-8220 data not shown). Such findings suggest a role for the MAPK
pathway, mTOR, and PKC in the control of PKC
87 formation
on cell passage. Inhibitors of protein kinase A, p38, and tyrosine
kinases had no effect on PKC
87 formation upon passage,
suggesting that these pathways are not important in this process. The
inhibitor data are summarized in Table
I.
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Fig. 5.
A, Western blot for PKC from 3T3
cells. Lane 1, quiescent; lane 2, passaged;
lane 3, passaged cells, Me2SO solvent control; lane
4, 3T3 cells passaged in the presence of 100 µM
PD98059. PKC
95 and PKC
87 are indicated.
B, Western blot for PKC
from 3T3 cells. Lane
1, quiescent; lane 2, passaged Me2SO
solvent control; lane 3, passaged in the presence of 500 nM rapamycin; lane 4, passaged in the presence
of 1 µM chelerythrine. PKC
95 and
PKC
87 are indicated. C, Western blot for
PKC
from quiescent 3T3 cells. Lane 1, treated for 15 min
with 250 nM 4
phorbol; lane 2, treated for
15 min with 250 nM TPA and 1 µM
chelerythrine; lane 3, treated for 15 min with 250 nM TPA. PKC
95 and PKC
87 are
indicated.
Effect of inhibitors on PKC87 formation on cell passage
87 Production in Quiescent
Cells--
TPA treatment of cells activates conventional and novel PKC
isoforms, causing their dephosphorylation and subsequent degradation (30, 31). When quiescent fibroblasts are treated with 250 nM TPA for 15 min, formation of PKC
87 is
detected, although this is at a reduced level compared with cell
passage (Fig. 5C). This TPA effect can be blocked with the PKC inhibitor chelerythrine and does not occur with the inactive 4
-phorbol (Fig. 5C). As with PKC
87
formation upon cell passage, this effect can be inhibited by preincubation with rapamycin and PD98059 (data not shown), suggesting that the MAPK and mTOR pathways are also important here.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and that this is
associated with a change in the intracellular localization of the
protein. PKC
95, predominant in quiescent cells, has a
perinuclear localization and is phosphorylated at Thr566,
Ser703, and Ser729. PKC
87 has a
cytosolic distribution and is phosphorylated at Thr566 and
Ser703 (26). We have suggested that on cell passage a
PKC
Ser729 phosphatase may be activated. The hydrophobic
(Ser729 in PKC
) site is conserved in most PKCs and in
the ACG group of kinases (10). It is therefore essential to understand
how phosphorylation and dephosphorylation in the hydrophobic domain is
controlled. Here we have examined some factors that are important in
the control of Ser729 phosphorylation status.
95 requires both serum and
adhesion. Fibroblasts do not proliferate in the absence of serum or
when prevented from adhering (28), and it is therefore likely that
PKC
87 formation is dependent upon re-entry of quiescent
or serum-starved cells into the cell cycle. However, we have shown that
re-entry into the cell cycle alone is not sufficient for this process
because if cells are serum-starved and then restimulated with serum
there is no formation of PKC
87. It is possible then that
PKC
87 formation is mediated through both disruption of
cell-substratum interactions and readhesion of the cells to the
substratum in the presence of serum. Our finding that cytochalasin D,
the microfilament-disrupting drug, stimulates the formation of
PKC
87 in quiescent cells supports this view. Readherence
of cells after passage involves reorganization of the cytoskeleton.
PKC
has an actin-binding domain (21, 22), and we therefore speculate that PKC
bound to actin may be activated by microfilament
disruption, thereby either stimulating phosphatase activity or allowing
access to a putative Ser729 phosphatase. Alternatively,
disruption of the actin cytoskeleton through passage or cytochalasin D
treatment could alter the localization of PKC
, making it susceptible
to attack by a Ser729 phosphatase.
87 upon passage is not dependent upon the more well
defined protein phosphatases, PP1, PP2A, and PP2C. However, we find
that the formation of PKC
87 upon cell passage is
inhibited by rapamycin, indicating involvement of mTOR. mTOR has
recently been implicated in the control of phosphorylation of the
hydrophobic site in PKC
and PKC
(10, 11). These authors speculate
that this is mediated through the activation via mTOR of a phosphatase
specific for the hydrophobic site rather than through regulation of a
kinase. Our data presented here support this view and also suggest that
this mTOR-controlled phosphatase is not PP1, PP2A, or PP2B.
87 detected upon cell passage. The most likely explanation
for these findings is that these inhibitors are blocking further
dephosphorylation and degradation of PKC
87, thereby
increasing PKC
87 levels. It seems that
PKC
95 dephosphorylation occurs in two stages; firstly
the removal of the phosphate at Ser729 by a specific
phosphatase, followed by dephosphorylation at Thr566 and
Ser703 by an OA-sensitive phosphatase. It has been shown
that dephosphorylated forms of PKC are more susceptible to subsequent
proteolytic degradation (13-15). PI 3-kinase inhibitors also increased
PKC
87 formation upon passage. This may be through
inhibition of a kinase that rephosphorylates PKC
Ser729,
possibly a complex including PKC
(9-11, 43).
95 becomes the
predominant form of PKC
within 1 h of passage. This
reappearance of PKC
95 may be mediated through
rephosphorylation at Ser729 of PKC
87, the
predominant form at earlier time points after passage. Our [35S]methionine-labeling experiments have shown that
there is no new synthesis of PKC
over this early time period,
suggesting that PKC
95 must be derived from the recycling
of PKC
87. Rephosphorylation of Ser729 could
potentially be mediated through autophosphorylation (4) or through the
activity of another kinase (10, 11).
activity and translocation also increased
PKC
87 detected upon passage suggesting that PKC
either inhibits PKC
dephosphorylation or promotes
PKC
87 dephosphorylation and degradation. Inhibition of
PKC
95 dephosphorylation may be mediated through
inhibiting a Ser729 phosphatase or through inhibiting the
activation of PKC
, hence its accessibility to phosphatase attack.
There is evidence in the literature of a yin-yang relationship between
PKC
and PKC
. For example, PKC
is growth promotory, whereas
PKC
inhibits cell growth (17). PKC
acts to prevent cells from
cardiac ischemia, whereas PKC
mediates cell death (44, 45). The role
of PKC
in the regulation of PKC
dephosphorylation in fibroblasts
needs to be further investigated.
87 formation upon passage is inhibited by PD98059, a
MEK inhibitor. This implicates the MAPK pathway in the control of
PKC
Ser729 dephosphorylation. Because serum is required
for this process, it is likely that the MAPK pathway is downstream of
any extracellular signals in the medium that lead to cell
proliferation. As already stated, it seems that signals that stimulate
cell proliferation are essential for PKC
87 formation
upon cell passage.
phosphorylation at Ser729 is PKC-sensitive because
we have demonstrated that PKC
87 production upon passage
can be inhibited by the PKC inhibitors chelerythrine and Ro 31-8220.
Also, PKC
87 production can be stimulated in quiescent
cells through the addition of 250 nM TPA for 15 min. This
suggests a role for another isoform of PKC in the regulation of PKC
dephosphorylation. Alternatively, PKC
may regulate the activity of a
Ser729 phosphatase. PKC inhibitors would therefore inhibit
this phosphatase activity through inhibition of PKC
.
regulation on passage is summarized in
Fig. 6. We have observed similar results
in 3T6 fibroblasts and C6 glioma cells suggesting that our findings are
not cell type-specific. Our data suggest that at least a two-stage
process is involved in regulating dephosphorylation of
PKC
95. Passage of cells or TPA treatment of quiescent
cells causes dephosphorylation at Ser729. It is probable
that PKC
is activated upon passage and readhesion. PKC
has been
shown to be important in the spreading of HeLa and other cells (46, 47)
where PKC
is activated upon cell matrix contact and is required for
actin polymerization. The loss of a phosphate at Ser729 is
sensitive to re-entry into the cell cycle from G0 and
readhesion of cells; it is also dependent upon the MAPK pathway. It has
been reported that, once in an activated conformation, PKC becomes susceptible to phosphatase attack (27) and that loss of the C-terminal
phosphorylations may regulate translocation of the kinase to the
cytoplasm after activation (48). The loss of a phosphate at
Ser729 is not mediated through PP1, PP2A, or PP2B.
PKC
87 could then be rephosphorylated at
Ser729 and recycled back to PKC
95. This may
involve a membrane-associated kinase complex including PKC
. (43)
Alternatively PKC
87 may be further dephosphorylated at
sites Ser703 and Thr566, perhaps mediated by an
OA-sensitive phosphatase. Complete dephosphorylation increases the
instability of PKC and targets the protein for degradation (13, 14,
15). It is therefore likely that once fully dephosphorylated PKC
is
sensitive to degradation via calpain and the proteasome.
View larger version (16K):
[in a new window]
Fig. 6.
Summary of the regulation of
PKC phosphorylation status in 3T3 cells at
quiescence and on cell passage.
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ACKNOWLEDGEMENTS |
---|
We thank Professor D. Mochly-Rosen (Stanford
University) for the PKC translocation inhibitor and activator peptides,
Professor S. Jaken (University of Vermont) for the pCH3 RD domain
vector, and Professor P. Parker (Imperial Cancer Research Fund,
London) for the PKC
clone.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by the Biotechnology and Biological Sciences
Research Council Integration of Cellular Responses Initiative. To whom correspondence should be addressed: Tumour Biology Laboratory, Department of Biochemistry, University College Cork, Lee Maltings Complex, Prospect Row, Cork, Republic of Ireland. E-mail:
kengland@ucc.ie.
§ Present address: Smith and Nephew Group Research Centre, Science Park, Heslington, York YO10 5DF, UK.
¶ Present address: Dept. of Life and Health Services, Aston Pharmacy School, Aston University, Aston Triangle, Birmingham B4 7ET, UK.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009421200
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ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; MAPK, mitogen-activated protein kinase; OA, okadaic acid; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PAGE, polyacrylamide gel electrophoresis; TPA, 12-O-teteradecanoylphorbol-13-acetate; PI, phosphatidylinositol.
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