From the Institute of Biomembranes, Department of
Molecular Cell Biology, Utrecht University, 3584 CH Utrecht, The
Netherlands and ¶ The Netherlands Cancer Institute, Department of
Cellular Biochemistry, 1066 CX Amsterdam, The Netherlands
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ABSTRACT |
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Protein kinase B (PKB), also known as Akt or
RAC-PK, is a serine/threonine kinase that can be activated by growth
factors via phosphatidylinositol 3-kinase. In this article we show that PKC Protein kinase B (PKB),1
also referred to as c-Akt or RAC-PK is a 60-kDa serine/threonine kinase
which is the cellular homologue of the viral oncogene v-Akt (1-3). So
far, three isoforms of PKB have been isolated: PKB PKB comprises a NH2-terminal Akt homology (AH) domain of
148 amino acids, a catalytic domain of 264 amino acids showing high homology with cyclic AMP-dependent protein kinase A (PKA)
and protein kinase C (PKC) and a short COOH-terminal tail of 68 amino acids. A pleckstrin homology (PH) domain of 106 amino acids is present
within the AH domain. Treatment of cells with different growth factors,
insulin, or phosphatase inhibitors results in rapid activation of PKB
(10, 14, 15). Also heat shock, hyperosmolarity stress, and
intracellular cAMP elevation were shown to activate PKB in
vivo (16, 17). Growth factor and insulin-induced activation is
almost completely prevented by overexpression of a dominant negative
form of phosphatidylinositol (PI) 3-kinase ( The mechanism of PKB activation through the PI 3-kinase signaling
pathway is not completely understood. In vitro,
phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), one of
the lipid products generated by PI 3-kinase, stimulates PKB activity by
binding to the PH domain (19, 20). Furthermore, PKB activation was
shown to be dependent on its phosphorylation of Thr308 and
Ser473 (21). Phosphorylation of Thr308 is
mediated by an upstream kinase, called phosphatidylinositol 3,4,5-triphosphate-dependent protein kinase-1 (PDK-1), while
the kinase responsible for phosphorylation of Ser473,
already designated as PDK-2, remains to be identified (22, 23). The
proposed mechanism for PKB activation is that PI(3,4)P2 and
phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3)
generated by PI 3-kinase recruit PKB to the plasma membrane where
Thr308 is phosphorylated by PDK-1 and Ser473 by
PDK-2 (24). The activation of PKB by both PI(3,4)P2 and PDK-1 and -2 makes the activation of PKB a multistep process.
Initial studies by Konishi and co-workers (25, 26) showed that the Expression Constructs--
The pSG5 (Stratagene, La Jolla, CA)
constructs containing HA-tagged wild-type bovine PKB Cell Culture, Transfections, and Immunoprecipitations--
COS-1
and CHO cells were grown in Dulbecco's modified Eagle's medium
supplemented with 7.5% fetal calf serum (Life Technologies, Inc.) at
37 °C in a humidified atmosphere with 7% CO2. Transient transfections in COS-1 cells were performed at 40% confluency by a
DEAE-dextran method. In short, DNA was diluted in 500 µg/ml DEAE-dextran (Sigma) in phosphate-buffered saline and added to the
cells. Following a 30-min incubation at 37 °C, medium containing 80 µM chloroquine (Sigma) was added and the cells were
incubated for 2.5-3 h at 37 °C and subsequently shocked with 10%
dimethyl sulfoxide (Sigma) for 2.5 min. Twenty-four hours after
transfection cells were serum starved for 16 h. Stimulated and
unstimulated cells were washed once with ice-cold phosphate-buffered
saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 40 mM
Western Blotting--
Cell extracts and immunoprecipitations
were separated on an 8% SDS-PAGE gel and transferred to polyvinylidene
difluoride membranes (Boehringer, Mannheim, Germany). Membranes were
blocked in 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in TBST
buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl,
and 0.1% Tween 20) for 1 h at room temperature. For detection of
the Myc-tagged or HA-tagged proteins the membranes were incubated with
the monoclonal 9E10 or 12CA5 antibody in 1% protifar in TBST buffer
subsequently followed by incubation with peroxidase-conjugated rabbit
anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA).
Detection of endogenous PKB was performed using the monoclonal PKB/Akt
antibody (Transduction Laboratories, Lexington, KY) or the polyclonal
Akt-C20 (Santa Cruz Biochemical Corp., Santa Cruz, CA) subsequently
followed by incubation with peroxidase-conjugated rabbit anti-mouse or donkey anti-goat (Jackson ImmunoResearch) secondary antibody, respectively. Endogenous PKC In Vitro Kinase Assays for PKC PKC
The binding of PKB with PKC PKB Activity Is Required for Complex Dissociation--
To
investigate the effect of PDGF on the PKB-PKC
As previously reported, PDGF induces the activation of PKB and, albeit
to a lesser extent, also of PKC PKC PKC
An important step in the full activation of PKB is the phosphorylation
of residues Thr308 and Ser473 by PDK1 and -2 (21). To establish whether the inhibition of PKB activation by PKC PKC GSK-3, a Downstream Target of PKB, Is Also Affected by
PKC PKC In this report we show that PKC As shown in this paper, the PKB-PKC An important observation of this study is the inhibitory effect of
PKC The question, however, remains how PKC Recently, PDK-1 has been described as the kinase that is responsible
for the PI 3-kinase-dependent phosphorylation and
activation of PKC The link between PKC In summary, we have shown that PKC but not PKC
and PKC
can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKC
was also found in COS-1 cells transiently expressing PKB and PKC
, and moreover we found that
this association is mediated by the AH domain of PKB. Stimulation of
COS-1 cells with platelet-derived growth factor (PDGF) resulted in a
decrease in the PKB-PKC
interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the
interacting kinases showed that PDGF-induced activation of PKC
was
not affected by co-expression of PKB. However, both PDGF- and
p110-CAAX-induced activation of PKB were significantly abolished in
cells co-expressing PKC
. In contrast, co-expression of a kinase-dead
PKC
mutant showed an increased induction of PKB activity upon PDGF
treatment. Downstream signaling of PKB, such as the inhibition of
glycogen synthase kinase-3, was also reduced by co-expression of
PKC
. A clear inhibitory effect of PKC
was found on the
constitutively active double PKB mutant (T308D/S473D). In summary, our
results demonstrate that PKB interacts with PKC
in vivo
and that PKC
acts as a negative regulator of PKB.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, PKB
, and
PKB
(1, 2, 4, 5). Overexpression of PKB family members has been
correlated with different cancers such as breast cancer and some
pancreatic and ovarian cancers (2, 6, 7). Recently, PKB has been found
to yield an anti-apoptotic signal, which is crucial for cell survival
in both fibroblasts and neuronal cells (8, 9). Other reports have
indicated a role for PKB in the regulation of glycogen synthesis by
inhibition of glycogen synthase kinase-3 (GSK-3) (10, 11). In addition,
glucose uptake and metabolism in 3T3-L1 adipocytes have been shown to
be regulated by PKB by mediating the translocation of the glucose
transporter GLUT4 to the plasma membrane (12, 13). Moreover, a role for
PKB has been described in the regulation of protein synthesis through indirect activation of the p70 ribosomal S6 kinase (p70S6K)
(14).
p85) or by pretreatment
of cells with the PI 3-kinase inhibitors wortmannin and LY294002 (14).
Furthermore, a PDGF receptor mutant that is not able to stimulate PI
3-kinase activity also fails to activate PKB (14, 18). These data
demonstrate that insulin and growth factor-induced signals leading to
PKB activation are transduced via the PI 3-kinase pathway. In contrast,
stress, okadaic acid, and cAMP induced activation of PKB is PI 3-kinase
independent since wortmannin is unable to block this pathway of PKB
activation (15-17). Thus, in vivo, PKB can be activated via
at least two pathways: a PI 3-kinase dependent and a PI 3-kinase
independent pathway.
,
, and
isoforms of PKC are able to interact with PKB in
vitro. In this paper we show that PKB can only be
co-immunoprecipiatated with PKC
and in addition we found that this
interaction is under control of PKB activity. To understand the
possible function of the PKB-PKC
association, we investigated
whether the interacting kinases regulate the activity of the respective
kinases. Although no effect was found of PKB on PKC
activity, both
PDGF- and p110-CAAX-induced activation of PKB is abolished by
co-expression of PKC
. The activity of GSK-3, a downstream target of
PKB is also affected by PKC
co-expression. Finally, we found that
the constitutive active PKB mutant (T308D/S473D) is inhibited by PKC
in a PDGF-independent fashion. The results obtained establish PKC
as
a negative regulator of PKB activity.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, PKB
"kinase dead" (K179A), PKBDD and PKBAA were
a gift from Dr. Paul Coffer (Department of Pulmonary Diseases, University Hospital Utrecht, The Netherlands). The DNA fragments encoding the AH domain of PKB (PKBAH) and PKB lacking the AH domain (PKB
AH) were amplified by polymerase chain reaction and subcloned as
a BamHI/KpnI fragment into the eukaryotic
expression vector pBK-CMV (Stratagene, La Jolla, CA) containing a HA
epitope tag (pBK-HA). p110-CAAX, p110-R916P-CAAX (PLAP-CAAX), and the
pMT2SM constructs containing Myc-tagged wild-type mouse PKC
and
Myc-tagged kinase-dead PKC
have been described earlier (27, 30).
-glycerophosphate, 1 mM sodium vanadate, 50 mM sodium fluoride, and 10 µg/ml aprotinin) and incubated
on ice for 5 min. Lysates were centrifuged and supernatants were
precleared with protein A-Sepharose beads (Pharmacia, Uppsala, Sweden)
for 1 h at 4 °C. HA-PKB was immunoprecipitated from aliquots
(200 µg of protein) of the precleared extracts using 6 µg of the
monoclonal anti-HA antibody (12CA5) coupled to protein G-Sepharose
beads (Sigma), whereas Myc-PKC
was immunoprecipitated from
precleared lysates by 1 µg of the monoclonal anti-Myc antibody (9E10)
(Boehringer, Mannheim, Germany) coupled to protein A-Sepharose beads.
Endogenous PKC
was immunoprecipitated from CHO cells using a
polyclonal PKC
antibody (27) coupled to protein G-Sepharose beads,
whereas PKC
and PKC
were immunoprecipitated by monoclonal PKC
and PKC
antibodies (Transduction Laboratories, Lexington, KY),
respectively. Normal rabbit serum was used as control antiserum for the
co-immunoprecipitation studies. Immunoprecipitations were washed twice
with lysis buffer and twice with low salt buffer (50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2) prior to Western
blot analysis or twice with high salt buffer (50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, and 0.5 M LiCl) and twice with low salt buffer prior to activity measurements.
, PKC
, and PKC
were detected using the monoclonal PKC
, PKC
(Transduction Laboratories), or
polyclonal PKC
antibody followed by incubation with
peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary
antibody (Jackson ImmunoResearch). For PKB detection with the
phospho-specific Akt (Ser473) antibody (New England
Biolabs, Beverly, MA) polyvinylidene difluoride membranes were
incubated with the polyclonal antibody followed by incubation with
peroxidase-conjugated goat anti-rabbit secondary antibody. Proteins
were visualized by Enhanced Chemiluminescence (Renaissance, NEN Life
Science Products Inc., Boston, MA). For quantification of protein
amounts a densitometer (Molecular Dynamics) and ImageQuant software
were used.
, PKB, and GSK-3--
PKC
activity was measured with the
-peptide (ERMRPRKRQGSVRRRV) as
substrate as described previously (27, 28). Immunoprecipitations were
incubated with 45 µl of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2) containing 50 µM
-peptide, 0.2 mM EGTA, 50 µM unlabeled ATP, and 3 µCi of
[
-32P]ATP (Amersham International, United Kingdom).
PKB activity was assayed with the Crosstide peptide (GRPRTSSFAEG) as a
substrate (10). Immunoprecipitations were incubated with 45 µl of
kinase assay mixture (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 30 µM Crosstide peptide, 1 µM of the specific
peptide inhibitor of cyclic AMP-dependent protein kinase
(PKI) (Bachem, Bubendorf, Switzerland), 50 µM unlabeled
ATP, and 3 µCi of [
-32P]ATP). GSK-3 activity was
measured using the GS peptide (YRRAAVPPSPSLSRHSSPHQSEDEEE) (29). Cell
lysates were incubated with 60 µM GS peptide, 2 mM MgCl2, 100 µM ATP, and 2 µCi
of [
-32P]ATP. After incubation for 20 min at 30 °C
under continuous shaking, reactions were stopped by addition of 200 mM EDTA. Proteins were precipitated by the addition of 25%
trichloroacetic acid and centrifuged for 1 min at 14,000 rpm.
Supernatants containing the phosphorylated peptide were spotted onto
p81 phosphocellulose filters (Whatman), washed three times with 1%
(v/v) orthophosphoric acid, and analyzed by Cerenkov counting. Control
experiments revealed that phosphorylation of the GS peptide is highly
specific for GSK-3
and that neither PKB nor PKC
is able to
phosphorylate the peptide.2
Under the conditions used the kinase assays are linear for at least 60 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Associates with PKB in CHO Cells--
In vitro
binding studies have recently shown that PKB associates with the
,
, and
isoforms of PKC (5). In order to investigate the possible
interaction of these PKC isoforms with PKB in vivo, we
performed co-immunoprecipitation studies using CHO cells. Endogenous
PKC
, PKC
, and PKC
were immunoprecipitated from cell lysates
and Western blot analysis shows that similar amounts of the three PKC
isoforms were precipitated (Fig.
1B). The presence of PKB was
analyzed using Western blot detection and, as shown in Fig. 1A, PKB is
present in the PKC
but not in the PKC
and PKC
immunoprecipitates. As a control, normal rabbit serum was incubated
with lysates of CHO cells and only a faint band is visible possibly
reflecting aspecific binding to the non-immune control (Fig. 1A).
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Fig. 1.
PKC associates with
PKB in CHO cells. A, CHO cells were lysed and incubated
with normal rabbit serum (non-immune) or antibodies against PKC
,
PKC
, and PKC
as described under "Materials and Methods."
Immunoprecipitates and a cell lysate (lysate) from CHO cells
were analyzed for the presence of PKB by Western blot using the
monoclonal PKB/Akt (lysate, non-immune, and
-PKC
) or the polyclonal Akt C-20 antibody
(
-PKC
and
-PKC
). IgG indicates the
heavy chain of the immunoglobulin. B, immunoprecipitates of
PKC
, PKC
, and PKC
were analyzed for the presence of these PKC
isoforms using Western blot analysis. C, COS-1 cells were
transiently transfected with HA-PKB and Myc-PKC
, HA-PKBAH, and
Myc-PKC
or HA-PKB
AH and Myc-PKC
as described under
"Materials and Methods." PKB, PKBAH, or PKB
AH were
immunoprecipitated from the lysates using the monoclonal HA antibody
12CA5 and subsequently separated by SDS-PAGE and immunoblotted onto
polyvinylidene difluoride. Co-immunoprecipitation of PKC
was
analyzed using the monoclonal Myc antibody 9E10. Aliquots of each cell
extract (30 µl) were separated by SDS-PAGE and immunoblotted using
both the monoclonal HA antibody 12CA5 and the monoclonal Myc antibody
9E10 to analyze both PKB and PKC
expression levels.
was subsequently investigated in more
detail by transient expression of HA-tagged PKB and Myc-tagged PKC
(Myc-PKC
) in COS-1 cells. HA-PKB was immunoprecipitated using a
monoclonal antibody against the HA-tag (12CA5) and
co-immunoprecipitation of Myc-PKC
was observed on Western blot using
a monoclonal antibody against the Myc-tag (9E10) (Fig. 1C).
To identify the domain of PKB that is necessary for the association
with PKC
in vivo, we generated HA-tagged PKB constructs
lacking the AH domain (HA-PKB
AH) or comprising the AH domain
(HA-PKBAH). Co-expression of these constructs with PKC
revealed that
the interaction of PKB with PKC
depends entirely on the presence of
the AH domain. This observation is in agreement with the in
vitro data obtained by Konishi and co-workers (26). PKC
could
not be observed on a Western blot when PKB
AH was immunoprecipitated
from cells expressing PKB
AH and PKC
(Fig. 1C). As a
control, the expression levels of the transiently expressed proteins
were analyzed in total cell lysates and similar expression levels were
found for all constructs (Fig. 1C). In conclusion, our
observations clearly demonstrate that PKC
associates with PKB
in vivo. Furthermore, the in vivo association of
PKB and PKC
is mediated via the AH domain of PKB.
complex, COS-1 cells
were transiently co-transfected with both HA-PKB and Myc-PKC
. The
cells were serum-starved overnight and either left untreated or
stimulated with 25 ng/ml PDGF for 10 min. PKB was immunoprecipitated
and co-immunoprecipitation of PKC
was determined by Western blot
analysis (Fig. 2A). Upon PDGF
treatment the interaction decreased with approximately 75% indicating
that PDGF induces the dissociation of the complex.
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Fig. 2.
Regulation of the PKB-PKC
complex. COS-1 cells were transiently transfected with
either wild-type PKB and wild-type PKC
, serum starved for 16 h
and left untreated or stimulated with 25 ng/ml PDGF-BB for 10 min as
described under "Materials and Methods" (A).
Alternatively, COS-1 cells were transiently transfected with wild-type
PKB and wild-type PKC
, wild-type PKB and kinase-dead PKC
, or
kinase-dead PKB and wild-type PKC
(B). PKB was
immunoprecipitated from the lysates using the monoclonal HA antibody
12CA5 and subsequently separated by SDS-PAGE and immunoblotted onto
polyvinylidene difluoride. Co-immunoprecipitation (IP) of
PKC
was analyzed using the monoclonal Myc antibody 9E10. The amount
of immunoprecipitated PKB was analyzed using the monoclonal HA antibody
3F10. Expression levels of PKB and PKC
were analyzed by Western blot
detection using the monoclonal 3F10 and 9E10 antibodies, respectively
(as described under "Materials and Methods").
(27). In order to establish whether
the activity of these kinases is involved in PKB·PKC
complex
formation, we analyzed the effect of kinase-dead mutants of both PKB
and PKC
. In repeated experiments expression of kinase-dead PKC
resulted in a reduction of complex formation which, however, can be
explained by the reduction in PKC
kd expression (Fig.
2B). In contrast, expression of kinase-dead PKB resulted in
a dramatic increase in the PKB-PKC
interaction (Fig. 2A).
This demonstrates that PKB activity induces the dissociation of the
complex, whereas PKC
activity seems not to be required for the
regulation of the complex. As a control experiment, we incubated the
same blot with anti-HA antibodies showing that similar amounts of
HA-PKB were precipitated (Fig. 2B).
Activity Is Not Affected by PKB in Vivo--
In order to
establish the physiological role for the PKB-PKC
interaction we
investigated the effect on the activity of both kinases. To test a
possible role for PKB on PKC
activity, COS-1 cells were transiently
transfected with PKC
alone or co-transfected with PKB. After
stimulation of the cells with PDGF, PKC
was immunoprecipitated and
its activity was measured by an in vitro kinase assay using the
-peptide as a substrate (28). PDGF stimulation resulted in an
increase of PKC
activity (Fig. 3)
which is in agreement with previous studies (27). Co-expression of PKB
did not affect activation of PKC
upon PDGF treatment, demonstrating
that PKB has no effect on the PDGF-induced activity of PKC
(Fig. 3).
To demonstrate that PKC
and not PKB activity accounts for the
observed change in
-peptide phosphorylation we co-transfected PKC
with kinase-dead PKB. Similar results were obtained as with wild-type PKB showing that PKB activity does not influence the observed change in
-peptide phosphorylation (Fig. 3).
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Fig. 3.
PKC activity is not
affected by PKB. COS-1 cells overexpressing wild-type PKC
(wt),
wild-type PKC
, and wild-type PKB or wild-type PKC
and kinase-dead
(kd) PKB were left untreated (gray bars) or
stimulated with 25 ng/ml PDGF-BB (black bars) for 10 min as
described under "Materials and Methods." PKC
was
immunoprecipitated from the lysates using the monoclonal Myc antibody
9E10 and its activity was assayed with the
-peptide as substrate
(see "Materials and Methods"). The results are presented as ± S.E. for six determinations (three independent experiments) related to
the activity of PKC
in unstimulated cells (100%).
Asterisks indicate p < 0.05 (Student's
t test).
Is a Negative Regulator of PKB Activity--
Using the same
approach, we investigated whether PKC
has an effect on PKB activity.
For these experiments, COS-1 cells were transiently transfected with
wild-type PKB or co-transfected with wild-type PKB and either wild-type
PKC
or kinase-dead PKC
. After PDGF treatment, PKB was
immunoprecipitated and its activity was measured by an in
vitro kinase assay using Crosstide as substrate (10). Activity
measurements showed that PKB activity was increased more than 3-fold
upon stimulation of the cells with PDGF (Fig. 4A). However, PDGF-induced PKB activation
was almost completely abolished when PKB was co-expressed with
wild-type PKC
(Fig. 4A). This indicates that PKC
is
able to inhibit PDGF induced activity of PKB. In contrast, PKB activity
was increased more than 7-fold by PDGF when the cells were
co-transfected with the kinase-dead mutant of PKC
(Fig.
4A). These data clearly show that PKB activity is negatively
regulated by PKC
.
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Fig. 4.
PDGF-induced PKB signaling is negatively
regulated by PKC . A, COS-1
cells overexpressing wild-type (wt) PKB, wild-type PKB and wild-type
PKC
, or wild-type PKB and kinase-dead (kd) PKC
were
serum starved for 16 h and left untreated (gray bars)
or stimulated with 25 ng/ml PDGF-BB (black bars) for 10 min
as described under "Materials and Methods." PKB was
immunoprecipitated from the lysates using the monoclonal HA antibody
12CA5 and its activity was assayed with Crosstide as substrate (see
"Materials and Methods"). The results are presented as average ± S.E. for six determinations (three separate experiments) related to
the activity of PKB in unstimulated cells overexpressing PKB (100%).
Asterisks indicate p < 0.05 (Student's
t test). B, corresponding GSK-3 activity
measurements. Total cell lysates of untreated and PDGF-stimulated cells
were analyzed for GSK-3 activity using a peptide phosphorylation assay
(see "Materials and Methods"). The results are presented as
average ± S.E. for four determinations (two independent
experiments) and PDGF-stimulated values were related to their own
control (100%).
is due to a reduced increase in the phosphorylation of PKB we used a
polyclonal antibody against PKB when phosphorylated on
Ser473. As shown in Fig. 5,
A and C, PDGF treatment induced an significant increase (p < 0.05) in Ser473
phosphorylation when PKB was the only transfected protein in COS-1
cells. In contrast, no significant increase in Ser473
phosphorylation was observed upon PDGF treatment when PKC
was co-expressed with PKB (Fig. 5, A and C). As a
control, expression levels of total PKB protein were determined and as
shown in Fig. 5B the amount of PKB in all lanes was equal
indicating that the observed differences in phosphorylated PKB on
Ser473 was not due to differences in PKB expression levels.
Taken together, from these experiments it can be concluded that PKC
is a negative regulator of PDGF-induced PKB activity.
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Fig. 5.
PKC attenuates
PDGF-induced phosphorylation of PKB-Ser473.
A, COS-1 cells overexpressing either wild-type
(wt) PKB or wild-type PKB and wild-type PKC
were left
untreated or stimulated with 25 ng/ml PDGF-BB for 10 min as described
under "Materials and Methods." Aliquots of each cell extract were
separated by SDS-PAGE and Ser473 phosphorylation was
analyzed using the polyclonal phospho-specific Akt (Ser473)
antibody. B, PKB expression levels were analyzed by Western
blot detection using the monoclonal HA antibody 12CA5. C,
Ser473 phosphorylation was quantified by densitometry and
presented as average ± S.E. for three determinations related to
the phosphorylation in unstimulated cells overexpressing PKB (100%).
Asterisk indicate p < 0.05 (Student's
t test).
Inhibits p110-CAAX-induced PKB Activity--
As already
mentioned, both PKB and PKC
are activated by PDGF most probably
through the PI 3-kinase signaling pathway. In order to find out whether
the negative regulation of PKB by PKC
is mediated by the PI
3-kinase/PKB signal transduction pathway we expressed a catalytically
active membrane-targeted PI 3-kinase (p110-CAAX) together with PKB in
COS-1 cells. p110-CAAX caused a significant, ligand-independent
increase in PKB activity (Fig. 6A). In contrast, a
catalytically inactive membrane-targeted PI 3-kinase (PLAP-CAAX) was
unable to do so (Fig. 6A), which is in agreement with the
work of Didichenko and co-workers (30). Co-expression of PKC
with
p110-CAAX and PKB reduced the p110-CAAX-induced PKB activity with
almost 80% (Fig. 6A). Interestingly co-expression of PKC
with PLAP-CAAX and PKB also resulted in a decrease in basal PKB
activity (Fig. 6A). These observations demonstrate that basal activity of PKC
is already sufficient to inhibit PKB.
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Fig. 6.
p110-CAAX induced PKB signaling is totally
abolished by PKC . A, COS-1
cells overexpressing wild-type PKB, wild-type PKB together with either
p110-CAAX or PLAP-CAAX in the presence or absence of PKC
were serum
starved for 16 h and left untreated. PKB was immunoprecipitated
from the lysates using the monoclonal HA antibody 12CA5 and its
activity was assayed with Crosstide as substrate (see "Materials and
Methods"). Results are presented as average ± S.E. for four
determinations (two independent experiments) related to the activity of
PKB in unstimulated cells overexpressing PKB (100%).
Asterisks indicate p < 0.05 (Student's
t test). The amount of immunoprecipitated PKB was analyzed
using Western blot detection (as described under "Materials and
Methods"). B, corresponding GSK-3 activity measurements.
Total cell lysates were analyzed for GSK-3 activity using a peptide phosphorylation assay (see "Materials
and Methods"). The results are presented as average ± S.E. for
four determinations (two independent experiments) and related to the
GSK-3 value of mock-transfected cells (100%). The expression levels of
PKB were analyzed by Western blotting of total cell lysates (as
described under "Materials and Methods").
--
It has previously been shown that GSK-3 is phosphorylated
and inactivated by PKB in vitro and in vivo (10,
11). In order to test whether this downstream effector of PKB is also
affected by co-expression of PKC
, we measured GSK-3 activities in
cells expressing PKB alone, PKB and PKC
, and PKB and kinase-dead
PKC
using a peptide phosphorylation assay (29). As expected, upon treatment of cells with PDGF the GSK-3 activity is decreased (Fig. 4B). However, when PKB is co-expressed with PKC
the
PDGF-induced reduction in GSK-3 activity is completely overcome (Fig.
4B). In contrast, cells co-expressing a kinase inactive
PKC
mutant exhibits a normal reduction in GSK-3 activity upon PDGF
treatment (Fig. 4B). In addition, similar results were
obtained when PKB was activated via the constitutively activated PI
3-kinase. While expression of p110-CAAX induces the activation of PKB,
a significant decrease was found in the GSK-3 activity. Conversely, the
expression of the catalytically inactive PLAP-CAAX did not induce a
reduction of GSK-3 activity (Fig. 6B). Expression of PKC
completely abolished the p110-CAAX-induced decrease in GSK-3 activity,
whereas co-expression of PKC
with PLAP-CAAX did not affect GSK-3 at
all (Fig. 6B). These results clearly show that both PDGF-
and p110-CAAX-induced PKB activity result in a decrease in GSK-3
activity. Furthermore, these experiments demonstrate that the
inhibitory effect of PKC
on PKB activity is also reflected in the
GSK-3 activity.
Acts Directly on PKB to Inhibit Its Activity--
The
question that remains is where PKC
exactly affects the PKB signaling
pathway. To find out whether PKC
might act on PKB itself, we
expressed an active PKB mutant (PKBDD) in which the two
phosphorylation sites (Thr308 and Ser473) that
are necessary for complete activation are replaced by aspartic acid
resulting in a constitutively active form of PKB (21). This mutant
exhibits high activity already in resting cells and as expected, PDGF
treatment did not increase the activity any further (Fig.
7). Co-expression of PKC
resulted in a
reduction of the ligand-independent activity of the PKBDD
mutant (Fig. 7). PDGF treatment did not contribute to this inhibitory effect since no difference could be observed between untreated and PDGF
stimulated conditions (Fig. 7). As a control, a PKB mutant (PKBAA) in which the two phosphorylation sites are mutated
into alanine was expressed and its activity was measured. Very little
activity was detected for this mutant and neither PDGF treatment nor
PKC
co-expression changed its activity any further (Fig. 7).
Together, these observations show that PKC
does not act upstream of
PKB in the PI 3-kinase signaling pathway to inhibit its activity but strongly suggest that PKC
acts at the level of PKB kinase causing the reduction of its activity. In addition, these data show that PI
3-kinase is not necessarily required for the inhibitory effect of PKB
by PKC
.
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Fig. 7.
Constitutively active PKB is inhibited by
PKC . A, COS-1 cells
overexpressing wild-type (wt) PKB, constitutively active
PKBDD, PKBDD, and PKC
, constitutively
inactive PKBAA or PKBAA and PKC
were serum
starved for 16 h and either left untreated or stimulated with 25 ng/ml PDGF-BB for 10 min. PKB was immunoprecipitated from the lysates
using the monoclonal HA antibody 12CA5 and its activity was assayed
with Crosstide as substrate (see "Materials and Methods"). Results
are presented as average ± S.E. for four determinations (two
independent experiments) related to the activity of PKB in unstimulated
cells overexpressing PKB (100%). Asterisks indicate
p < 0.05 (Student's t test).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
binds in vivo to the
serine/threonine kinase PKB (c-Akt, RAC-PK). The PKB-PKC
interaction was demonstrated by co-immunoprecipitation studies of both endogenous and transiently expressed PKC
and PKB proteins. In contrast, we were
not able to detect association of PKB with endogenous PKC
and PKC
in vivo in CHO cells. This latter observation is in
disagreement with binding studies of Konishi and co-workers (5). These
studies, however, were performed by in vitro binding studies
using the PH domain of PKB fused to GST and lysates of COS-7 cells
containing transiently expressed PKC isoforms. In addition, the PKC
association has only been demonstrated in heat-treated cells (16).
These results suggest that the affinity of PKB for PKC
is higher
than for PKC
or PKC
. Although we were not able to detect the
association of PKB with PKC
or PKB
by co-immunoprecipitation studies we cannot exclude the association of these kinases in the
in vivo situation. The association between PKB and PKC
was observed in both serum-starved and PDGF-stimulated cells.
Stimulation of the cells with PDGF resulted in a reduction in complex
formation. An important question is how the association between PKB and
PKC
is regulated.
interaction is mediated by the
AH domain of PKB. This was concluded from the observation that PKC
co-precipitated with the AH domain while no co-precipitation was
observed between PKC
and the kinase-tail domain of PKB (PKB
AH). The AH domain of PKB largely consists of a PH domain, which has previously been identified as a lipid-binding domain (31). PH domains,
however, including the PH domain of PKB, have also been described to
bind to proteins such as PKC and the
subunit of the
heterotrimeric G-protein (5, 32). The
subunit has been shown to
bind to the carboxyl-terminal
-helix region of the
ARK PH domain
(33). In contrast, different isoforms of PKC including the
Ca2+-dependent (
,
I, and
II) and
Ca2+-independent (
and
) isoforms have been shown to
interact with the second and third
-sheet of the PH domain of the
tyrosine kinase Bruton tyrosine kinase (Btk) (34, 35). PKC
has been shown to bind to the first and second
-sheet of PKB (26). The
-sheets are part of the binding pocket of the PH domain for
phosphoinositides. This raises the possibility that the PKB-PKC
interaction is regulated by the PDGF-induced generation of
D3-phosphoinositide lipids as they may compete with PKC
for binding
to the PKB-AH domain. This mechanism was implicated for the binding of
PKC
I to the PH domain of Btk (35). Alternatively, PKB itself may
regulate the dissociation of the complex. A huge increase was observed
in the binding of PKC
to a kinase-dead PKB mutant while no
difference was found in the association with a PKC
kinase-dead
mutant. This indicates that PKB activity is very important for
dissociation of the complex. The most straightforward explanation for
this effect is that PKC
is a substrate for PKB. In this situation
the phosphorylation of PKC
by PKB after stimulation of the cell with
a growth factor would result in the dissociation of the complex.
Alternatively, it is equally possible that another unknown protein in
the complex, which is a substrate for PKB, is mediating this
interaction. Current research is aimed at the determination of the role
of inositol lipids and PKB substrates in complex formation between PKB
and PKC
.
on the PDGF- and p110-CAAX-induced activation of PKB. This
effect is both measured on PKB activity and on the phosphorylation of
serine 473 of PKB. Also the downstream signaling to GSK-3 is inhibited
by PKC
. These findings can be explained either by the inhibition of
the upstream signaling of PKB or by a direct effect of PKC
on PKB
itself. Our results do not support the possibility that PKC
inhibits
PI 3-kinase, since PKC
was shown to inhibit the constitutive active
PKB mutant (PKBDD) even without stimulation of the cell
with a growth factor. The PKBDD mutant is no longer a
substrate for the PDK-1 and -2 kinases and is active without
stimulation of the cell by growth factors. A possible PI 3-kinase
independent mechanism for inhibition of PKB by PKC
could be that
PKC
activates PP2A, the serine/threonine-specific phosphatase. PP2A
activity has been described to inhibit PKB activity (15). However, the
fact that PKC
inhibits the activity of the PKBDD mutant
that cannot be dephosphorylated does not favor this model. In
conclusion, our data strongly suggest that the inhibiting activity of
PKC
is expressed directly on PKB.
negatively regulates PKB
activity. One of the first steps in the activation process of PKB is
the translocation of PKB to the membrane. This process could be
sensitive to PKC
activity. Our data show that PKC
inhibits the
constitutively active PKB mutant already in quiescent cells, making
this possibility unlikely. On the other hand, we have observed that a
kinase-dead PKC
normally binds to PKB but fails to inhibit PKB
activity. This suggests that PKB is inhibited by phosphorylation rather
than binding. An obvious model is that PKB is a direct substrate of
PKC
and that phosphorylation of PKB results in the inhibition of
enzyme activity. A similar mechanism has been found for the regulation
of Btk activity by PKC. Btk has been shown to bind to different PKC
isoforms and they inhibit Btk autophosphorylation activity by direct
phosphorylation (34). Preliminary experiments in our laboratory indeed
show that there is a PKC
-dependent phosphorylation of
PKB in vitro when PKB was immunoprecipitated from cells
expressing both PKB and PKC
.2 More research is required
to completely understand the mechanism by which PKB is inhibited by
PKC
.
(36, 37). PDK-1 was also found to associate with
PKC
in unstimulated cells (36, 37). This, together with our
observation that PKB associates with PKC
in quiescent cells,
suggests that PDK-1 can form complexes with both PKB and PKC
. This
is an intriguing situation given the fact that PDK-1 stimulates the
activation of PKB by direct phosphorylation and induces the
inactivation of PKB via PKC
. As shown in this paper, the
dissociation of the PKB·PKC
complex depends upon PKB activity,
suggesting that the binding of the inositol lipids
PI(3,4)P2 and PI(3,4,5)P3 to PKB results in the
partial activation of PKB and subsequently in the dissociation of the
complex. The same lipids stimulate PDK-1 resulting in the activation of
PKC
and the subsequent inactivation of PKB. On the other hand, the
fraction of PKB that is not in complex with PKC
can become
completely activated also by PDK-1. This implies that both activity
states of PKB, the inactive (PKB in complex with PKC
) and the active
state (PKB associated to the membrane), may be under the control of
PDK-1 activity.
and the PKB pathway also has implications for
the downstream effectors of PKB. As shown in this paper, the decrease
in PKB activity by PKC
leads to changes in GSK-3 activity.
Furthermore, it has recently been shown that C2-ceramides inhibit
PKB/Akt activity and induce apoptosis through an unknown mechanism (38,
39). In addition, C2-ceramides have also been shown to decrease glucose
uptake through inhibition of PKB activity (40). Since ceramides have
been described to activate atypical PKC
(41), it is tempting to
speculate that these effects are mediated by PKC
. In addition,
PKC
has been described to regulate MAPK activity through Raf-1 (42).
So, it seems that PKC
may regulate the activity of different
proteins from different signaling pathways.
associates in vivo
with PKB through its AH domain. Both PDGF- and p110-CAAX-induced
activation of PKB was inhibited by co-expression of PKC
, which was
also reflected in GSK-3 activity. Our data demonstrate that PKC
negatively regulates PKB signaling, an effect that is regulated by
direct action on PKB.
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ACKNOWLEDGEMENTS |
---|
We gratefully thank Dr. Paul Coffer for kindly providing HA-tagged PKB cDNAs. Dr. Adri Thomas is thanked for help with the GSK-3 activity measurements. Dr. Marcus Thelen is acknowledged for kindly providing p110-CAAX and PLAP-CAAX cDNAs. Finally, we also thank Jose van der Wal for technical assistance and Drs. Jord Stam and Johannes Boonstra for critically reading the manuscript.
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FOOTNOTES |
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* 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.
§ Contributed equally to the results of this work.
To whom correspondence should be addressed: Institute of
Biomembranes, Dept. of Molecular Cell Biology, Utrecht University, 3584 CH Utrecht, The Netherlands. Tel.: 31-30-2533349; Fax: 31-30-2513655; E-mail: bergenp{at}bio.uu.nl.
2 R. P. Doornbos, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PKB, protein kinase B; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PI, phosphatidylinositol; GSK-3, glycogen synthase kinase-3; GS, glycogen synthase; PH, pleckstrin homology; AH, Akt homology; PDK, phosphatidylinositol-3,4,5-triphosphate-dependent protein kinase; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; Btk, Bruton tyrosine kinase.
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REFERENCES |
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