From the Third Department of Internal Medicine,
Yamaguchi University School of Medicine, Minami-Kogushi, Ube,
Yamaguchi 755-8505, the § Department of Internal Medicine,
Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8566, the ¶ Department of Molecular Biology and
Medicine, Research Center for Advanced Science and Technology,
University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, and the
Institute for Adult Disease, Asahi Life Foundation,
1-9-14 Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan
Received for publication, June 28, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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To investigate the role of
3-phosphoinositide-dependent protein kinase 1 (PDK1) in the
Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead
(kd) mutant were expressed using an adenovirus gene transduction system
in Chinese hamster ovary cells stably expressing insulin receptor.
Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308
already to be maximally phosphorylated at 1 min but completely
dephosphorylated at 5 min, with insulin stimulation, whereas
insulin-induced Akt1 activation was maintained even after
dephosphorylation of Thr-308. Overexpression of wt-PDK1 further
increased insulin-stimulated phosphorylation of Thr-308, also followed
by rapid dephosphorylation. The insulin-stimulated Akt1 activity was
also enhanced by wt-PDK1 expression but was maintained even at 15 min.
Thus, phosphorylation of Thr-308 is not essential for maintaining the
Akt1 activity once it has been achieved. Interestingly, the
insulin-stimulated phosphorylation state of Thr-308 was maintained even
at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a
dominant negative effect on dephosphorylation of Thr-308 of Akt1.
Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the
insulin-stimulated phosphorylation state of Thr-308. In addition,
in vitro experiments revealed PP2A, but not PP1, to
dephosphorylate completely Thr-308 of Akt1. These findings
suggest that a novel pathway involving dephosphorylation of Akt1 at
Thr-308 by a phosphatase, possibly PP2A, originally, identified as is
regulated downstream from PDK1, an Akt1 kinase.
Akt, also known as protein kinase B or Rac-PK, is a Ser/Thr kinase
that was originally identified as a transforming oncogene in a
retrovirus from a spontaneous thymoma in an AKR mouse (1). Activation
of Akt1 by growth factors, such as insulin and
IGF-1,1 has been shown to be
necessary for various cellular processes including cell growth,
differentiation, metabolism, and apoptosis (2-6). To date, two
potential in vivo substrates of Akt1 have been identified,
namely glycogen synthase kinase-3 (GSK3) and BAD (4). GSK3 is inhibited
by Akt1, which is thought to contribute to the insulin-induced
dephosphorylation (activation) of glycogen synthase and protein
synthesis initiation factor eIF2B and thereby to the stimulation of
glycogen synthesis and protein synthesis (4). In addition, one of the
cellular targets that Akt1 may phosphorylate to protect cells from
apoptosis is BAD (7, 8). This protein, in its dephosphorylated form,
interacts with the Bcl family member BclXL and induces apoptosis of
some cells; however, BAD, which is phosphorylated by Akt1,
dissociates from BclXL, instead of interacting with 14-3-3, to prevent
apoptosis (9). Furthermore, in transfection-based experiments,
constitutively active Akt1 also mimics other actions of insulin, such
as the enhancement of glucose uptake in 3T3-L1 adipocytes (10) and L6
myotubes (11) that results in the translocation of GLUT4 from an
intracellular compartment to the plasma membrane.
Although several lines of evidence suggest that Akt1 may be activated
by products of phosphoinositide 3-kinase, such as phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol
3,4,5-triphosphate (PtdIns(3,4,5)P3) (12), other studies
have shown that the activation of this enzyme results primarily from
phosphorylation (13). Akt1 has an N-terminal pleckstrin homology (PH)
domain and two major phosphorylation sites, Thr-308 and Ser-473.
PtdIns(3,4)P2 and PtdIns(3,4,5)P3, which are
produced by growth factor-activated phosphoinositide 3-kinase, induced
membrane localization of Akt1 via its PH domain. This event allows
phosphorylation of Akt1 at Thr-308 and Ser-473 by the upstream kinase
(14), which are reportedly required for full activation of Akt1
(13).
Recently, a Ser/Thr protein kinase
(3-phosphoinositide-dependent protein kinase-1; PDK1) was
identified as an enzyme that phosphorylates Akt1 at Thr-308. PDK1,
which is expressed ubiquitously in human tissue, is composed of an
N-terminal catalytic domain and a noncatalytic C-terminal tail
containing a PH domain (15, 16). Recombinant PDK1 phosphorylates
Thr-308 of Akt1 directly, in a reaction that is almost completely
dependent on PtdIns(3,4,5)P3 (15, 17). In previous studies,
effects of PDK1 on Akt1 kinase activity were well documented (14, 16,
18, 19), but effects of PDK1 on phosphorylation of Akt1 at Thr-308 and
Ser-473 in vivo have not been fully examined.
To investigate the role of PDK1 in the insulin-stimulated
phosphorylation of Akt1 at Thr-308 and Ser-473 in vivo,
wild-type PDK1 (wt-PDK1) or its kinase dead mutant (kd-PDK1) was
expressed using an adenovirus gene transduction system in Chinese
hamster ovary cells stably expressing insulin receptor (CHO-IR). We
report herein a novel mechanism of Akt1 dephosphorylation in which
PDK1, an Akt1 kinase, is involved.
Antibodies--
The rabbit polyclonal anti-phospho-Akt1
(Thr-308) and anti-phospho-Akt1 (Ser-473) antibodies were purchased
from New England Biolabs, and sheep polyclonal anti-PDK1 antibody was
from Upstate Biotechnology, Inc. The rabbit polyclonal anti-HA antibody
and the rabbit polyclonal anti-Myc antibody were purchased from Santa Cruz Biotechnology.
Cell Culture--
Chinese hamster ovary cells in which the
insulin receptor is stably overexpressed (CHO-IR cells) were maintained
in Ham's F-12 medium (Life Technologies, Inc.) containing 10% fetal
calf serum in an atmosphere of 5% CO2 at 37 °C.
Cloning and Construct--
Polymerase chain reaction (PCR) was
performed to amplify cDNA of PDK1 using the human liver cDNA as
a substrate and oligonucleotides based on its reported sequence (15) as
primers, yielding cDNA of PDK1 encompassing the entire coding
region. cDNA corresponding to HA epitope (YPYDVPDYASL) was ligated
to cDNA of PDK1 to tag it with the HA epitope at its N terminus.
Mutant (K111A) of PDK1 was generated by standard PCR-based strategies.
By using, as a probe, amplified cDNA fragments that had been
generated by PCR based on the reported sequence of mouse Akt1 (20),
full-length mouse Akt1 was isolated by screening a cDNA library
from MIN6 cells (21). cDNA corresponding to the Myc epitope
(MEQKLISEEDLEF) was ligated to cDNA of Akt1 to tag it with the Myc
epitope at the C termini.
Gene Transduction--
The epitope-tagged PDK1, its mutant, and
the epitope-tagged Akt1 were constructed by homologous recombination
between the expression cosmid cassette and the parental virus genome as
described previously (22, 23). CHO-IR cells were incubated in Ham's F-12 medium containing the adenoviruses at a multiplicity of infection (m.o.i.) of 10-30 plaque-forming units/cell for 1 h at 37 °C, and the growth medium was then added. Experiments were performed 48-72
h after infection. Exogenous protein expression was observed in more
than 90% of CHO-IR cells.
Immunoblotting--
Cells, which had been serum-starved and then
incubated with or without 1 µM insulin, were lysed and
boiled in Laemmli buffer containing 10 mM dithiothreitol,
subjected to SDS-polyacrylamide gel electrophoresis, and transferred
onto nitrocellulose filters. In some experiments, cells were pretreated
with 25 nM calyculin A as indicated in the text. The
filters were incubated with the indicated antibodies and then with
anti-rabbit or anti-sheep immunoglobulin G coupled to horseradish
peroxidase. The immunoblots were visualized with an enhanced
chemiluminescence detection kit (Amersham Pharmacia Biotech). The
intensities of bands were quantified by NIH Image 1.62 program. Values
presented are the means ± S.D. of three or four separate experiments.
PDK1 Activity Assay--
Cells were solubilized in ice-cold
lysis buffer A containing 50 mM Tris, pH 7.4, 100 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM
sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 40 mM Akt1 Activity Assay--
Cells were serum-starved, incubated
with or without 1 µM insulin for indicated periods, and
solubilized in ice-cold lysis buffer B containing 50 mM
Tris, pH 7.4, 100 mM NaCl, 10 mM EDTA, 10%
glycerol, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 40 mM
PP1 and PP2A Activity Assays--
Cells were serum-starved,
incubated with or without 1 µM insulin for 5 min, and
resuspended in ice-cold phosphatase extraction buffer containing 20 mM imidazole HCl at pH 7.2, 2 mM EDTA, 0.2% In Vitro Dephosphorylation of Thr-308 of Akt1 by PP1 or
PP2A--
Cells were serum-starved, incubated with 1 µM
insulin for 1 min, and solubilized in ice-cold lysis buffer B without
sodium orthovanadate, To investigate the effects of PDK1 activity on Akt1
phosphorylation at Thr-308 and Ser-473, the wild-type (wt) PDK1 or the kinase-dead (kd) mutant of PDK1 was expressed into CHO-IR cells using
an adenovirus gene transduction system, followed by immunoblotting using Thr-308 and Ser-473 phospho-specific antibodies. In parental CHO-IR cells, however, it was difficult to detect constantly the phosphorylation of endogenous Akt1 at Thr-308 with immunoblotting using
anti-phospho-Akt1 (Thr-308) antibody (data not shown). Therefore, the
experiments were performed in CHO-IR cells coexpressing the wild-type
Akt1 tagged with Myc epitope using an adenovirus gene transduction
system. First, in CHO-IR cells expressing wt-Akt1 alone (A cells),
those expressing wt-PDK1 and wt-Akt1 (wtPA cells), and those expressing
kd-PDK1 and wt-Akt1 (kdPA cells), the Ser/Thr kinase activity of PDK1
was measured in the immunoprecipitates with anti-tag antibody (Fig.
1A) or anti-PDK1 antibody
(Fig. 1B). Exogenous PDK1 proteins were also expressed at
similar levels in wtPA cells and kdPA cells (Fig. 1C).
Insulin did not stimulate either endogenous or exogenous PDK1 (Fig. 1,
A and B). Overexpression of wt-PDK1 markedly
increased kinase activity to 6-fold (Fig. 1B), which was
consistent with the marked increase (approximately 8-fold) in the
expression level of wt-PDK1 as demonstrated by immunoblotting with
anti-PDK1 antibody (Fig. 1D). In contrast, kd-PDK1 exhibited
no significant kinase activity (Fig. 1A). In addition,
expression of kd-PDK1 did not affect endogenous PDK1 activity (Fig.
1B), demonstrating that the mutant does not have a dominant
inhibitory effect on the Ser/Thr kinase activity of endogenous
PDK1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, and 50 mM NaF.
Lysates were extracted by centrifugation at 15,000 × g
for 10 min followed by immunoprecipitation with anti-PDK1 antibody or
anti-HA antibody. The Ser/Thr kinase activities of the wild-type PDK1
and its mutant were assayed in the immunoprecipitates as previously
reported (24) using a peptide sequence derived from the activation loop
of Akt1 (KTFCGTPEYLAPEVRR) as a substrate (24).
-glycerophosphate, and 50 mM NaF. Lysates were extracted
by centrifugation at 15,000 × g for 10 min followed by
immunoprecipitation with anti-Myc antibody. The Ser/Thr kinase activity
of the wild-type Akt1 was assayed in the immunoprecipitates as
previously reported (25) using a peptide sequence derived from GSK3
(GRPRTSSFAEG) as a substrate (25).
-mercaptoethanol, 2 mg/ml glycogen, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of
aprotinin, leupeptin, antipain, soybean trypsin inhibitor, and
pepstatin A. The cells were homogenized with by 8 passages through a
29-gauge needle and centrifuged at 1,000 × g for 5 min. Supernatants were centrifuged at 100,000 × g for
30 min at 4 °C to separate the particulate fractions from cytosolic
fractions (26). PP1 and PP2A activities were assayed in the particulate
fractions and cytosolic fractions using a protein serine/threonine
phosphatase assay system (New England Biolabs). The activities of PP1
and PP2A were distinguished by their differential sensitivities to
okadaic acid (OA). PP1 activity was defined as the activity that was
insensitive to 5 nM OA. PP2A and PP2A-like activities were
defined as the activity that was inhibited by 5 nM OA. As a
control, protein phosphatase activity was also measured in the presence
of 1 µM OA, which completely inhibits both PP1 and PP2A
(27).
-glycerophosphate, and NaF. Lysates were
extracted by centrifugation at 15,000 × g for 10 min
followed by immunoprecipitation with anti-Myc antibody.
Immunoprecipitates were incubated with 1.7 units/ml PP1 or PP2A for
1 h at 30 °C, followed by electrophoresis and immunoblotting
with anti-phospho-Akt1 (Thr-308) antibody as described above. The
recommended reaction buffer was used for each phosphatase as follows:
phosphatase 1 buffer containing 50 mM Tris, pH 7.0, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij and
phosphatase 2A buffer containing 50 mM Tris, pH 7.5, 1%
mercaptoethanol, 1 mM MnCl, 1 mM benzamidine,
and 0.5 mM phenylmethylsulfonyl fluoride (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PDK1 activity in the absence or presence of
insulin. A and B, CHO-IR cells expressing
wt-Akt1 alone (A cells), those expressing wt-PDK1 and wt-Akt1 (wtPA
cells), and those expressing kd-PDK1 and wt-Akt1 (kdPA cells) were
incubated with or without 1 µM insulin for 15 min.
Lysates were immunoprecipitated (IP) with anti-HA
(A) or anti-PDK1 (B) antibody, and PDK1 activity
in the immunoprecipitates was assayed, as described under
"Experimental Procedures." C and D, the
expression levels of PDK1 proteins. Lysates were immunoblotted
(Blot) with anti-HA (C) or anti-PDK1 antibody
(D), and densitometry was performed on the original blots as
described under "Experimental Procedures." The graphs
show the intensities of bands, and data are expressed as percentages of
the intensities in kdPA cells. Values presented are the means ± S.D. of three separate experiments. The representative immunoblotting
figures are also presented (upper panels).
Next, we examined the effects of expression of wt-PDK1 or kd-PDK1 on
the phosphorylation states of Akt1 at Thr-308 and Ser-473 after 15 min
of insulin stimulation. By adjusting the transfection conditions
including m.o.i., wt-Akt was expressed at a level similar to that in A
cells, wtPA cells, and kdPA cells, as demonstrated by immunoblotting
with anti-tag antibody (Fig.
2C). Insulin-induced phosphorylation of Akt1 at Thr-308 was not observed in A cells (Fig.
2A). Overexpression of wt-PDK1 slightly enhanced
insulin-stimulated Thr-308 phosphorylation of Akt1 (Fig.
2A). To our surprise, a marked increase (an approximately
4-fold increase compared with the value in wtPA cells) was observed in
the insulin-induced phosphorylation at Thr-308 in kdPA cells 15 min
after insulin addition (Fig. 2A), despite the absence of
activity of kd-PDK1 (Fig. 1A). In contrast to the change in
Thr-308 phosphorylation state, insulin-stimulated Ser-473
phosphorylation of Akt1 was clearly observed in A cells, and
overexpression of wt-PDK1 or kd-PDK1 had virtually no effect on its
change in phosphorylation (Fig. 2B). To confirm the
involvement of kd-PDK1 in the enhancement of Thr-308 phosphorylation,
we examined Thr-308 phosphorylation in CHO-IR cells with different
kd-PDK1 expression levels after 15 min of insulin stimulation.
Different levels of kd-PDK1 expression were achieved by infection of
CHO-IR cells with kd-PDK1 adenovirus at different multiplicities (Fig. 3B), whereas wt-Akt1 was
expressed at similar levels in these cells in response to infection
with Akt1 adenovirus at similar multiplicities (Fig. 3C).
The levels of Thr-308 phosphorylation were dependent on those of
kd-PDK1 expression (Fig. 3, A and B). These
findings indicate that kd-PDK1 expression enhances Thr-308 phosphorylation after 15 min of insulin stimulation. An approximately 3-fold increase in the level of Thr-308 phosphorylation was observed at
30 m.o.i. as compared with that at 20 m.o.i., whereas kd-PDK1 expression was increased less than 1.5-fold. This may be attributable to the mechanism whereby kd-PDK1 functions as a dominant inhibitory mutant for dephosphorylation of Akt1 at Thr-308.
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Contrary to our expectation, expression of kd-PDK1, which exhibited no
kinase activity (Fig. 1A), resulted in further enhancement of Thr-308 phosphorylation compared with expression of wt-PDK1 at 15 min of insulin addition (Fig. 2A). To clarify the mechanism underlying this unexpected observation, we examined the phosphorylation states of Thr-308 and Ser-473 at earlier time points, 1, 3, 5, 10, and
15 min after insulin addition. Immunoblotting study revealed that
Thr-308 was already maximally phosphorylated at 1 min after insulin
addition but almost completely dephosphorylated by 5 min in A cells
(Fig. 4A). In wtPA cells,
phosphorylation of Thr-308 was markedly increased (~1.8-fold) at 1 min after insulin addition, compared with A cells, but Thr-308
phosphorylation was also rapidly reversed (Fig. 4B). Akt1
phosphorylation at Thr-308 occurred in kdPA cells and was similar to
that observed in A cells, at 1 min after insulin addition. However, the
phosphorylation was maintained at 15 min (Fig. 4C) with even
higher Thr-308 phosphorylation in kdPA cells (~3.8-fold) than in wtPA
cells. These results suggest that overexpression of wt-PDK1 enhances
Thr-308 phosphorylation of Akt1 at earlier time points, such as 1 min,
and that expression of kd-PDK1 inhibits dephosphorylation but does not
affect phosphorylation.
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On the other hand, phosphorylation of Ser-473, which was observed at 1 min after insulin addition, was maintained even at 15 min of insulin stimulation in A cells (Fig. 4D). Overexpression of wt-PDK1 or kd-PDK1 had no significant effects on insulin-induced Ser-473 phosphorylation throughout 15 min of insulin addition (Fig. 4, E and F). Thus, PDK1 is clearly involved in Thr-308 phosphorylation, whereas Ser-473 phosphorylation appears to be accomplished through a different mechanism in vivo.
Recent findings have shown that phosphorylation of both Thr-308 and
Ser-473 is necessary for full activation of Akt1 (13). To investigate
the relationship between the phosphorylation states of these residues
and the Ser/Thr kinase activity of Akt1, we examined the time course of
Akt1 activity in these cells. In A cells, activation of Akt1 was
observed at 1 min after insulin addition and was maintained even at 15 min (Fig. 5, dotted line). Overexpression of wt-PDK1 enhanced insulin-induced activation of Akt1,
and this enhanced activity was maintained for 15 min (Fig. 5,
dashed line), whereas overexpression of kd-PDK1 did not appear to affect insulin-stimulated Akt1 activity (Fig. 5, solid line); Akt1 activity was increased to similar extents in A cells and kdPA cells. Comparison of the time course of Akt1 activation (Fig.
5) with that of Thr-308 phosphorylation (Fig. 4, A-C) shows clearly that phosphorylation of Thr-308 is not essential for
maintaining Akt1 activity once it has been initiated.
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To unravel the mechanism whereby Thr-308 is dephosphorylated, we
examined the effects of a Ser/Thr protein phosphatase inhibitor, calyculin A, on the Thr-308 phosphorylation of Akt. Calyculin A is
reportedly cell-permeable and equally inhibits protein phosphatase (PP)
1 and PP2A in vivo (29). When A cells were pretreated with 25 nM calyculin A for 15 min, Thr-308 phosphorylation of
Akt1 was prolonged even at 15 min after insulin addition (Fig.
6A), which resembled the
observation in kdPA cells (Fig. 4C). These findings suggest
that expression of kd-PDK1 inhibits protein phosphatase activity,
resulting in the maintenance of Thr-308 phosphorylation.
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Inhibition of Thr-308 dephosphorylation by calyculin A indicates that PP1 and/or PP2A is involved in Thr-308 dephosphorylation. We examined whether PP1 or PP2A activity is affected by insulin treatment and expression of wt-PDK1 or kd-PDK1. PP1 or PP2A activity was estimated in particulate and cytosolic fractions from CHO-IR cells, those expressing wt-PDK1, and those expressing kd-PDK1, with or without insulin using appropriate concentrations of okadaic acids as described under "Experimental Procedures." No significant differences in PP1 or PP2A activity were observed in each fraction either among these cells or between the basal state and insulin-stimulated states (data not shown).
Then, we further examined which phosphatase was capable of
dephosphorylating Akt1 at Thr-308 in vitro. The
phosphorylated Akt1, stimulated by insulin, was incubated with purified
PP1 or PP2A in vitro, followed by immunoblotting with
anti-phospho-Akt1 (Thr-308) antibody. Thr-308 phosphorylation
completely abolished by treatment with PP2A, whereas it persisted,
although at a decreased level, with PP1 treatment (Fig. 6B).
Thus, phosphorylated Thr-308 of Akt1 is a more preferable substrate for
PP2A than for PP1. Taken together with the findings obtained using
in vivo calyculin A treatment and in vitro
dephosphorylation experiments, insulin-stimulated phosphorylation of
Akt1 at Thr-308 might be reversed by PP2A, although we cannot rule out
the possibility that another unknown phosphatase, which is inhibited by
calyculin A, is involved.
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DISCUSSION |
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In response to growth factor stimulation, Akt1 becomes phosphorylated at two major sites, Thr-308 in the kinase domain and Ser-473 in the C-terminal tail (13). PDK1 is a recently identified protein kinase that phosphorylates Akt1 at Thr-308 in lipid vesicles containing PtdIns(3,4,5)P3 or PtdIns(3,4)P2 (18). Overexpression of PDK1 in 293 cells reportedly potentiated the IGF-1-induced phosphorylation of Akt1 at Thr-308 in vivo (15). In the present study, we showed overexpression of wt-PDK1 to potentiate the insulin-stimulated phosphorylation of Akt1 at Thr-308 at 1 min, but this was rapidly followed by dephosphorylation. In contrast, wt-PDK1 overexpression did not affect Ser-473 phosphorylation in the basal or insulin-stimulated condition. Furthermore, kd-PDK1 expression did not apparently affect Ser-473 phosphorylation. Thus, PDK1 is clearly involved in Thr-308 phosphorylation, whereas Ser-473 phosphorylation appears to occur through a different mechanism in vivo. Contrary to our expectation, expression of the kinase dead mutant of PDK1 did not inhibit but rather maintained the insulin-stimulated phosphorylation of Thr-308. In addition, the levels of Thr-308 phosphorylation paralleled those of kd-PDK1 expression. Furthermore, calyculin A, an inhibitor of both PP1 and PP2A, prolonged the phosphorylation state of Thr-308. Thus, kd-PDK1 expression has a dominant inhibitory effect on Ser/Thr protein phosphatase for Thr-308 of Akt1, suggesting that PDK1, an Akt kinase, also regulates dephosphorylation of Akt1 at Thr-308, resulting in a transient Thr-308 phosphorylation in CHO-IR cells. It was reported that phosphorylation of Thr-308 of Akt1 was observed at 10 min after insulin (13) and IGF-1 (13, 15) stimulation in 293 cells. We also observed the persistent phosphorylation of Thr-308 in 3T3-L1 adipocytes at 15 min after insulin stimulation (data not shown). However, in CHO-IR cells, at a more modest insulin concentration (10 nM), phosphorylation of Thr-308 was rapidly reversed, and kd-PDK1 expression prolonged Akt1 phosphorylation at Thr-308 (see Supplemental Material) in a fashion similar to that observed at 1 µM insulin (Fig. 4, A and C). Thus, it is unlikely that the markedly transient phosphorylation of Akt1 at Thr-308 involves inappropriate coupling of insulin receptors to intracellular targets and/or effects of insulin binding nonspecifically to related receptors. The discrepancy among cell types might be due to differences in the levels of expression and/or subcellular distribution of putative signaling molecules that transduce signals from PDK1 to the downstream phosphatase.
It appears that overexpression of PDK1 is less effective in the dephosphorylation of Thr-308 than in the phosphorylation of this residue, since the levels of Akt1 phosphorylation at Thr-308 were higher, at all times after insulin stimulation, in wtPA cells (Fig. 4B) than in A cells (Fig. 4A). Whereas PDK1 directly phosphorylates Thr-308 of Akt1, dephosphorylation of Thr-308 perhaps occurs via endogenous phosphatase activation. Therefore, this discrepancy between the effects of PDK1 on phosphorylation and dephosphorylation of Thr-308 might be attributable to endogenous phosphatase activity possibly being rate-limiting for dephosphorylation of Akt1 at Thr-308, under conditions of wt-PDK1 overexpression.
Based on biochemical characteristics, Ser/Thr protein phosphatases were
initially divided into two classes; type 1 phosphatases (PP1) are
inhibited by two heat-stable proteins, termed inhibitor-1 and
inhibitor-2, and preferentially dephosphorylate the -subunit of
phosphorylase kinase, whereas type-2 phosphatases are insensitive to
heat-stable inhibitors and preferentially dephosphorylate the
-subunit of phosphorylase kinase (30, 31). Type 2 phosphatases can
be further subdivided into spontaneously active (PP2A),
Ca2+-dependent (PP2B), and
Mg2+-dependent (PP2C) classes (32). More than
30 protein kinase activities are known to be modulated by PP2A in
vitro (33), suggesting that PP2A plays a major role in kinase
regulation. In fact, extensive biochemical, pharmacological, and
genetic evidence suggests that PP2A controls the activities of several
major protein kinase families, particularly those that belong to the
AGC subgroup, the calmodulin-dependent kinases (34-36),
extracellular signal-regulated kinase/mitogen-activated protein kinases
(37), cyclin-dependent kinases (38, 39), and I
B kinase
(40). It is noteworthy that Akt1, protein kinase C, and p70 S6 kinase,
which belong to AGC subgroup, are all reportedly substrates of PDK1
(18, 41, 42).
Although previous studies have shown treatment of Akt1 with PP2A in vitro to inactivate kinase (28), its exact phosphorylation states of Thr-308 and Ser-473 in vivo remain unclear. In the present study, we showed calyculin A to inhibit dephosphorylation of Akt1 at Thr-308, which occurred in a fashion similar to that of kd-PDK1 expression. In addition, under conditions of identical enzyme activity, PP2A completely dephosphorylated Akt1 at Thr-308 in vitro, whereas Thr-308 of Akt1 was not completely dephosphorylated by PP1 despite the loose substrate specificity of the catalytic subunit of Ser/Thr protein phosphatases (32). These results suggest that phosphorylated Akt1 at Thr-308 is a more preferable substrate for PP2A than for PP1. These findings are in agreement with the recent report that PP2A is the major protein phosphatase responsible for hyperosmotic stress-induced dephosphorylation of Akt1 at both Thr-308 and Ser-473 (43). We measured the PP2A activity, but we detected no insulin stimulation of PP2A activity in CHO-IR cells. In addition, overexpression of the wild-type or kinase-dead PDK1 also had no effect on PP2A activity (data not shown). However, Begum and co-workers (44, 45) have recently shown that addition of insulin to rat skeletal muscle cells decreases PP2A activity by 40-80% (44), whereas in fetal chick neurons, insulin increases PP2A activity (45). Although differences in these results may depend on cell types, these reports suggest that PP2A activity can be modulated by extracellular signals such as insulin. PP2A is a multimeric protein, and the catalytic subunit of PP2A forms a complex with an array of regulatory subunits that modulate its activity, substrate specificity, and subcellular localization (33). The phosphatase activity assay in extracts from whole cells does not necessarily reflect the PP2A activity in the specific subcellular loci. Thus, the lack of a significant change in the PP2A activity of whole cytosolic and particulate fractions does not exclude the possibility that, upon insulin stimulation, PP2A in specific subcellular loci is activated downstream from PDK1 in response to insulin in vivo. The proportion of the regulatable PP2A might be different in other cell types. Alternatively, it is also possible that, without altering phosphatase activity, a portion of PP2A changes subcellular localization upon insulin stimulation, thereby gaining access to the phosphorylated Akt1 at Thr-308, and this cellular process is inhibited by kd-PDK1.
It was reported that mutation of either Thr-308 or Ser-473 to Ala
greatly decreased the activation of transfected Akt1 by insulin. In
addition, Akt1 became partially active in vitro when either
Thr-308 or Ser-473 was changed to Asp, and far more active when Thr-308
and Ser-473 were both mutated to Asp, suggesting a critical role of
phosphorylation of both the Thr-308 and the Ser-473 residues in full
activation of Akt1 (13). However, in the present study, Akt1 activation
was prolonged even after dephosphorylation of Thr-308, although basal
and insulin-stimulated Akt1 activities were enhanced by overexpression
of wt-PDK1. Thus, a discrepancy was observed between the Akt1
phosphorylation at Thr-308 and Akt1 activity. In contrast,
phosphorylation of Ser-473 was prolonged in a fashion similar to that
of Akt1 activity. These findings suggest that initial activation of
Akt1 depends on its phosphorylation states, including that of Thr-308,
whereas the maintenance of once activated Akt1 activity is via a
mechanism different from that of initial activation. Thr-308
phosphorylation is not essential for maintenance of the Akt1 activity,
in which Ser-473 phosphorylation might be involved. PDK1 reportedly
phosphorylates the activation loop of PKC and PKC
II, which is
homologous to the sequence around Thr-308 of Akt1, and this
phosphorylation is a step required for the maturation of PKC
and
PKC
II. This phosphorylation serves as a trigger to induce the two
C-terminal autophosphorylations that are required to lock PKC in a
catalytically competent conformation, resulting in full activation.
Once this mature conformation is achieved, the phosphate group on the
activation loop does not regulate the activity of the enzyme (46).
Thus, phosphorylation of Thr-308 of Akt1, like phosphorylation of the
activation loop of PKC
and PKC
II by PDK1, may serve as a trigger
for maturation of Akt1, although phosphorylation of Thr-308 of Akt1
does not lead to autophosphorylation of its Ser-473 (13).
It was reported that kd-PDK1 does not interfere with PKC activator
(phosphatidylserine, diacylglycerol, and
Ca2+)- induced PKC and PKC
II activation (46).
On the other hand, kd-PDK1 has a dominant negative effect on
PKC activator (phosphatidylserine, phosphatidylcholine, and
PtdIns(3,4,5)P3)-induced PKC
(41) and insulin-induced
p70 S6-kinase activation (42). In the present study, kd-PDK1 was shown
to have a dominant inhibitory effect on dephosphorylation of Akt1 but
not on phosphorylation of the same molecule or on endogenous PDK1
activity. These observations suggest that mechanisms whereby PDK1
transduces signals to downstream targets depend on the target molecule.
In summary, we have reported herein that, upon insulin stimulation,
phosphorylated Thr-308 of Akt1 is rapidly dephosphorylated downstream
from PDK1, an Akt kinase, and that phosphorylation of Thr-308 is not
essential for maintaining the Akt1 activity once it has been achieved.
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FOOTNOTES |
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* This work was supported by Grant-in-aid for Creative Research (10NP0201) and Grant-in-aid for Scientific Research (B2, 11470234) from the Ministry of Education, Science, Sports and Culture of Japan (to Y. O.).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.
The on-line version of this article (available at
http://www.jbc.org) contains Fig. 1S.
** To whom correspondence should be addressed. Fax: 81-836-22-2256; E-mail: oka-y@po.cc.yamaguchi-u.ac.jp.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M005685200
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ABBREVIATIONS |
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The abbreviations used are: IGF1, insulin-like growth factor 1; PDK1, 3-phosphoinositide-dependent protein kinase-1; wt, wild type; kd, kinase dead; GSK3, glycogen synthase kinase-3; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; PH, pleckstrin homology; HA, hemagglutinin; PCR, polymerase chain reaction; CHO-IR, Chinese hamster ovary cells stably expressing insulin receptor; m.o.i., multiplicity of infection; OA, okadaic acid; PP, protein phosphatase; PKC, protein kinase C.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Staal, S. P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5034-5037[Abstract] |
2. | Toker, A., and Cantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve] |
3. | Hawkins, P. T., Welch, H., McGregor, A., Eguinoa, A., Gobert, S., Krugmann, S., Anderson, K., Stokoe, D., and Stephens, L. (1997) Biochem. Soc. Trans. 25, 1147-1151[Medline] [Order article via Infotrieve] |
4. | Alessi, D. R., and Cohen, P. (1998) Curr. Opin. Genet. & Dev. 8, 55-62[CrossRef][Medline] [Order article via Infotrieve] |
5. | Coffer, P. J., Jin, J., and Woodgett, J. R. (1998) Biochem. J. 335, 1-13[Medline] [Order article via Infotrieve] |
6. | Downward, J. (1998) Curr. Opin. Cell Biol. 10, 262-267[CrossRef][Medline] [Order article via Infotrieve] |
7. | Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve] |
8. |
del Peso, L.,
Gonzalez-Garcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689 |
9. | Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996) Cell 87, 619-628[Medline] [Order article via Infotrieve] |
10. |
Kohn, A. D.,
Summers, S. A.,
Birnbaum, M. J.,
and Roth, R. A.
(1996)
J. Biol. Chem.
271,
31372-31378 |
11. | Hajduch, E., Alessi, D. R., Hemmings, B. A., and Hundal, H. S. (1998) Diabetes 47, 1006-1013[Abstract] |
12. |
Franke, T. F.,
Kaplan, D. R.,
Cantley, L. C.,
and Toker, A.
(1997)
Science
275,
665-668 |
13. | Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., and Hemmings, B. A. (1996) EMBO J. 15, 6541-6551[Abstract] |
14. |
Stokoe, D.,
Stephens, L. R.,
Copeland, T.,
Gaffney, P. R.,
Reese, C. B.,
Painter, G. F.,
Holmes, A. B.,
McCormick, F.,
and Hawkins, P. T.
(1997)
Science
277,
567-570 |
15. | Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., Norman, D. G., Gaffney, P., Reese, C. B., MacDougall, C. N., Harbison, D., Ashworth, A., and Bownes, M. (1997) Curr. Biol. 7, 776-789[Medline] [Order article via Infotrieve] |
16. |
Stephens, L.,
Anderson, K.,
Stokoe, D.,
Erdjument-Bromage, H.,
Painter, G. F.,
Holmes, A. B.,
Gaffney, P. R.,
Reese, C. B.,
McCormick, F.,
Tempst, P.,
Coadwell, J.,
and Hawkins, P. T.
(1998)
Science
279,
710-714 |
17. | Belham, C., Wu, S., and Avruch, J. (1999) Curr. Biol. 9, R93-R96[CrossRef][Medline] [Order article via Infotrieve] |
18. | Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997) Curr. Biol. 7, 261-269[Medline] [Order article via Infotrieve] |
19. | Anderson, K. E., Coadwell, J., Stephens, L. R., and Hawkins, P. T. (1998) Curr. Biol. 8, 684-691[Medline] [Order article via Infotrieve] |
20. | Bellacosa, A., Franke, T. F., Gonzalez-Portal, M. E., Datta, K., Taguchi, T., Gardner, J., Cheng, J. Q., Testa, J. R., and Tsichlis, P. N. (1993) Oncogene 8, 745-754[Medline] [Order article via Infotrieve] |
21. |
Katagiri, H.,
Terasaki, J.,
Murata, T.,
Ishihara, H.,
Ogihara, T.,
Inukai, K.,
Fukushima, Y.,
Anai, M.,
Kikuchi, M.,
Miyazaki, J.,
Yazaki, Y.,
and Oka, Y.
(1995)
J. Biol. Chem.
270,
4963-4966 |
22. | Kanegae, Y., Lee, G., Sato, Y., Tanaka, M., Nakai, M., Sasaki, T., Sugano, S., and Saito, I. (1995) Nucleic Acids Res. 23, 3816-3821[Abstract] |
23. |
Katagiri, H.,
Asano, T.,
Ishihara, H.,
Inukai, K.,
Shibasaki, Y.,
Kikuchi, M.,
Yazaki, Y.,
and Oka, Y.
(1996)
J. Biol. Chem.
271,
16987-16990 |
24. |
Dong, L. Q.,
Zhang, R. B.,
Langlais, P.,
He, H.,
Clark, M.,
Zhu, L.,
and Liu, F.
(1999)
J. Biol. Chem.
274,
8117-8122 |
25. | Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995) Nature 378, 785-789[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Begum, N.
(1995)
J. Biol. Chem.
270,
709-714 |
27. |
Yan, Y.,
and Mumby, M. C.
(1999)
J. Biol. Chem.
274,
31917-31924 |
28. |
Andjelkovic, M.,
Jakubowicz, T.,
Cron, P.,
Ming, X. F.,
Han, J. W.,
and Hemmings, B. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5699-5704 |
29. | MacKintosh, C., and MacKintosh, R. W. (1994) Trends Biochem. Sci. 19, 444-448[CrossRef][Medline] [Order article via Infotrieve] |
30. | Cohen, P. (1989) Annu. Rev. Biochem. 58, 453-508[CrossRef][Medline] [Order article via Infotrieve] |
31. | Ingebritsen, T. S., and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261[Abstract] |
32. | Wera, S., and Hemmings, B. A. (1995) Biochem. J. 311, 17-29[Medline] [Order article via Infotrieve] |
33. | Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. (1999) Trends Biochem. Sci. 24, 186-191[CrossRef][Medline] [Order article via Infotrieve] |
34. |
DeRemer, M. F.,
Saeli, R. J.,
Brautigan, D. L.,
and Edelman, A. M.
(1992)
J. Biol. Chem.
267,
13466-13471 |
35. | Barnes, G. N., Slevin, J. T., and Vanaman, T. C. (1995) J. Neurochem. 64, 340-353[Medline] [Order article via Infotrieve] |
36. |
Park, I. K.,
and Soderling, T. R.
(1995)
J. Biol. Chem.
270,
30464-30469 |
37. | Anderson, N. G., Maller, J. L., Tonks, N. K., and Sturgill, T. W. (1990) Nature 343, 651-653[CrossRef][Medline] [Order article via Infotrieve] |
38. | Lee, T. H., Solomon, M. J., Mumby, M. C., and Kirschner, M. W. (1991) Cell 64, 415-423[Medline] [Order article via Infotrieve] |
39. | Poon, R. Y., and Hunter, T. (1995) Science 270, 90-93[Abstract] |
40. | DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-554[CrossRef][Medline] [Order article via Infotrieve] |
41. | Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H., Chen, C. S., Newton, A. C., Schaffhausen, B. S., and Toker, A. (1998) Curr. Biol. 8, 1069-1077[Medline] [Order article via Infotrieve] |
42. |
Pullen, N.,
Dennis, P. B.,
Andjelkovic, M.,
Dufner, A.,
Kozma, S. C.,
Hemmings, B. A.,
and Thomas, G.
(1998)
Science
279,
707-710 |
43. |
Chen, D.,
Fucini, R. V.,
Olson, A. L.,
Hemmings, B. A.,
and Pessin, J. E.
(1999)
Mol. Cell. Biol.
19,
4684-4694 |
44. |
Srinivasan, M.,
and Begum, N.
(1994)
J. Biol. Chem.
269,
12514-12520 |
45. | Begum, N., Robinson, L. J., Draznin, B., and Heidenreich, K. A. (1993) Endocrinology 133, 2085-2090[Abstract] |
46. | Dutil, E. M., Toker, A., and Newton, A. C. (1998) Curr. Biol. 8, 1366-1375[Medline] [Order article via Infotrieve] |