From the Department of Molecular Oncology, Genentech, Inc., South San Francisco, California 94080
Received for publication, February 12, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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Phospholipid-dependent kinase 1 (PDK 1) is a 3'-phospholipid-responsive serine/threonine kinase
that plays a critical role in cell survival by phosphorylating and
activating the anti-apoptotic AKT/PKB kinase. While PDK 1 is clearly an
important component of the cell survival machinery, the potential for
phospholipid-independent activation of the AKT/PKB survival pathway has
not been extensively examined at the molecular level. We have
identified a second form of PDK 1 in the nematode Caenorhabditis
elegans that we have termed PIAK
(phospholipid-independent AKT/PKB
kinase). PIAK is highly homologous to C. elegans and mammalian PDK 1 with the exception that the novel
kinase lacks a phospholipid binding pleckstrin homology domain. The
domain structure of PIAK suggests that it might be a
phospholipid-independent kinase, and PIAK phosphorylates mammalian
AKT/PKB at the activating Thr308 residue in the presence of
the phosphatidylinositol (PI) 3-kinase inhibitors as well as in the
absence of growth factors. In addition, PIAK is capable of inducing the
phospholipid-independent, AKT/PKB-induced phosphorylation of the
AFX-type forkhead transcription factor, resulting in its cytoplasmic
localization. Because the nuclear localization of this transcription
factor induces an apoptotic state, this PIAK-mediated cytoplasmic
sequestration allows for cell survival. Finally, PIAK activity appears
to be induced by various inhibitors of cell cycle G1
progression. These data suggest an alternate,
phosphatidylinositol 3-kinase-independent mechanism for the
activation of the AKT/PKB survival pathway that may be utilized during
periods of cellular quiescence.
PI1 3-kinases are
important conduits, which funnel information from the cell surface to
downstream survival pathways by producing various
3'-phosphorylated phospholipids, including phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol
3,4,5-trisphosphate (PtdIns(3,4)P2 and
PtdIns(3,4,5)P3, respectively). Activation of the
PI 3-kinase pathway by normal (i.e. growth factor) or
oncogenic (i.e. HER 2 overexpression) cell surface events
results in the production of PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 (1). These specific phospholipids can then
act as membrane binding sites for the lipid-binding pleckstrin homology
(PH) domains of two classes of downstream kinases,
3-phospholipid-dependent kinase 1 (PDK 1) and the
AKT/PKB kinases (2-8). The PH domain-induced membrane association of
these kinases results in physical interactions between these two types
of enzymes, so that the PDK 1 enzyme mediates the phosphorylation of a
specific threonine residue (Thr308) in the activation loop
of the AKT/PKB kinases (8-12). The PDK 1-activated AKT/PKB can
subsequently phosphorylate a number of substrates involved with cell
survival, including BAD (13) and a subset of the forkhead transcription
factors (14). The phosphorylation of a number of sites in these
forkhead transcription factors results in their transport from the
nucleus (14). This cytoplasmic localization is an important aspect of
PI 3-kinase-mediated cell survival, as nuclear localization of the
forkhead transcription factors in cultured cells rapidly results in
cell death, which is in part mediated by the activation of caspases
(15, 14).2 This
survival pathway can be down-regulated by the catalytic activity of
PTEN/MMAC, a lipid phosphatase with specificity for the 3 position of
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (16).
The importance of the PI 3-kinase pathway to oncogenesis is emphasized by the finding that PTEN/MMAC is a tumor suppressor gene, which is
homozygously lost in a high percentage of a variety of tumors (17, 18).
Examination of tumors and cell lines lacking PTEN/MMAC has demonstrated
increased AKT/PKB activity, which presumably results in pro-survival
signals (19, 20). Thus, the PI 3-kinase-mediated activation of AKT/PKB
is likely to be a major component of cell survival in both normal as
well as oncogenic situations.
The significance of the PI 3-kinase pathway to cell survival as well as
to the regulation of metabolic processes is emphasized by genetic and
molecular studies in the nematode, Caenorhabditis elegans.
The dauer larval stage is a reversible larval arrest pathway where
worms do not feed, and their metabolism is shifted toward energy
storage rather than energy production. Genetic analysis of this
pathway, together with molecular analysis of the genes involved with
dauer formation, have demonstrated that it appears to be the worm
homologue of the mammalian insulin signaling pathway. Activation of a
membrane-associated homologue of the insulin/IGF-1 receptor (DAF-2)
appears to be the initiator of an inhibitory cascade that blocks dauer
formation. Importantly, a worm homologue of the PI 3-kinase is found
downstream of this receptor tyrosine kinase (21). Constitutive
activation of this kinase also inhibits dauer formation. Interestingly,
two different AKT/PKB kinases are found to be downstream of the PI
3-kinase, and it appears that mutation of these AKT/PKB kinases results
in constitutive dauer formation, suggesting that the role of these
kinases is to block dauer formation in response to activation of the PI
3-kinase by the DAF-2 receptor (22). The dauer pathway appears to
converge upon the DAF-16 transcription factor, which is the worm
homologue of the mammalian forkhead proteins known to be phosphorylated by AKT/PKB (22, 23). Recently, this pathway has been further elucidated
with the discovery that DAF-18 mutations result in a constitutive block
in dauer formation. Examination of the DAF-18 gene revealed that it is
the worm homologue of mammalian PTEN, consistent with the
interpretation that loss of PTEN results in irreversible activation of
the worm AKT/PKBs and inhibition of the activity of the worm homologue
of mammalian forkhead proteins (24). Finally, recent data have shown
that a worm homologue corresponding to mammalian PDK 1 appears to be
downstream of the DAF 2 receptor and PI 3-kinase but upstream of the
AKT/PKB kinases, consistent with biochemical observations in the
mammalian system (24). An important finding is that this pathway also
appears to be conserved in the fruit fly Drosophila
melanogaster, where it appears to regulate cell number and cell
size (25). Together, these data suggest that the dauer pathway is a
homologue of the mammalian insulin pathway. In addition, because worms
with mutations in components of this pathway show body size, egg
laying, and neurological phenotypes, the results also suggest that
other aspects of cell regulation mediated by the PI 3-kinase/PDK 1/AKT
pathway may also be conserved between worms and mammals.
While the majority of literature suggests that the AKT/PKB pathway is
regulated by phospholipids, a number of reports suggest that other
mediators, such as isoproteronol (26) and cAMP (27, 28), may also be
involved with the activation of this pathway in a PI
3-kinase-independent fashion. In addition, during periods of cellular
quiescence, for example when nutrients are limiting and the PI 3-kinase
pathway is down-regulated, it seemed likely that additional protective
mechanisms might be operative. Examination of the C. elegans
genome revealed a kinase, which was homologous to PDK 1, but which
lacked the phospholipid-binding PH domain. Here we report that this
novel PDK 1-related enzyme is capable of activating the AKT/PKB kinase
in a manner that is consistent with a PI 3-kinase-independent pathway.
Materials--
LY 294002 and wortmannin were purchased from
Sigma. Olomoucine and roscovitine were obtained from Biomol
Research Laboratories. IGF-1 was purchased from Roche Molecular
Biochemicals. The anti-AKT, anti-phospho-AKT
(Thr308), and anti-phospho-AKT (Ser473)
antibodies were from New England Biolabs. The anti-GFP,
anti-V5, and anti-phospho-AFX (Ser193)
were obtained from CLONTECH, Invitrogen, and
Upstate Biotechnology, respectively.
Constructs--
Dr. M. Yan and Dr. T. Tang (Genentech, Inc.)
kindly provided the FLAG-tagged AKT in PRK5 and the EGFP-AFX
vector, respectively. The FLAG- Cell Culture--
The 293E human epithelial cell line was
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum (Life Technologies, Inc.) and antibiotics (50 units/ml
penicillin and 50 µg/ml streptomycin). The MCF7 human breast
carcinoma cells were cultured in RPMI 1640 medium supplemented
with 10% heat-inactivated fetal calf serum and antibiotics. Cells were
transfected using the FuGENETM 6 reagent (Roche Molecular
Biochemicals). The T98G human glioblastoma cells were obtained from
ATCC and cultured in Eagle's minimal essential medium
supplemented with 10% fetal calf serum (Life Technologies, Inc.),
antibiotics, 1.0 mM sodium pyruvate, and 0.1 mM
nonessential amino acids.
Expression of GST-AKT in 293E Cells--
The 293E cells were
transfected with the plasmid encoding GST-AKT using the
FuGENETM 6 reagent (Roche Molecular Biochemicals). 12 h after transfection, the cells were serum-starved for 24 h. The
cells were lysed in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM sodium Immunoprecipitation and Western Blot Analysis--
293E cells
were seeded in six-well dishes at the density of 5 × 105 cells/well and then transiently transfected with the
indicated plasmids. Cells were harvested 36 h post-transfection
and lysed in 200 µl of lysis buffer (50 mM Tris-HCl, pH
7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM sodium glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 µM
microcystin-LR, 0.27 M sucrose, and protease
inhibitors). Cell lysates were subjected to immunoprecipitation with
the indicated antibodies. Immunoprecipitates were washed in lysis
buffer, resolved by 4-20% SDS-polyacrylamide gel electrophoresis, and
subsequently analyzed by protein immunoblotting.
In Vitro Phosphorylation of GST-AKT by PIAK and PDK
1--
1 × 107 293E cells were transfected with
plasmids encoding wild type or kinase dead (KA) mutant of
V5-PIAK. Cells were harvested 36 h post-transfection
and lysed in buffer A. V5-PIAK proteins were
immunoprecipitated from the lysate proteins with V5
antibody. The precipitated proteins were washed twice in kinase assay
buffer containing 50 mM Tris-HCl, pH 7.5, 2.5 mM protein kinase C inhibitor, 1 µM
microcystin-LR, 10 mM Mg(Ac)2, 100 µM ATP. The resulting PIAK proteins were resuspended in
the kinase assay buffer plus 100 µM
phosphatidylserine, 100 µM
phosphatidylcholine in the presence or absence of 10 µM PtdIns(3,4,5)P3. The reaction was
initiated by the addition of 2 µg of purified GST-AKT. After
incubation for 30 min at 30 °C, the reaction was terminated by 1%
SDS. The assay was performed in a similar manner using PDK 1.
A Novel C. elegans Kinase That Phosphorylates AKT/PKB on
Thr308--
While examining the C. elegans
genome for a homologue of the PDK 1 kinase that might be involved with
the phosphorylation of the second (Ser473) activation site
on AKT/PKB, we observed a kinase that was highly related to both
C. elegans (24) and mammalian (3) PDK 1. Fig.
1 illustrates that this kinase (PIAK)
contained a kinase domain that was conserved with the kinase domains of
mammalian (41% identity, 57% similarity) and C. elegans
(32% identity, 56% similarity) PDK 1. In addition, this figure
illustrates that the novel kinase lacked the lipid-binding PH
domain, which is found in both the PDK 1 as well as the AKT/PKB
kinases. In addition, scrutiny of the C. elegans genome did
not reveal any phospholipid-binding PH encoding exons 3' of the gene
encoding this novel kinase. Examination of the non-kinase sequences in
the novel protein did not demonstrate any known membrane association
motifs. These data suggested that the novel C. elegans
kinase could have substrate specificity that was similar to PDK 1, but
might be regulated in a phospholipid-independent manner.
To determine whether the C. elegans PDK 1 can phosphorylate
AKT/PKB at the activating Thr308 residue in a heterologous
system, we cotransfected the novel kinase together with human AKT/PKB
into 293 cells in the presence of serum and examined the
phosphorylation of this residue using a phospho-specific antibody. Fig.
2A illustrates that the
C. elegans PDK 1 is capable of phosphorylating human AKT/PKB
at Thr308, thus confirming the genetic data suggesting a
role for this kinase in AKT/PKB activation in the dauer pathway (24).
Importantly, removal of the PH domain of this kinase resulted
in a loss of Thr308 phosphorylation, suggesting that the
AKT/PKB phosphorylating activity of C. elegans PDK 1 kinase
requires an interaction with cellular 3'-phospholipids mediated
by this domain. This figure also illustrates that the novel kinase is
capable of phosphorylating the Thr308 residue as
efficiently as the known PDK 1 kinase, despite the fact that the novel
kinase lacks a PH domain. In addition, the figure illustrates that
the kinase activity of the novel enzyme is required for this
phosphorylation event, since the ATP binding mutant (KA) shows little,
if any, activity. Finally, Fig. 2B shows that, while the
PDK 1 kinase of C. elegans requires a PH domain on the
AKT/PKB substrate for Thr308 phosphorylating activity, the
novel kinase does not require this domain. These data suggest that,
while the C. elegans PDK 1 appears to have fastidious
requirements for phospholipid interaction domains, the novel kinase is
capable of phosphorylating the activation site of AKT/PKB in the
absence of a phospholipid-binding domain on either protein.
The Novel Kinase Phosphorylates AKT/PKB Thr308 in a PI
3-Kinase-independent Manner--
The above data were consistent with
the hypothesis that the novel C. elegans PDK 1-like kinase
might be active in the absence of the 3'-phospholipids produced
by PI 3-kinase activity. To examine this possibility, human and
C. elegans PDK 1 as well as the novel C. elegans
kinase were analyzed for AKT/PKB Thr308 phosphorylating
activity in the presence of the PI 3-kinase inhibitor, wortmannin. Fig.
3 illustrates that, in contrast to human
and C. elegans PDK 1, PIAK is able to phosphorylate AKT at
Thr308 in the absence of the IGF-1 growth factor. The
figure also illustrates that the IGF-1-mediated activation of human and
C. elegans PDK 1 is strongly inhibited by wortmannin, while
the constitutive activity of PIAK is not. Fig. 3B further
shows that in the presence or absence of PtdIns(3,4,5)P3,
purified GST-AKT can be phosphorylated by PIAK in vitro.
Fig. 4 illustrates that, as expected, LY
294002 strongly inhibits the ability of both human as well as C. elegans PDK 1 kinases to phosphorylate human AKT/PKB at the
Thr308 residue in response to serum. Importantly, this
figure also depicts that the activity of the PIAK kinase is not
inhibited by LY 294002, but is instead enhanced, suggesting that the
activity of this kinase is independent of PI 3-kinase. To further
examine the possible mechanism for this enhanced activity, we analyzed
the ability of the PDK 1 kinases to interact physically with AKT/PKB in
a coprecipitation assay. Fig. 4 illustrates that both human and C. elegans PDK 1 can be coprecipitated with human AKT/PKB,
and this interaction appears to depend upon the presence of
serum-induced PI 3-kinase activity. One interpretation of these results
is that membrane association of the two kinases via PH-mediated binding to 3'-phospholipids is in part responsible for their physical interaction (2-7). In contrast to this result, the association between
the human AKT/PKB and the novel kinase appears to be strengthened by
the inhibition of PI 3-kinase activity. A possible explanation for this
result is that loss of 3-phospholipids after inhibition of PI
3-kinase activity by LY 294002 releases the membrane-associated AKT/PKB, thus allowing for an interaction with the presumably nonmembrane-associated novel kinase (29). Together, these data strongly
suggest that the novel kinase is able to phosphorylate AKT/PKB in a
phospholipid-independent manner. Thus, we have chosen to name the novel
kinase PIAK (phospholipid independent
AKT/PKB kinase).
PIAK Induces the PI 3-Kinase-independent Cytoplasmic Localization
of the Forkhead Transcription Factor, AFX--
While these data
suggested that PIAK was capable of inducing the phosphorylation of the
AKT/PKB activating site, they did not address the functional
implications of this phosphorylation event. Both genetic and
biochemical data suggest that the phosphorylation of a subset of
forkhead transcription factors by AKT/PKB mediates the cytoplasmic
localization of these factors (14, 15, 22, 23). In addition, it has
been established that the nuclear localization of these transcription
factors induces cell death via apoptosis in cultured cells, presumably
because the transcription factors bring about the expression of genes
that precipitate the apoptotic cascade (14).2 Thus, a major
activity of AKT/PKB is to sequester these death-inducing transcription
factors in the cytoplasm, where they are inactive. We therefore sought
to examine the phosphorylation of one of these transcription factors,
AFX, in response to human and C. elegans PDK 1 as well as to
PIAK in the absence and presence of another specific PI 3-kinase
inhibitor, wortmannin.
Fig. 5A illustrates that both
human as well as C. elegans PDK 1 are able to stimulate the
AKT/PKB-dependent phosphorylation of AFX as detected by a
phospho-specific antibody. In addition, this figure shows that this
phosphorylation is inhibited in the presence of wortmannin and is
dependent upon serum, again consistent with the hypothesis that the
phosphorylation of AKT/PKB by these kinases is contingent upon
phospholipids produced by PI 3-kinase. Analysis of the ability of PIAK
to induce AFX phosphorylation reveals that this occurs both in the
absence of serum as well as in the presence of wortmannin, consistent
with the suggestion that PIAK is able to induce the activation of
AKT/PKB in the absence of phospholipids produced by PI 3-kinase. To
examine whether the AFX phosphorylation was of functional importance,
we analyzed the subcellular localization of a green fluorescent protein
chimera of AFX (AFX-GFP). Fig. 5, B and C,
illustrate that AFX-GFP is found to be cytoplasmically localized in the
presence of both human and C. elegans PDK 1 as well as PIAK.
However, when transfected cells are exposed to wortmannin, the AFX-GFP
is found in the nucleus of the human and C. elegans PDK
1-transfected cells, but is still sequestered predominately in the
cytoplasm of PIAK-transfected cells. Because nuclear localization of
AFX is lethal (15, 14),2 these results suggest that PIAK is
capable of inhibiting AFX-induced apoptosis in the absence of PI
3-kinase activity.
CDK Inhibitors Induce PIAK Activity--
Because LY 294002 is
capable of inducing G1 arrest (30), we next decided to
examine whether inhibitors of cell cycle progression could induce the
AKT/PKB phosphorylating activity of PIAK. Fig. 6A illustrates that olomoucine
and roscovitine, specific inhibitors of cyclin-dependent
kinases (31), are able to increase the level of AKT/PKB phosphorylation
in response to transfected PIAK, but not to either human or C. elegans PDK 1. This increase in AKT/PKB activity was not observed
with other types of inhibitors such as nocodazole or aphidicolin (data
not shown), suggesting that it may be a specific effect of inhibiting
cyclin-dependent kinases. Interestingly, the enhancement of
phosphorylation by olomoucine was similar to that observed with the
protein phosphatase 2A inhibitor okadaic acid (data not shown),
confirming the suggestion that protein phosphatase 2A dephosphorylates
the Thr308 site on AKT/PKB in vivo (32) and
suggesting that the observed olomoucine-induced enhancement was
significant. Finally, Fig. 6B shows that these CDK
inhibitors also activate endogenous AKT/PKB in T98G human glioblastoma
cells. Together, these data suggest that PIAK may be activated by
inhibition of cell cycle progression, particularly by the arrest of
G1 progression.
The results reported here are consistent with the proposal that a PI
3-kinase-independent pathway can activate the AKT/PKB survival kinase,
at least in the nematode C. elegans. This conclusion is
important, because it provides the first detailed molecular evidence
that this critical cytoprotective pathway can be regulated by
nonphospholipid-dependent mechanisms. Importantly, the
results are completely consistent with the domain structure of PIAK,
which lacks the phospholipid binding PH motif found in PDK 1. The
results also suggest that the activation of PIAK induces the
cytoplasmic localization of the apoptotic transcription factor, AFX.
This latter result is significant, because it suggests that the
activation of this novel kinase is functionally important to the
maintenance of cell survival. Finally, the activation of this kinase by
induction of cell cycle arrest, particularly in the G1
phase of the cell cycle, suggests that PIAK may be functionally
important during periods of cellular inactivity induced by
growth factor starvation.
While these results argue for a significant role for PIAK in
cytoprotection, a number of questions remain. The mechanism by which
PIAK interacts with and activates AKT/PKB remains unresolved. The fact
that the two proteins can be efficiently coprecipitated, especially in
the absence of PIP3 phospholipids, suggests that these proteins are directly associated. In addition, while previous data suggested that the AKT/PKB kinase maintains a structure that is
resistant to Thr308 phosphorylation in the absence of
3-phospholipids (2-7), the current data suggest that PIAK may interact
with AKT/PKB in a manner that exposes this critical residue to the
enzyme's kinase activity. A second question concerns the conservation
of this kinase in other species. While examination of mammalian data
bases has not revealed an obvious homologue of PIAK, examination of the
D. melanogaster genome revealed that the inaC
(33) protein kinase, a homologue of protein kinase C, was the kinase
most closely related to PIAK. While it is unlikely that the
inaC kinase is a functional homologue of PIAK, it is of
course possible that a more distantly related kinase is in fact the
functional homologue of the nematode kinase. No apparent homologue of
PIAK in Homo sapiens has yet been found, although a
definitive answer awaits the release of the complete human genome
sequence. In one human glioblastoma cell line, T98G, the
phosphorylation of endogenous AKT at Thr308 was found to be
stimulated by Cdk and the inhibitors olomoucine and roscovitine
(Fig. 6B). Because only PIAK, but not PDK 1, was found to be
stimulated by Cdk inhibitors, this observation suggests that there is
at least a functional homologue of PIAK in humans. Alternatively, it is
possible that PIAK functions in a pathway that is specific for C. elegans. For example, PIAK might be activated during the dauer
stage of the larval nematode to enable cytoprotection under low
nutrient conditions where cells are presumably quiescent. It will of
course be important to examine the effects of mutations of PIAK in
worms to more accurately elucidate its functional significance. In
addition, it will be of significant interest to examine the ability of
PIAK to phosphorylate other PDK 1 substrates, such as SGK, P70S6K,
P90RSK, and various PKC isoforms (8). Finally, while the exact
mechanism by which PIAK is itself activated remains to be elucidated,
the fact that inhibitors of GI progression result in PIAK-induced
Thr308 phosphorylation is also consistent with the
hypothesis that this activation pathway may be operable during periods
of cell quiescence. One possibility is that low
PIP3 levels during cellular quiescence might
release membrane-associated AKT/PKB, thus allowing for an enhanced
interaction with PIAK. Together, these data suggest that disparate
mechanisms may operate to ensure that the apoptotic cascade initiated
by forkhead transcription factors is inhibited under diverse stressful conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
PH-AKT encodes amino acid
residues 118-480 of AKT, thus excluding the NH2-terminal
PH domain. Plasmids for expression of GST-AKT in 293E cells were
constructed in the vector pEBG-2T. Using standard polymerase chain
reaction protocols, full-length cDNA encoding PIAK or CEPDK1
was cloned from a C. elegans cDNA library (Stratagene),
and hPDK1 was cloned from a human fetal brain cDNA library.
Expression vectors for PIAK, CEPDK1, and hPDK1 were constructed in
pcDNA3.1/V5-His TOPO vector (Invitrogen). The
V5-
PH-CEPDK1 encodes amino acid residues 1-480
of CEPDK1, thus excluding the COOH-terminal PH domain. The PIAK
K59M, CEPDK1 K98M, and hPDK1 K111M
mutants were made by site-directed mutagenesis using the
QuickchangeTM kit from Stratagene. The presence of the
introduced mutation and fidelity of polymerase chain reaction reactions
were confirmed by sequence analysis.
-glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 µM
microcystin-LR, 0.27 M sucrose, and protease inhibitors). The resulting lysate was centrifuged at 4 °C for 10 min
at 13,000 × g, and the supernatant was incubated for
2 h with glutathione-Sepharose beads. The beads were collected by centrifugation for 1 min at 3000 × g and then washed
three times in buffer A. The beads were further washed five times in
buffer B (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA,
0.03% Brij-35, 0.27 M sucrose to remove Triton
X-100, which might interfere with the activation of AKT by PIAK.
GST-AKT was eluted from the beads with buffer B containing 20 mM glutathione, pH 8.0.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Comparison of the primary structures
of PIAK, CEPDK1a, and human PDK1. A, alignment of the
deduced amino acid sequence of PIAK (GenBankTM
accession number T33662) with the human PDK1 sequence
(hPDK1, GenBankTM accession number
AAC51825) and the C. elegans PDK1 sequence
(CEPDK1a, GenBankTM accession number
AAD42307), carried out using the CLUSTAL W program. The ATP binding
site, the active site, the substrate recognition site, and the
PH domain are indicated. The catalytic lysine is indicated by an
asterisk. B, schematic diagram of the domain
structures of PIAK, C. elegans PDK1, and human PDK1.
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Fig. 2.
PIAK phosphorylates human AKT at
Thr308. A, 293E cells were transiently
transfected with DNA constructs expressing FLAG-AKT with wild type or
kinase dead (KA) mutant of V5-CEPDK1,
V5- PH-CEPDK1, or V5-PIAK. 36 h
after transfection, cell extracts were prepared, immunoprecipitated
with anti-AKT antibody, and then immunoblotted with a polyclonal
antibody (New England Biolabs) to detect the
phospho-Thr308 of AKT, the phospho-Ser473 of
AKT, or the total AKT. 20 µg of cell lysate proteins was also blotted
with anti-V5 antibody (Invitrogen). B, 36 h
after transfected with constructs expressing FLAG-
PH-AKT
with V5-CEPDK1 or V5-PIAK, 293E cells were
analyzed for the phosphorylation of AKT at Thr308 as in
A.
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Fig. 3.
PIAK phosphorylates human AKT at
Thr308 in a PI 3-kinase-independent manner.
A, 293E cells were transiently transfected with DNA
constructs expressing FLAG-AKT with wild type or kinase dead
(KA) mutant of V5-hPDK1, -CEPDK1, or -PIAK.
12 h after transfection, cells were starved for 18 h. The
starved cells were then left untreated, stimulated with IGF-1 (100 ng/ml) for 10 min, or treated with 200 nM wortmannin for 30 min before the stimulation of IGF-1 (100 ng/ml) for 10 min.
Phosphorylation of AKT was detected by immunoprecipitation with AKT
antibody, followed by immunoblotting with anti-phospho-AKT
(Thr308) or anti-AKT antibodies. B, purified
GST-AKT was incubated for 30 min at 30 °C with the
immunoprecipitated wild type or kinase dead (KA) mutant of
V5-PIAK in the presence of 100 µM ATP and
phospholipid vesicles with or without PtdIns(3,4,5)P3.
Phosphorylation of AKT was detected by immunoblotting with
anti-phospho-AKT (Thr308). C, the assay was
repeated with purified PDK 1. Note the lipid dependence of PDK 1 activity versus the lipid independence of PIAK
activity.
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Fig. 4.
PIAK interacts with AKT more efficiently in
the presence of LY 294002. 293E cells were transiently transfected
with plasmids expressing FLAG-AKT with V5-hPDK1,
V5-CEPDK1, or V5-PIAK. 12 h after
transfection, the cells were left untreated or treated with 20 µM LY 294002 for 16 h. Cell lysate proteins were
immunoprecipitated with anti-AKT antibody and then immunoblotted with
anti-V5 antibody, anti-phospho-AKT (Thr308)
antibody, or anti-AKT antibody. 10% of the lysate used in the
immunoprecipitation was blotted with anti-V5 antibody
showing the input proteins.
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Fig. 5.
PIAK induces the PI 3-kinase-independent
phosphorylation and cytoplasmic localization of AFX. MCF7 cells
were transfected with EGFP-AFX, FLAG-AKT, and V5-PDK
(hPDK1, CEPDK1, or PIAK). 12 h after transfection, cells were
untreated or treated with 20 µM LY 294002 for 16 h.
A, cell lysate proteins were immunoblotted with an antibody
against phospho-AFX (Ser193), GFP, AKT, or V5.
Localization of EGFP-AFX was detected by direct fluorescence.
Representative pictures are shown in B, and quantification
of the fluorescence data is shown in C. For each condition,
100-200 cells were scored. Cells were scored as having fluorescence
that was stronger in the nucleus, equal in nucleus and cytoplasm, or
stronger in cytoplasm.
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Fig. 6.
PIAK is activated by the inhibitors of
Cdks. A, 293E cells were transiently transfected with
plasmids expressing FLAG-AKT with V5-hPDK1,
V5-CEPDK1, or V5-PIAK. 24 h after
transfection, the cells were left untreated or treated with 200 µM olomoucine or 30 µM roscovitine for
2 h. Cell lysate proteins were immunoprecipitated with anti-AKT
antibody and then immunoblotted with anti-phospho-AKT
(Thr308) antibody or anti-AKT antibody. 20 µg of the
lysate protein was also blotted with anti-V5 antibody to
show the expressed PDKs. B, the inhibitors of Cdks stimulate
the phosphorylation of endogenous AKT at Thr308 in the T98G
human glioblastoma cells. 3 × 107 T98G cells were
left untreated or treated with 200 µM olomoucine or 30 µM roscovitine for 2 h. Cell lysate proteins were
prepared, immunoprecipitated with anti-AKT antibody, and then
immunoblotted with anti-phospho-AKT (Thr308) antibody or
anti-AKT antibody.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Yan for providing the FLAG-AKT construct, Dr. T. Tang for providing the EGFP-AFX vector, and J. Lee for human brain cDNA library. We also appreciate the help from the DNA sequencing laboratory and the oligonucleotide synthesis laboratory at Genentech.
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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.
To whom all correspondence should be addressed. Tel.:
650-225-1123; Fax: 650-225-6127; E-mail: lal@gene.com.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M101309200
2 T. Tang and L. Lasky, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphatidylinositol; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PIP3, phosphatidylinositol 1,4,5-trisphosphate; PH, pleckstrin homology; PDK 1, phospholipid-dependent kinase 1; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; IGF, insulin-like growth factor; V5 epitope, Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Arg-Ser-Thr.
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