From the Investigative Treatment Division, National
Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa,
Chiba 277-8577, Japan and the ¶ Queensland Institute of Medical
Research and the Department of Surgery, University of Queensland,
Herston, Brisbane, Queensland 4029, Australia
Received for publication, June 17, 2002, and in revised form, October 28, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We identified a novel human AMP-activated protein
kinase (AMPK) family member, designated ARK5, encoding 661 amino acids
with an estimated molecular mass of 74 kDa. The putative amino
acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK- AMPK1 is a mammalian
homologue of sucrose non-fermenting protein kinase (SNF-1), which
belongs to a serine/threonine protein kinase family, and its activation
is well documented in cells under metabolic stress, hypoxia, heat
shock, and ischemia (1, 2). The SNF-1/AMPK family is highly conserved
in several species including mammals (3-8), and the AMPK is activated under various stress conditions where the cellular
ATP concentration decreases and plays a key role in cellular adaptive
responses to maintain energy balance. The well known targets of AMPK
belong to those involved in energy metabolism including
3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA), acyl-CoA
decarboxylase, and glucose and amino acid transporters (15). However,
the cellular mechanism for survival under stress conditions is complex
and includes cell cycle regulation, repair of cellular components,
remodeling tissue components, and regulation of the cell death pathway.
Targets of AMPK are not fully described to date. Recently, we
demonstrated that some tumor cells, such as the pancreatic cancer cell
line PANC-1, showed high tolerance against glucose starvation, which is
compatible with hypovascular findings on clinical angiography (16),
although most cell lines, including a human hepatoma cell line and the
human normal fibroblast cell line, underwent cell death under glucose
starvation (16, 17). Similar tolerance to glucose starvation was
induced by hypoxic conditions, and tolerance was found to be dependent
on AMPK activity as well as Akt (16-18). During glucose starvation, a
G1 phase cell cycle delay occurs with a concomitant
increase in p53 phosphorylation, and AMPK appeared to be involved in
this process (19).
The tumor suppressor gene ATM has been identified as
the gene defective in the human genetic disorder ataxia-telangiectasia, which is characterized by neurological degeneration and cancer predisposition (20-22). ATM is a member of the phosphatidylinositol 3-kinase family that activates the tumor suppressor p53 during the
cellular response to DNA double strand break (23-25). Although several
studies have reported a close involvement of the ATM/p53 pathway during
the cellular response to DNA damage, the regulation of this pathway,
particularly by non-DNA damage events, has not been well described, and
it remains unclear how ATM is activated.
In the current study, we identified a novel AMPK family member through
an investigation of human SNARK and designated it ARK5. ARK5, the
activation of which was observed during glucose starvation, caused
cells to survive in an Akt-dependent manner. Furthermore, Akt-activated ARK5 phosphorylated ATM both in vivo and
in vitro and led to the phosphorylation of p53. We report
here that a novel AMPK family member, ARK5, is a new target molecule of
Akt and transduces a signal to activate ATM during nutrient starvation.
Preparation of FLAG-tagged ARK5--
FLAG-tagged ARK5 was
prepared from full-length KIAA0537-ligated pBluescript II
SK+ vector supplied from KAZUSA DNA Research
Institute with LA PCR (Takara Biomedicals, Kyoto, Japan); the
up-stream primer was
5'-AAGCTTATGGATTATAAAGATGATGATGATAAAGAAGGGGCCGCCGCGCCTGTGGCGGGG-3' and
the down-stream primer was 5'-TCTAGACTAGTTGAGCTTGCTGCAGATCTCCAG-3'. The PCR product was ligated into pT7-Blue T vector for
subcloning, and then insert cDNA digested by HindIII and
XbaI was re-ligated into the pcDNA3.1(+) expression
vector. Insert cDNA was also ligated into pcDNA3.1(
The FLAG-ARK5 protein was prepared by immunoprecipitation technology
using anti-FLAG-conjugated agarose. A HepG2 cell line overexpressing
FLAG-ARK5 (H/ARK) was lysed with PBS containing 0.1% Nonidet P-40, and
then immunoprecipitation was performed. Immunoprecipitates were washed
eight times with PBS containing 0.1% Nonidet P-40, and purification
was then examined with SDS-PAGE and silver staining.
Antibodies and Vectors--
Monoclonal antibody against FLAG and
His6, anti-FLAG-conjugated agarose, recombinant active
Akt1, and dominant negative and dominant active Akt1 expression vectors
were purchased from Upstate Biotechnology. Polyclonal antibody against
total Akt and phosphorylated Akt (Ser473) were purchased
from Cell Signaling Technology.
Cell Line and Culture--
Cell lines used for current
experiments were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Sigma). Cell viability was
assessed by the Hoechst 33342/PI staining procedure. Hoechst 33342 and
PI were purchased from Molecular Probes. After treatment, cells were
collected and stained with Hoechst 33342 and PI and then observed by
fluorescence microscopy. Cell induction ratio was indicated by the
ratio of cells carrying PI-stained nuclei to total cells (about 1000 cells).
Akt1 Phosphorylation Assay--
FLAG-tagged proteins were
isolated from cell lysate by anti-FLAG-conjugated agarose and suspended
in kinase buffer containing 25 mM Tris (pH 7.5), 2 mM dithiothreitol, and 10 mM MgCl2.
Subsequently, 100 µM [ Preparation of Anti-human SNARK Antibody--
Antibody to human
SNARK was raised in rabbits by immunizing them against a peptide based
on the predicted amino acid sequence of human SNARK
(KKPRQRESGYYSSPEPS). The peptide was synthesized and coupled to keyhole
limpet hemocyanin (KLH) at the C terminus. Japanese White rabbits were
immunized with 0.5 mg of peptide conjugate by initially injecting it in
50% Freund's complete adjuvant (Sigma) and in 50% Freund's
incomplete adjuvant (Sigma) for subsequent immunizations. One week
after the booster injection of 0.5 mg of peptide conjugate, polyclonal
anti-human SNARK antiserum was collected and used for immunoblotting analysis.
GST-SAMS Phosphorylation Assay--
Crude cell extracts and
immunoprecipitates were used to assay the phosphorylation of the SAMS
peptide fused to glutathione S-transferase. An enzyme assay
was performed at 30 °C in buffer containing 15 mM HEPES
(pH 7.0), 200 mM 5'-AMP, 200 mM GST-SAMS, cell
lysate, 0.01% Briji 35, 0.3 mM dithiothreitol, 15 mM MgCl2, and 50 mM
[ ARK5 Is the Novel AMPK Family Protein Kinase Leading Cells to
Survival--
Recently, a novel AMPK family member, SNARK, was
identified from rat (13). On the basis of the reported amino acid
sequence, BLAST search analysis revealed a human cDNA encoding
SNARK (the amino acid sequence is listed in Fig.
1B), and we prepared a
polyclonal antibody against the SNARK peptide. Using this antibody, the
endogenous expression of SNARK in several human cell lines including
lung carcinoma (PC-10 and A549), pancreatic cancer (PANC-1 and ASPC-1), hepatoma (HepG2 and HLE), and fibroblast (HF) was examined. As shown in Fig. 1A, Western blotting revealed the expression
of an unknown cross-reacting protein at 74 kDa in some of cell lines tested (intense expression in PC-10, PANC-1, ASPC-1 and HLE; weak expression in A549 and HF; no expression in HepG2). Because the calculated molecular mass of human SNARK is 69.5 kDa, we tried to
identify this 74-kDa unknown protein by BLAST search analysis using
human SNARK amino acid sequence or the peptide sequence by which the
anti-human SNARK antibody was prepared and a human protein data base,
and we found a putative protein encoded by a KIAA0537 cDNA clone.
As shown in Fig. 1B, the putative catalytic domain of SNARK
and the KIAA0537 protein showed 84% similarity, and the peptide
sequence used for the preparation of the anti-human SNARK antibody also
showed strong similarity with the KIAA0537 protein. To investigate
whether this KIAA0537 cDNA clone encodes the unknown 74-kDa protein
detected by the SNARK antibody, we prepared a FLAG-tagged KIAA0537
cDNA. When the cDNA was expressed in HepG2 cells, Western
blotting using monoclonal and polyclonal antibodies against FLAG and
SNARK gave a common 74-kDa band, suggesting that KIAA0537 cDNA
encodes the above mentioned unknown 74-kDa protein (Fig.
1C). Because four factors, AMPK-
Homology search analysis of the ARK5 amino acid sequence revealed that
it has 55.0% overall homology to human SNARK (data not shown). It also
shows 47.0, 45.8, and 42.4% homology to AMPK-
We have proposed that tolerance to nutrient starvation is one of the
determinants of tumor malignancy (16, 18), and tolerance to glucose
starvation can be induced by the activation of AMPK (18) or the hypoxic
condition (17). In this context, the involvement of ARK5 in the
mechanism for tolerance to glucose starvation was examined. When a
human hepatoma cell line, HepG2, was subjected to glucose starvation
under normoxic (21% O2) conditions, more than 90% of the
cells underwent necrotic cell death during 24 h treatment, but
only 40% of H/ARK cells showed cell death during the same treatment
(Fig. 2C). We prepared an antisense RNA expression vector of
ARK5 (ARK5(AS)). When HepG2 or PANC-1 cells were transiently transfected with ARK5(AS), mRNA expression of ARK5 was specifically suppressed (Fig. 2D). When cells were subjected to glucose
starvation at 1% oxygen tension, >90% of HepG2 cells survived, but
~50% cells died when ARK5(AS) was transfected into HepG2 cells (Fig.
2E). These results clearly showed that ARK5 is involved in
tolerance to glucose starvation.
ARK5 activity in whole cell extracts and anti-FLAG immunoprecipitates
was measured as the GST-SAMS phosphorylation during glucose starvation.
As shown in Fig. 2F, increased activity was observed in cell
extracts and immunoprecipitates from H/ARK cells but not from HepG2
cells. In addition, more increased activity was detected when H/ARK
cells were subjected to glucose-free medium for 1 h (Fig.
2F).
ARK5 Is Activated by Akt through Phosphorylation at
Ser600--
We have previously found that another protein
kinase, Akt, is activated immediately after nutrient starvation in
HepG2 cells (16). As is evident in Fig
3A, Akt was phosphorylated
rapidly. Amino acid sequence analysis of ARK5 revealed that there is a perfectly conserved putative Akt phosphorylation site in the C-terminal portion as shown in Fig. 3B. However, the putative Akt
phosphorylation site was not detected in AMPK-
To determine whether enzymatic activation of ARK5 is really required
for tolerance to glucose starvation, the effects of transfection of
ARK5 mutants and the dominant active and dominant negative forms of Akt
as well as the chemical inhibitor of Akt activation on HepG2 survival
during glucose starvation were examined. As shown in Fig.
4A, >90% of HepG2 cells
underwent necrotic cell death during 24 h of glucose starvation.
Necrotic cell death was markedly suppressed to ~40% by transfecting
wild-type ARK5 but not ARK5(S600A) or ARK5( ATM Is a Candidate for Being the Target of ARK5--
Tumor
tolerance against nutrient starvation is found to be modulated by AMPK
(18, 29), and AMPK activation induces a G1 cell cycle delay
as a result of p53 stabilization (19). Because ATM is well known as a
p53 activator, we suspected that ATM might be a target of AMPKs. As
shown in Fig. 5A, amino acid
sequence analysis revealed that ATM contains some putative
phosphorylation sites for Akt (two sites) and AMPK (three sites). Using
the PANC-1 cell in which His-tagged ATM (30) is stably expressed
(P/ATM), an in vivo 32P-labeling study was
performed. 32P incorporation into His-ATM was detected in
P/ATM cells cultured in ordinary medium, and an increase in ATM
phosphorylation was detected when P/ATM cells were subjected to glucose
starvation (Fig. 5B). Interestingly, ATM phosphorylation was
completely suppressed by the transient expression of an antisense RNA
expression vector of ARK5, and an increased phosphorylation of ATM was
detected in cells transiently expressing ARK5 (Fig. 5B),
suggesting that ARK5 phosphorylates ATM during glucose starvation. To
further confirm this possibility, in vitro phosphorylation
was examined. As shown in Fig. 5C, ARK5- or PDK1-activated
Akt1 alone did not phosphorylate ATM; however, ATM phosphorylation was
detected in the presence of both ARK5 and active Akt1, indicating that
ARK5 phosphorylates ATM directly. To determine that this
phosphorylation of ATM was activating ATM kinase, we assayed for
phosphorylation of p53, which has been shown to be a downstream
effector of ATM (23-25). The results in Fig. 5D reveal that
p53 is phosphorylated in the presence of both ATM and ARK5, suggesting
that ARK5-phosphorylated ATM is functionally active. When HepG2 cells
were subjected to glucose-free medium for 1 h, an accumulation of
p53 protein was observed (Fig. 5E). This p53 accumulation
induced by glucose starvation was completely suppressed by the
transient transfection of ARK5(AS) (Fig. 5E). These results
indicate that ARK5 activated during glucose starvation induces p53
accumulation through the functional phosphorylation of ATM in tumor
cells.
We recently found that some tumor cells have a strong tolerance to
nutrient starvation; tolerance to glucose starvation can be induced by
hypoxia in normal human fibroblasts and the human hepatoma HepG2 cells,
which are otherwise quite sensitive to glucose starvation. Akt and AMPK
appear to be involved closely in the mechanism of tolerance (16-18).
Taking all these findings into consideration, we suspected that there
might be a connection between Akt and AMPK signaling pathways. The
consensus sequence of the Akt phosphorylation is conserved in several
species. ARK5, but not other members of AMPK family, contains this
sequence at amino acids 595-600, and direct activation by Akt was
demonstrated by phosphorylation in vitro in this study. This
observation strongly indicates that ARK5 is a novel target of Akt.
During glucose starvation, cell survival was induced by ARK5, and ARK5
mutants blocked DA-Akt1-induced cell survival, indicating that ARK5
acts as the tumor cell survival factor down-stream to Akt in HepG2
cells. In previous studies, we found that the antisense RNA expression
constructs for AMPK- Recently, we demonstrated that the AMPK family is involved in cell
cycle arrest via p53 accumulation (19). ATM is a member of the
phosphatidylinositol kinase family (20-22) and in response to
radiation exposure activates the G1/S checkpoint via p53
phosphorylation (23, 24). In response to DNA damage, activated ATM
phosphorylates p53 directly on Ser15 and indirectly through
Chk2 on Ser20, causing cell cycle arrest or cell death
(23-25). In addition to acting as a sensor of DNA damage, it has been
suggested that ATM is also a sensor of oxidative stress (31, 32).
Evidence for increased oxidative stress in A-T cells and in ATM
gene-disrupted mice supports this suggestion (33, 34). Nutrient
starvation induces necrotic cell death in HepG2 cells, and a comet
assay revealed that necrotic cell death is not due to DNA double strand break (data not shown). Recently, we found ATM-induced
phosphorylation and accumulation of p53 following p21 up-regulation and
cell cycle arrest at the G1 phase in HepG2 cells during
glucose starvation.2 Our
current observations that ARK5 phosphorylated ATM in vitro and in vivo in response to glucose starvation together with
an involvement of the ATM/p53 pathway in maintaining cell survival during glucose deprivation provide additional support for a more general role for ATM in intracellular signaling. Amino acid sequence analysis revealed that ATM contained consensus sequences phosphorylated by AMPK and Akt. Other evidence of a role for ATM in non-damage signaling has been provided (35) where insulin activated ATM kinase to
phosphorylate 4E-BPI (PHAS-1), a regulator of protein synthesis. The
results described here are pertinent to that report (Ref. 35) in that
we have demonstrated that insulin treatment activates Akt, which in
turn activates ARK5, and this may lead to activation of ATM, which we
also showed is a downstream effector of ARK5 under conditions of
glucose starvation. The activation of ATM through this pathway would
add an additional pathway to the scheme proposed after insulin
treatment (21).
In the current study, we identified ARK5 as a novel member of the AMPK
family. ARK5 activation is regulated by Akt, and activated ARK5, in
turn, phosphorylated ATM during glucose starvation. Because phosphorylated ATM induces cell cycle arrest at the G1
phase via p53 phosphorylation and accumulation (23-25), the present
findings are consistent with our previous notion that the AMPK family
is involved in p53 accumulation during glucose starvation (19). ARK5
mRNA expression in human normal tissue was observed in the heart,
brain, skeletal muscle, kidney, and ovary, but not in the liver,
pancreas, lung and intestine (official data of KIAA0537 on the KAZUSA
DNA Research Institute homepage); however, protein and mRNA
expression of ARK5 was observed in some tumor cell lines derived from
hepatoma and pancreatic cancer, suggesting a possible involvement of
ARK5 in tumorigenesis. On the basis of our current results, we propose
here that ARK5 plays a key role in the tumor tolerance to nutrient
starvation related to tumor malignancy. Therefore, ARK5 is a new target
for cancer therapy.
1, AMPK-
2, MELK, and SNARK, respectively, suggesting that it is a new
member of the AMPK family. It has a putative Akt phosphorylation motif
at amino acids 595-600, and Ser600 was found to be
phosphorylated by active Akt resulting in the activation of kinase
activity toward the SAMS peptide, a consensus AMPK substrate. During
nutrient starvation, ARK5 supported the survival of cells in an
Akt-dependent manner. In addition, we also demonstrated
that ARK5, when activated by Akt, phosphorylated the ATM protein that
is mutated in the human genetic disorder ataxia-telangiectasia and also
induced the phosphorylation of p53. On the basis of our current
findings, we propose that a novel AMPK family member, ARK5, is the
tumor cell survival factor activated by Akt and acts as an ATM kinase
under the conditions of nutrient starvation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of
AMPK has been shown to be the catalytic subunit (2). Four proteins,
AMPK-
1 and AMPK-
2 (9-12), MELK (5), and SNARK (13), have been
identified as catalytic subunits of the AMPK family to date. Although
it has been reported that AMPK activation is initiated by
phosphorylation at Thr172 (14), how the phosphorylation
is initiated remains unclear.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
expression vector for the preparation of antisense RNA expression vector.
-32P]ATP (20 µCi) and recombinant active Akt1 were added to a kinase buffer
containing recombinant proteins and incubated for 30 min at 30 °C.
After incubation, the agarose was washed six times with PBS containing
0.1% Nonidet P-40 and then re-suspended in PBS, and radioactivity was
measured with a scintillation counter. Furthermore, measured samples
were separated on 7.5% SDS-PAGE for autoradiography.
-32P]ATP (10 mCi). After incubation, the fusion
protein was purified with glutathione-Sepharose (Amersham Biosciences)
and counted in scintillation counter (Beckman Coulter). There was no
significant difference in Km value between SAMS
peptide and GST-SAMS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, AMPK-
2, MELK, and
SNARK, have been identified as the catalytic subunits of the AMPK
family (Fig. 1D), we tentatively designated KIAA0537 as
ARK5.
View larger version (73K):
[in a new window]
Fig. 1.
Identification of KIAA0537 as ARK5.
A, cell extracts from several lines were Western blotted
with SNARK antibody. Asterisk shows the position of an
unknown 74 kDa protein. B, putative amino acid sequence of
KIAA0537 (top lines) and human SNARK
(bottom lines). Box A, the
region of putative catalytic domain. Box B, amino
acid sequence used for the preparation of SNARK antibody. C,
cell extracts from HepG2 transfected with (+) or without ( ) an
expression vector of FLAG-KIAA0537 were Western blotted with antibody
against FLAG (
FLAG) or SNARK (
SNARK).
D, evolutional tree of molecules belonging to AMPK
family.
1, AMP-
2, and MELK
respectively (data not shown). These data strongly suggest that ARK5 is
a serine/threonine protein kinase activated by AMP. This possibility
was directly examined by transfecting an ARK5 expression vector into
HepG2 cells (H/ARK), which do not otherwise show any cross-reactivity
to anti-SNARK for the 74-kDa band (Figs. 1A and
2A). As shown in Fig.
2A, H/ARK showed a distinct band at 74 kDa. This was further
confirmed by the immunoprecipitation method. Cellular extracts from
parental HepG2 and H/ARK cell lines were immunoprecipitated with an
anti-FLAG antibody first, and both the supernatant and the
immunoprecipitates were subjected to Western blot analysis using
anti-SNARK antiserum. Only immunoprecipitates from H/ARK cells gave
strong reactivity (Fig. 2A). In vitro kinase activity showed a clear increase using GST-SAMS as a substrate after
transfection. The activity was further stimulated in the presence of
200 µM AMP (Fig. 2B), indicating strongly that
ARK5 is a novel member of the AMPK family.
- and
-subunits of
AMPK have been well known as the binding proteins of AMPK-
1 and
AMPK-
2 (12, 26). In the previous report (13), it appeared
that rat SNARK showed the enzyme activity without any binding protein. In the current study, immunoprecipitation and silver staining technologies showed that binding proteins were not required for the
enzymatic activity of ARK5 (data not shown).
View larger version (44K):
[in a new window]
Fig. 2.
Characterization of ARK5 as an AMPK family
member. A, Western blotting analysis with SNARK
antibody was performed on cell extracts (Ext.) or
immunoprecipitates (IP.) with FLAG antibody
(sup., supernatant; ppt., immunopellet) from
HepG2 or H/ARK cells. B, SAMS phosphorylation by cell
extracts (HepG2 and H/ARK) or recombinant ARK5
(rARK5) was measured in the presence (+) or absence ( ) of
200 µM AMP. Enzyme activity is shown as means of three
experiments, and the bars represent S.E. value.
Asterisks show the statistical significance
p < 0.01 (t test). C, cell death
induction was measured in HepG2 or H/ARK cells subjected to medium with
(+) or without (
) glucose for 24 h. Ratio of cell death
induction is shown as means of three experiments, and the
bars represent S.E. value. Asterisks show the
statistical significance p < 0.01 (t test).
D, expressions of ARK5, SNARK, AMPK-
1, and AMPK-
2 were
examined with the reverse transcription PCR method to total RNA (0.5 µg) from HepG2 (HG) or PANC-1 (P-1) cell lines
transiently transfected with ARK5(AS). E, HepG2 cells
transfected with (filled bars) or without
(open bars) ARK5 antisense were subjected to
medium with (+) or without (
) glucose under normoxic (21%) or
hypoxic (1%) oxygen tension for 24 h. F, SAMS
phosphorylation activity of cell extracts (WCE) or
immunoprecipitates (IP) from HepG2 (HG) or H/ARK
(HA) cells cultured in medium with (+) or without (
)
glucose for 1 h was measured. Enzyme activity is shown as means of
three experiments and the bars represent S.E. value.
Asterisks show the statistical significance
p < 0.01 (t test).
1, AMPK-
2, and
SNARK (data not shown). These observations strongly indicate that ARK5
is activated by Akt through phosphorylation at Ser600. This
possibility was directly examined in vitro. A point mutant of ARK5 at Ser600 was constructed by substituting Ser with
Ala, and an expression vector with a FLAG tag was constructed
(ARK5(S600A)). Both wild-type and ARK5(S600A) were transfected into
HepG2 cells, and FLAG-tagged ARK5 and ARK5(S600A) were purified by
immunoprecipitation with anti-FLAG antibody-conjugated agarose. ARK5
phosphorylation was examined after incubation with or without
PDK1-activated Akt1 in vitro. As shown in Fig.
3C, only ARK5 but not ARK5(S600A) was phosphorylated by Akt.
In addition, an increased phosphorylation of GST-SAMS was induced by
ARK5 but not by ARK5(S600A) in the presence of active Akt1 (Fig.
3D), suggesting that ARK5 phosphorylated by active Akt1 is
enzymatically "active". To further examine the activation of ARK5
by Akt, physiological interaction of these components in
vivo was examined by ARK5 and Akt co-immunoprecipitation. The
results clearly showed that ARK5 and Akt were in association with each
other, but they dissociated just after Akt activation either by glucose
starvation or insulin treatment that activates Akt (Fig.
3E), which is consistent with the case of Mdm2
phosphorylation by Akt (27). No Akt was co-immunoprecipitated
from HepG2 cells with anti-FLAG antibody (data not shown).
View larger version (69K):
[in a new window]
Fig. 3.
ARK5 is a novel substrate of Akt.
A, cell extracts from HepG2 or H/ARK cells subjected to
glucose starvation for the indicated periods were Western blotted with
polyclonal antibody against phospho Akt (pAkt) or total Akt
(tAkt). B, comparison of Akt phosphorylation
sites in ARK5 and other substrates. C, ARK5 or ARK5(S600A)
was incubated with active Akt1 (act.Akt1) in
vitro. After incubation, immunoprecipitates with anti-FLAG
antibody were separated on SDS-PAGE. Immunoprecipitate with anti-FLAG
antibody from H/ARK cells labeled with [35S]methionine
was also separated on SDS-PAGE. D, ARK5 and ARK5(S600A) was
were incubated with (+) or without ( ) active Akt1
(act.Akt) in vitro. After incubation, SAMS
phosphorylation was measured. 32P incorporation is shown as
means of three experiments, and the bars represent S.E.
value. Asterisks show the statistical significance
p < 0.01 (t test). E, H/ARK
cells were subjected to glucose starvation or insulin stimulation for
0-60 min, and whole cell extracts (WCE) or
immunoprecipitates with antibody against FLAG (FLAG-IP) or
Akt (Akt-IP) were blotted with antibody for total Akt
(tAkt), phospho Akt (pAkt) or FLAG.
APM) (ARK5 lacking 66 amino acids in the C-terminal region including the Akt phosphorylation
site). When dominant active (DA-Akt), but not dominant negative,
(DN-Akt) Akt was transfected into HepG2, cell death induction was also suppressed significantly. The combined expression of ARK5 and dominant
active Akt suppressed cell death further, down to ~20%, but dominant
negative Akt and/or mutants of ARK5 failed to do so (Fig.
4A). These results clearly indicated that ARK5 activated by
active Akt is essential for tolerance to glucose starvation in HepG2
cells. These observations were further confirmed using H/ARK cells
(Fig. 4B). When dominant active Akt is introduced into H/ARK
cells, cell death was remarkably suppressed (Fig. 4B). However, when dominant negative Akt was used, induction of cell death
occurred to a greater extent (Fig. 4B). When an inhibitor of
phosphatidylinositol-3 kinase, LY294002 (28), was included, activation
of Akt was clearly inhibited with a concomitant increase in cell death.
These results are consistent with those for transient transfections.
View larger version (27K):
[in a new window]
Fig. 4.
ARK5 mediates Akt-induced cell survival
signaling. A, HepG2 cells transiently transfected with
(+) or without ( ) ARK5, dominant active Akt
(DA-Akt), dominant negative Akt (DN-Akt),
ARK5-deletion mutant (ARK(
APM)) and/or ARK5-point
mutant (ARK(S600A)) were subjected to glucose starvation for
24 h. Ratio of cell death induction is shown as means of three
experiments, and the bars represent S.E. value.
B, H/ARK cells transiently transfected with or without
DA-Akt or DN-Akt were subjected to glucose starvation in the presence
or absence of LY294002 (20 µM) for 24 h.
View larger version (57K):
[in a new window]
Fig. 5.
ARK5-induced ATM activation.
A, schematic model of phosphorylation site by AMPK or Akt
was indicated by arrowhead on ATM sequence. B,
P/ATM cells were transfected with (+) or without ( ) ARK5 or ARK5(AS)
for 24 h. After transfection, in vivo labeling of
32P was performed, and cells were incubated in media with
or without glucose for 1 h. His-ATM was collected with
nickel-agarose and phosphorylated ATM (pATM;
32P-autoradiography) and total ATM (tATM;
Western blotting of His-ATM with anti-human ATM antibody) were
detected. C, His-ATM (2 µg) was reacted with or without
ARK5 (2 µg) in the presence or absence of active Akt1
(act.Akt1; 2 µg). D, ATM kinase assay was
performed with full-length recombinant protein of human p53 in the
presence or absence of His-ATM (2 µg) and/or ARK5 (2 µg).
E, HepG2 cells transiently transfected with (+) or without
(
) ARK5(AS). After 24 h, cells were subjected to medium with (+)
or without (
) glucose for 1 h, and then Western blotting using
antibodies for p53 and actin was performed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and AMP-
2 subunits suppressed
hypoxia-induced tolerance to glucose starvation in HepG2 cells (17) and
constitutive tolerance in PANC-1 cells (29), indicating a close
involvement of AMPK-
1 and AMP-
2 in tumor cell tolerance during
glucose starvation. AMPK-
1 appeared to be more responsive to hypoxic
conditions than AMPK-
2 (17). In our previous reports (16,
17), we also demonstrated an important role of Akt in tumor cell
tolerance induced by hypoxia. The Akt phosphorylation was suppressed by the PI3K inhibitor LY294002 and wortmannin, but not by the AMPK inhibitor AraA (16, 17), suggesting an involvement of PI3K but not AMPK
in the phosphorylation of Akt during glucose starvation and hypoxia.
Because only ARK5 but not any other member of the AMPK family is
responsive to Akt, it appears that ARK5 acts as the key mediator of Akt
in tumor cell tolerance during glucose starvation. ARK5, the
activation of which is induced by Akt during glucose starvation and
hypoxia, may influence AMPK-
1 and AMPK-
2 to induce tumor cell
tolerance to glucose starvation under the hypoxic condition.
![]() |
ACKNOWLEDGEMENT |
---|
We thank KAZUSA DNA Research Institute for KIAA0537 cDNA clone (GenBankTM accession number AB011109).
![]() |
FOOTNOTES |
---|
* This work was partly supported by a second-term comprehensive 10-year Strategy of Cancer Control grant from the Ministry of Health, Welfare, and Labor and a Medical Frontier Program grant from the Ministry of Health, Welfare, and Labor.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.
§ Recipient of a research resident fellowship award from the Foundation for the Promotion of Cancer Research.
To whom correspondence should be addressed. Tel.:
81-4-7134-6880; Fax: 81-4-7134-6859; E-mail:
hesumi@east.ncc.go.jp.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M206025200
2 G. Kusakai, A. Suzuki, A. Kishimoto, J. Lu, T. Ogura, M. Lavin, and H. Esumi, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: AMPK, AMP-activated protein kinase; PI, propidium iodide; ATM, ataxia-telangiectasia mutated; H/ARK, HepG2 cells with transfected ARK5 expression vector; PBS, phosphate-buffered saline; GST, glutathione S-transferase; ARK5(AS), antisense RNA expression vector of ARK5; ARK5(S600A), ARK5 point mutant; PDK1, phosphoinositide-dependent kinase 1; DA-Akt, dominant active Akt; DN, dominant negative Akt; P/ATM, PANC-1 cell in which His-tagged ATM is stably expressed.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Carlson, M. (1999) Curr. Opin. Microbiol. 2, 202-207[CrossRef][Medline] [Order article via Infotrieve] |
2. | Hardie, G. D., Carling, D., and Carlson, M. (1998) Annu. Rev. Biochem. 67, 821-855[CrossRef][Medline] [Order article via Infotrieve] |
3. | Becker, W., Heukelbach, J., Heiner, K., and Joost, H.-G. (1996) Eur. J. Biochem. 235, 736-743[Abstract] |
4. | Gardner, H. P., Wertheim, G. B., Ha, S. I., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Marquis, S. T., and Chodosh, L. A. (2000) Genomics 63, 46-59[CrossRef][Medline] [Order article via Infotrieve] |
5. | Heyer, B. S., Warsowe, J., Solter, D., Knowles, B. B., and Ackerman, S. L. (1997) Mol. Reprod. Dev. 47, 148-156[CrossRef][Medline] [Order article via Infotrieve] |
6. | Inglis, J. D., Lee, M., and Hill, R. E. (1993) Mamm. Genome 4, 401-403[Medline] [Order article via Infotrieve] |
7. | Ruiz, J. C., Conlon, F. L., and Robertson, E. J. (1994) Mech. Devel. 48, 153-164[CrossRef][Medline] [Order article via Infotrieve] |
8. | Wang, Z.-N., Takemori, H., Halder, S. K., Nonaka, Y., and Okamoto, M. (1999) FEBS Lett. 453, 135-139[CrossRef][Medline] [Order article via Infotrieve] |
9. | Aguan, K., Scott, J., See, C. G., and Sarkar, N. H. (1994) Gene 149, 345-350[Medline] [Order article via Infotrieve] |
10. | Carling, D., Clarke, P. R., Zammit, V. A., and Hardie, D. G. (1989) Eur. J. Biochem. 186, 129-136[Abstract] |
11. | Davies, S. P., Sim, A. T., and Hardie, D. G. (1989) Eur. J. Biochem. 186, 123-128[Abstract] |
12. |
Stapleton, D.,
Mitchelhill, K. I.,
Gao, G.,
Widmer, J.,
Michell, B. J.,
Teh, T.,
House, C. M.,
Fernandez, C. S.,
Cox, T.,
Witters, L. A.,
and Kemp, B. E.
(1996)
J. Biol. Chem.
271,
611-614 |
13. | Lefebvre, D. L., Bai, Y., Shahmolky, N., Poon, R., Drucker, D. J., and Rosen, C. F. (2001) Biochem. J. 355, 297-305[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Hawley, S. A.,
Davidson, M.,
Woods, A.,
Davies, S. P.,
Beri, R. K.,
Carling, D.,
and Hardie, D. G.
(1996)
J. Biol. Chem.
271,
27879-27887 |
15. | Sullivan, J. E., Carey, F., Carling, D., and Beri, R. K. (1994) Biochem. Biophys. Res. Commun. 200, 1551-1556[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Izuishi, K.,
Kato, K.,
Ogura, T.,
Kinoshita, T.,
and Esumi, H.
(2000)
Cancer Res.
60,
6201-6207 |
17. |
Esumi, H.,
Izuishi, K.,
Kato, K.,
Hashimoto, K.,
Kurashima, Y.,
Kishimoto, A.,
Ogura, T.,
and Ozawa, T.
(2002)
J. Biol. Chem.
277,
32791-32798 |
18. | Hashimoto, K., Kato, K., Imamura, K., Kishimoto, A., Yoshikawa, H., Taketani, Y., and Esumi, H. (2002) Biochem. Biophys. Res. Commun. 290, 263-267[CrossRef][Medline] [Order article via Infotrieve] |
19. | Imamura, K., Ogura, T., Kishimoto, A., Kaminishi, M., and Esumi, H. (2001) Biochem. Biophys. Res. Commun. 287, 562-567[CrossRef][Medline] [Order article via Infotrieve] |
20. | Lavin, M. F., and Shiloh, Y. (1997) Annu. Rev. Immunol. 15, 177-202[CrossRef][Medline] [Order article via Infotrieve] |
21. | Lavin, M. F. (2000) Nat. Cell Biol. 2, 215-217 |
22. | Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L., Tagle, D. A., Smith, S., Uziel, T., Sfez, S., Ashkenazi, M., Pecker, I., Frydman, M., Harnik, R., Patanjali, S. R., Simmons, A., Clines, G. A., Sartiel, A., Gatti, R. A., Chessa, L., Sanal, O., Lavin, M. F., Jaspers, N. G. J., Malcolm, A., Taylor, R., Arlett, C. F., Miki, T., Weissman, S. M., Lovett, M., Collins, F. S., and Shiloh, Y. (1995) Science 268, 1749-1753[Medline] [Order article via Infotrieve] |
23. |
Banin, S.,
Moyal, L.,
Shieh, S.,
Taya, Y.,
Anderson, C. W.,
Chessa, L.,
Smorodinsky, N. I.,
Prives, C.,
Reiss, Y.,
Shiloh, Y.,
and Ziv, Y.
(1998)
Science
281,
1674-1677 |
24. |
Canman, C. E.,
Lim, D. S.,
Cimprich, K. A.,
Taya, Y.,
Tamai, K.,
Sakaguchi, K.,
Appella, E.,
Kastan, M. B.,
and Siliciano, J. D.
(1998)
Science
281,
1677-1679 |
25. | Sheih, S. Y., Ikeda, M., Taya, Y., and Prives, C. (1997) Cell 91, 325-334[Medline] [Order article via Infotrieve] |
26. | Hardie, D. G., and Carling, D. (1997) Eur. J. Biochem. 246, 259-273[Abstract] |
27. |
Mayo, L. D.,
and Donner, D. B.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
11598-11603 |
28. |
Vlahos, C. J.,
Matter, W. F.,
Hui, K. Y.,
and Brown, R. F.
(1994)
J. Biol. Chem.
269,
5241-5248 |
29. | Kato, K., Ogura, T., Kishimoto, A., Minegishi, Y., Nakajima, N., Miyazaki, M., and Esumi, H. (2002) Oncogene 21, 6082-6090[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Zhang, N.,
Chen, P.,
Khanna, K. K.,
Scott, S.,
Gatei, M.,
Kozlov, S.,
Watters, D.,
Spring, K.,
Yen, T.,
and Lavin, M. F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
8021-8026 |
31. | Rotman, G., and Shiloh, Y. (1997) Cancer Surv. 29, 285-304[Medline] [Order article via Infotrieve] |
32. | Pearce, A. K., and Humphrey, T. C. (2001) Trends Cell Biol. 11, 426-433[CrossRef][Medline] [Order article via Infotrieve] |
33. | Barlow, C., Brown, K. D., Deng, C. X., Tagle, D. A., and Wynshaw-Boris, A. (1997) Nat. Genet. 17, 453-456[Medline] [Order article via Infotrieve] |
34. | Gatei, M., Shkedy, D., Khanna, K. K., Uziel, T., Shiloh, Y., Pandita, T. K., Lavin, M. F., and Rotman, G. (2001) Oncogene 20, 289-294[CrossRef][Medline] [Order article via Infotrieve] |
35. | Yang, D. Q., and Kastan, M. B. (2000) Nat. Cell Biol. 2, 893-898[CrossRef][Medline] [Order article via Infotrieve] |