From the Buck Institute for Age Research, Novato,
California 94945-1400 and the § Laboratory of Cell
Signaling, Graduate School, Tokyo Medical and Dental University, 1-5-45 Bunkyo-ku, Tokyo 113-8549, Japan
Received for publication, November 7, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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The type 1 insulin-like growth factor receptor
(IGF-IR) is a receptor-tyrosine kinase that plays a critical role in
signaling cell survival and proliferation. IGF-IR binding to its
ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins
such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP
kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal
kinase (JNK) and p38 activation in response to Fas and tumor necrosis
factor (TNF) receptor stimulation, and for oxidative stress- and
TNF The type 1 insulin-like growth factor receptor
(IGF-IR)1 is a
receptor-tyrosine kinase that plays a critical role in signaling cell
survival and proliferation. Cells lacking this receptor cannot be
transformed by most oncogenes, with the exception of v-Src (1). In
addition, IGF-I can stimulate proliferation of a variety of cell types
in culture in the absence of other growth factors. Gene knockout
experiments in mice (2-4) and flies (5) have demonstrated that the
IGF-I axis is required for normal growth at the organismal level as
well. In mammalian cells, the assembly of a signaling complex at the
cytoplasmic domain of IGF-IR results in the activation of
phosphoinositide 3-kinase (PI3K) and its target, Akt/PKB (6) and
promotes cell proliferation, and in some cases differentiation, by
engaging the mitogen-activated protein kinase (MAPK) cascade (7, 8) and
the Ras pathway (9). The inactivation of the pro-apoptotic Bcl-2 family
member BAD through phosphorylation by Akt/PKB (10) is thought to
underlie the strong anti-apoptotic activity of IGF-I and IGF-II.
Activation of IGF-IR by its ligand also initiates metabolic cascades
that result in the stimulation of protein synthesis, glucose intake, glycogen synthesis, and lipid storage.
The MAPK signaling cascade is conserved in evolution and controls
transcriptional responses to mitogenic or stress stimuli by the
sequential activation of protein kinases. There are at least 6 independent MAPK signaling units described in mammals (11). The
pathways that culminate in the activation of the extracellular signal-regulated kinases (ERKs) and stress-activated protein kinases (SAPK/JNK and p38) have been characterized in some detail. Apoptosis signal-regulating kinase (ASK1) is a MAPKKK that activates the JNK/p38
pathway in response to proinflammatory cytokines such as TNF ASK1 selectively activates the SEK1-JNK1 and MKK3/MAPKK6-p38 pathways
(16) and is required for the sustained activation of p38 in response to
TNF Withdrawal of growth factors present in serum (15) or withdrawal of
nerve growth factor (25) has been shown to lead to a severalfold
increase in ASK1 activity. Also, ASK1-induced death in cultured cells
requires either the deletion of its N-terminal domain (19) or low serum
conditions (16). We hypothesized that a pathway activated by trophic
factor(s) could thus negatively regulate ASK1 activity. Given that
IGF-I is a potent activator of cell proliferation, and that in many
cases it can replace serum to stimulate growth of cells in culture, we
sought to investigate whether IGF-I signaling modulates ASK1 activity.
The results presented here indicate that IGF-IR signaling inhibits ASK1
and relieves ASK1-induced apoptotic cell death. We found that ASK1
formed a complex with IGF-IR both in the presence and in the absence of exogenously added IGF-I and became phosphorylated on tyrosine residue(s) on its regulatory N-terminal domain in a manner dependent on
IGF-IR kinase activity.
IGF-IR signaling inhibited ASK1 activation in the presence of TNF- Cell Culture and Transient Transfections--
Unless indicated
otherwise, human embryonic kidney 293 and L929 cells were grown in 60-, 100-, or 150-mm dishes at 37 °C under 5% CO2 in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and penicillin/streptomycin (Cellgro). 293 cells grown in 60- or 100-mm
dishes were transiently transfected with 3-5 or 6-8 µg of the
indicated plasmid constructs, respectively, using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions and
maintained in serum-free medium for the duration of the experiment.
Lysates were collected 13-15 h after transfection. 8-10-h
post-transfection, cultures of 293 cells were treated with 100 ng/ml of
recombinant TNF DNA Constructs and Antibodies--
Hemagglutinin-tagged
wild-type and kinase-mutant human ASK1 (ASK1-HA and ASK1KD-HA) and the
glutathione S-transferase fusion of MKK6KD were described
previously (16). The ASK1Y574A-HA construct was generated by
site-directed mutagenesis using primers
CCAACCAAAATCTATCAACCTTCTGCTTTGTCTATCAACAATGAAGTTGAGG and
CCTCAACTTCATTGTTGATAGACAAAGCAGAAGGTTGATAGATTTTGGTTGG and a modification
of the Stratagene's QuikChange protocol. The p-EGFP vector was
purchased from Clontech. pCEP-IGF-IR encodes the
full-length IGF-IR precursor under the control of the cytomegalovirus
promoter. pCDNA-IGF-IRIC and
pCDNA-IGF-IRKM encode the intracellular domain of the
IGF-IR Immunoprecipitation and Immunoblotting--
Cells were
collected, washed once in phosphate-buffered saline, and disrupted in
lysis buffer (1 mM EDTA, 20 mM Tris-HCl, pH
7.5, 12 mM In Vitro Kinase Assays--
Cells were disrupted in lysis buffer
as described above and clarified with proteinA/G PLUS-agarose. Proteins
of interest were immunoprecipitated as described above. The beads were
washed three times in kinase buffer and incubated in the presence of 20 µM ATP, 3 µCi of [ In Vitro Phosphorylation--
Immunoprecipitated ASK1-HA or
ASK1KM-HA were incubated with 20 µM ATP, 3 µCi of
[ Immunohistochemistry and Deconvolution of Confocal
Images--
293 cells were grown on 22-mm square coverslips and
treated as indicated. Cells were then fixed by immersion in Assessment of Apoptotic Cell Death--
Percentages of apoptotic
cells in cultures were determined by standard propidium iodide (PI)
staining protocols and flow cytometry (27) 60 h after
transfection. IGF-IR-expressing cells were treated with 100 ng/ml IGF-I
and 200 nM wortmannin 12 h after transfection as
indicated and maintained in their presence for the duration of the
experiment. Fresh IGF-I was added to the cultures every 8 h.
IGF-I and the Activated IGF-IR Inhibit ASK1 Activation--
Given
that maximal activation of ASK1 in cultured cells is achieved when
cultures are maintained in the absence of serum, we hypothesized that a
pathway activated by trophic factor(s) could negatively regulate ASK1
activity. IGF-I is a potent activator of cell proliferation and in many
cases it can replace serum to stimulate growth of cells in culture. We
therefore asked whether IGF-I would be sufficient to decrease ASK1
activity in serum-deprived L929 cells. The activity of ASK1 was reduced
40% in L929 cells grown in 10% serum in comparison to L929 cells
maintained in the absence of serum (Fig.
1, panels A and B).
IGF-I treatment of serum-starved L929 cells decreased the activity of
ASK1 to levels identical to those observed in cultures treated with
10% serum, implying that the inhibition of ASK1 activity by serum may
be attributed to the effect of IGF-I. Recently, Chao and co-workers (28) showed that ASK1 can be inhibited by Akt, a serine/threonine kinase that is activated by PI3K and has a crucial role in the signal
transduction pathway initiated by IGF-I. To determine the contribution
of Akt to the decrease in ASK1 activity induced by IGF-I, we incubated
serum-starved, IGF-I-stimulated cells in the presence of wortmannin, a
potent PI3K inhibitor. Inhibition of PI3K signaling did not have a
significant effect on the decrease in ASK1 activity resulting from
IGF-I stimulation of serum-starved cells (Fig. 1, panel B).
In some experiments, we observed a moderate decrease in ASK1 activity
in the presence of wortmannin (Fig. 1, panel A). This could
be caused by a general effect of wortmannin on protein synthesis, even
at the relatively short incubation times used in these experiments.
Given that Akt activation was effectively inhibited by wortmannin in
IGF-I-stimulated L929 cells (not shown), we concluded that the
inhibition of ASK1 by IGF-I occurred, at least partially, upstream or
independently of PI3K.
Next, we transiently expressed a human ASK1 complementary DNA
(cDNA) tagged with a hemagglutinin epitope (ASK1-HA) in the presence of a kinase-competent, full-length human IGF-IR cDNA (IGF-IR), a kinase-competent, myristylated intracellular domain of the
IGF-IR ASK1 Is Phosphorylated on Tyr in the Presence of IGF-IR--
Given
that the inhibition of ASK1 by IGF-I stimulation in L929 cells was only
partially abolished by treatment with the PI3K inhibitor wortmannin, we
hypothesized that ASK1 was inhibited at a step upstream or
independently of PI3K, possibly by the activated IGF-IR itself. To test
this idea, we determined whether ASK1 was phosphorylated on Tyr. A
decrease in the electrophoretic mobility of the ASK1-HA band in
gradient gels was observed in the presence of kinase-active forms of
the IGF-IR, but not when a kinase-inactive form of IGF-IR was used
(Fig. 2, panel A). The slower migrating ASK1-HA forms were
phosphotyrosine immunoreactive (Fig. 2, panel B). No
phosphotyrosine immunoreactivity could be observed in ASK1-HA when it
was expressed in the presence of a kinase-inactive form of the
IGF-IR
To determine whether ASK1 is a direct substrate for IGF-IR, we
immunoprecipitated ectopically expressed IGF-IR, the intracellular domain of the IGF-IR ASK1 Is Found in a Complex with IGF-IR--
Because the detection
of a complex formed by IGF-IR and ASK1 would provide additional support
for the notion that ASK1 is a substrate of the IGF-IR, we performed
reciprocal immunoprecipitation experiments using lysates from L929
cells maintained in the presence of 10% serum or deprived of serum
with and without IGF-I stimulation. We found that the
Given that ASK1 is a cytoplasmic protein, we next asked whether the
interaction of ASK1 with IGF-IR involved the cytoplasmic portion of the
receptor's ASK1 and IGF-IR Colocalize in a Compartment Juxtaposed to the
Plasma Membrane and in Structures Adjacent to the Nucleus--
We then
sought to determine the subcellular localization of the interaction
between ASK1 and IGF-IR. The two proteins were found in structures
adjacent to the nucleus of 293 cells ectopically expressing ASK1 and
the IGF-IR precursor (Fig. 3D, upper panels). The
presence of ASK1/IGF-IR complexes in perinuclear structures may reflect
the accumulation of immature forms of IGF-IR in the endoplasmic
reticulum (ER) compartment and their interaction with ASK1, which is
associated with components of the unfolded protein response machinery
that assembles at the ER membrane (31). In agreement with the
observation that the intracellular domain of IGF-IR mediates its
interaction with ASK1, we found that IGF-IRIC colocalized
with ASK1 in a compartment juxtaposed to the cytoplasmic face of the
plasma membrane in 293 cells in a manner independent of its kinase
activity (Fig. 3D, middle and lower
panels).
The N-terminal Domain of ASK1 Is Tyr-phosphorylated--
It has
been shown that the N-terminal domain of ASK1 contains regulatory
domains that affect the activity of the kinase through phosphorylation
(28) and protein-protein interactions (14, 15). To determine whether
phosphorylation by the IGF-IR occurs at the N-terminal regulatory
domain of ASK1 we transiently expressed a mutant ASK1-HA construct in
which the N-terminal 648 amino acids of the kinase had been deleted
(ASK1 IGF-IR Inhibits ASK1 Downstream of TNF-R and Blocks JNK1
Activation--
ASK1 is activated by TNF-R (13, 14) and by cellular
stress (11, 20), and it activates JNK and the p38 MAP kinase pathways (16). The possibility that transiently overexpressed IGF-IR may be able
to signal in the absence of exogenous ligand stimulation allowed us to
examine the effect of IGF-IR signaling in 293 cells in serum-free
conditions, thus avoiding the stimulation of endogenous IGF-IR, which
is sufficient to inhibit ASK1 activation (Fig. 1) and ASK1-induced cell
death (Ref. 16 and data not shown). We therefore sought to determine
whether IGF-IR signaling inhibits (a) ASK1 activation by
TNF Signaling from IGFIR Suppresses ASK1-induced Apoptotic Cell
Death--
ASK1 is required for TNF The response of cells to changes in their physical and chemical
environment is key to the control of many aspects of cellular function.
Environmental changes happen in ranges of intensities and duration and
in a combinatorial fashion in most biological settings. It is therefore
likely that most signaling pathways have evolved in the context of
their interconnection in an entire network, and thus the investigation
of how individual signaling pathway cross-talk is crucial for our
understanding of cellular responses.
Signaling through IGF-IR is required for growth of most cell types, and
is sufficient to support survival of a variety of cells in culture in
the absence of other growth factors. IGF-I was shown to suppress
apoptosis through the activation of the PI3K/Akt axis, which results in
the phosphorylation and inhibition of BAD, a pro-apoptotic member of
the Bcl-2 family (10). IGF-I was also shown to suppress apoptosis
downstream of TNF-R activation (23) and to block the activation of JNK
by the toxic fragment derived from the amyloid precursor protein,
amyloid In the present work we have addressed the modulation of one of the
apical stress-activated MAP kinases, ASK1, by the IGF-IR signaling
pathway. Our findings indicate that ASK1 is inhibited by IGFIR and
becomes phosphorylated on Tyr residue(s) on its regulatory N-terminal
domain in a manner dependent on IGFIR activity. IGF-IR and ASK1 formed
a complex in cells irrespective of the functionality of either kinase,
and irrespective of IGF-IR ligand stimulation. This interaction
involved the C-terminal domain of ASK1 and the intracellular domain of
the IGF-IR Our observation that transiently expressed IGF-IR may be active in the
absence of ligand binding allowed us to examine signaling by the
receptor in the absence of IGF-I stimulation of the endogenous IGF-IR,
which is sufficient to inhibit ASK1 activation and ASK1-induced cell
death. Our results demonstrate that ASK1-dependent JNK
activation and cell death (which occur only in the absence of IGF-I)
were inhibited in a manner dependent on the presence of IGF-IR and independent of PI3K function.
In this experimental system, TNF The observation that ASK1 and IGF-IR were present in a complex in L929
cells irrespective of IGF-I stimulation and that this interaction was
independent of either IGF-IR or ASK1 kinase activity in 293 cells may
indicate that ASK1 molecules bind the intracellular portion of IGF-IR
heterodimers in a constitutive fashion. ASK1 has been shown to signal
in the cytoplasmic compartment (16) and to be activated by recruitment
to the membrane-associated signaling complexes such as TNF-R/TRAF2,
Fas/Daxx (12, 14), and IRE1/TRAF2 (31). Formation of these complexes is
thought to favor the homo-oligomerization and transphosphorylation of the kinase (12, 33). Our observation that a kinase-inactive form of
IGF-IR partially retained the ability to inhibit ASK1 suggests that
IGF-IR may also inhibit ASK1 activation by interfering with its
homo-oligomerization through protein-protein interactions. It is
possible that a membrane-associated pool of ASK1 molecules exists in
equilibrium between active and inactive forms, which could be shifted
in either direction by selective ligand binding of activators such as
TNF-R or of inhibitors such as IGF-IR.
Our results indicate that ASK1 is phosphorylated on multiple Tyr
residues at its N-terminal domain in a manner dependent on IGF-IR
activity and suggest that ASK1 is a bona fide substrate of
IGF-IR. The N-terminal domain of ASK1 contains 23 Tyr residue(s) of
which 5 are predicted as candidate phosphorylation sites by NetPhos 2.0 (Tyr-95, Tyr-210, Tyr-390, Tyr-574, and Tyr-625). One of these five Tyr
residues is phosphorylated in the presence of kinase-competent
IGF-IR,2 but the additional
phosphorylated Tyr residue(s) remain to be identified. The
identification of all phosphorylated Tyr residues in the N-terminal
domain of ASK1 is currently underway and will allow us to further our
understanding of the physiological significance of the phosphorylation
of ASK1 by the IGF-IR.
Kim et al. (28) recently showed that PKB/Akt, a downstream
effector of the IGF-I signaling pathway, phosphorylates and inhibits ASK1. These observations, together with the results presented here,
suggest that the IGF-I signaling pathway can antagonize the activation
of ASK1 through two different kinases. It is possible that inhibition
of ASK1 by IGF-IR and its effector PKB/Akt occurs in a stepwise fashion
and that phosphorylation, and/or protein-protein interactions with both
kinases is required for complete inhibition of ASK1. This hypothesis is
supported by our observation that inhibition of PI3K activity by
wortmannin only partially reversed IGF-I-dependent
inhibition of ASK1. Alternatively, phosphorylation and/or
protein-protein interactions by either IGF-IR or PKB/Akt may be
sufficient to inhibit ASK1 activity to levels below those required for
activation of JNK/p38. The observation that two different kinases in
the same pathway inhibit ASK1 may indicate that the metabolic responses
triggered by IGF-I stimulation require that the inhibition of the
stress-activated arm of the MAPK cascade be ensured.
-induced apoptosis. The results presented here indicate that ASK1
forms a complex with the IGF-IR and becomes phosphorylated on tyrosine
residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling
inhibited ASK1 irrespective of TNF
-induced ASK1 activation and
resulted in decreased ASK1-dependent JNK1 stimulation.
Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell
death in a manner independent of PI3K activity. These results
indicate that IGF-IR signaling suppresses the ASK-1-mediated
stimulation of JNK/p38 and the induction of programmed cell death. The
simultaneous activation of MAP kinases and the inhibition of the
stress-activated arm of the cascade by IGF-IR may constitute a potent
proliferative signaling system and is possibly a mechanism by which
IGF-I can stimulate growth and inhibit cell death in a wide variety of
cell types and biological settings.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
FasL (12-14). As for other MAPKKK, deletion of the N-terminal domain
of ASK1 results in its constitutive activation, suggesting that the N
terminus contains a regulatory domain (15). Consistent with this
observation, Daxx (12), TRAF2 (14), and reduced thioredoxin (15) have
been shown to interact with sequences in the ASK1 N-terminal domain.
and oxidative stress (17). The biological outcome of JNK/p38
activation is largely dependent on cell type and cellular context (18),
and in some situations leads to the activation of programmed cell death
(19-21). TNF
-induced JNK activation can be blocked by IGF-II in
neuronal cell lines (22). Also, IGF-I can suppress apoptosis downstream
of TNF-R activation (23) and block JNK activation by amyloid-
(24).
and attenuated ASK1-dependent JNK1 stimulation. We propose that one of the mechanisms underlying the cytoprotective activity of
IGF-I signaling may involve the down-regulation of the stress-activated arm of the MAP kinase cascade through the phosphorylation and inhibition of ASK1.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Sigma) for 20 min. In all transfection experiments,
the amount of plasmid DNA transfected was equalized by the addition of
pcDNA3.1 vector DNA. For the analysis of endogenous proteins, 8-10
150-mm dishes of L929 cells were incubated in serum-free media for
2 h and then either left untreated or treated with 200 nM wortmannin (Sigma) 20 min prior and during treatment
with 100 ng/ml purified IGF-I (Sigma) for 10 additional minutes.
-chain (residues 930-1337 in the precursor) carrying a
myristylation sequence at their N termini and a Lys to Arg mutation at
position 1003 for pCDNA-IGFIRKM. Plasmids containing
the FLAG-tagged human JNK1 open reading frame under the regulation of
the cytomegalovirus promoter have been described (26) and were
generously provided by Dr. Roya Koshravi-Far. Anti-HA antibodies
(HA.11) were purchased from BabCO. Anti-IGF-IR (C-20), anti-ASK1
(N-19), anti-ASK1 (H-300), and protein A/G-Sepharose were purchased
from Santa Cruz Biotechnology. Purified anti-human JNK1/JNK2 and
anti-JNK1 were purchased from BD PharMingen. All horseradish
peroxidase-conjugated secondary antibodies, FITC-conjugated anti-goat
and Texas Red-conjugated anti-rabbit were purchased from Jackson
ImmunoResearch Laboratories and AP-conjugated anti-rabbit and
AP-conjugated anti-mouse were purchased from Promega.
Antiphospho-SAPK/JNK(Thr-183/Tyr-185) and antiphospho-tyrosine were
purchased from Cell Signaling.
-glycerophosphate, 1 mM sodium
orthovanadate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 0.5% sodium deoxycholate, 3 mM dithiothreitol with the addition of Mini protease
inhibitors (Roche Molecular Biochemicals)). In immunoprecipitation
experiments involving L929 cells, 1% Triton X-100 was replaced by
0.2% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% Triton X-100.
Lysates were spun at 16,000 × g, supernatants were
precleared by incubation with protein A/G-agarose for 30 min at
4 °C, and proteins were immunoprecipitated by incubation in
appropriate antibodies followed by protein A/G PLUS-agarose. Pellets
were washed three times with lysis buffer and then twice with kinase
buffer (25 mM HEPES pH 7.6, 20 mM MgCl, 2 mM dithiothreitol, 1 mM sodium orthovanadate, and 20 mM
-glycerophosphate). Complexes were disrupted
in Laemmli buffer (Invitrogen) and resolved in 7%, 3-8% Tris acetate
gels or in 10% NuPage gels (Invitrogen). The separated polypeptides were transferred to polyvinylidene difluoride membranes (Schleicher and
Schuell), blocked in 5% nonfat milk, and immunoblotted with the
indicated antibodies. Immunoreactive proteins were visualized by
enhanced chemiluminescence (Amersham Biosciences) and detected using
Kodak Biomax MR film (Kodak Eastman).
-32P]ATP, and 2.5 µg of either GST-MKK6KM or myelin basic protein (Sigma). Reactions
were incubated for 30 min at 30 °C. The reaction was stopped by
addition of Laemmli buffer, and the products were resolved in gels,
which were dried and exposed to film or analyzed in a Typhoon
PhosphorImager (Molecular Dynamics). In all experiments, both the
extent of phosphorylation and the amount of protein determined by
Western blotting were determined with ImageQuant software. GST-MKK6KM
was expressed in BL21 cells together with a thioredoxin-expressing vector to enhance solubility of the fusion protein and purified using
glutathione-Sepharose 4B (Amersham Biosciences) according to the
manufacturer's instructions.
-32P]ATP, and immunoprecipitated IGF-IR in kinase
buffer for 30 min at 30 °C. 32P incorporation was
assessed by gel electrophoresis and PhosphorImager analysis. Proteins
were visualized using Coomassie Blue staining.
20 °C
methanol for 20 min and blocked in 10% normal donkey serum (Jackson
ImmunoResearch Laboratories) in 1% bovine serum albumin in 1×
phosphate-buffered saline for 1 h at room temperature. Cells were
then incubated in goat anti-ASK1 (N-19) and rabbit anti-IGF-IR (C-20)
in 1% bovine serum albumin for 1 h at room temperature, washed
three times with phosphate-buffered saline, and incubated with
FITC-conjugated, donkey anti-goat and Texas Red-conjugated donkey
anti-rabbit antibodies for 1 h at room temperature. Cells were
then washed extensively (8-10 times, 15 min) with phosphate-buffered
saline at room temperature and mounted in DAPI-Vectashield (Vector
Laboratories). Wide-field images were acquired using a Nikon
Eclipse-800 microscope and appropriate filters and collected using
Compix Simple PCI software. Images were then processed in a SGI Octane
R12 computer running Bitplane's Advanced Imaging Software suite.
Briefly, deconvolution was done using a maximum likelihood estimation
algorithm with Huygens software and then processed by the Imaris
imaging interphase (Bitplane AG).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
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Fig. 1.
IGF-I inhibits ASK1 activity.
A, ASK1 was immunoprecipitated from L929 cells treated as
indicated. Complexes were resolved in gels and immunoblotted
(upper panel) or used in kinase assays with GST-MKK6KD as a
substrate in the presence of [ -32P]ATP. The products
of the reaction were resolved in gels and detected by phosphorimager
analysis (lower panel). IP, immunoprecipitation;
WB, Western blot; KA, kinase assay. B,
the activity of ASK1 in L929 cells treated as indicated was determined
by phosphorimager analysis and densitometry as described under
"Experimental Procedures." The fold activation of ASK1 was
determined in reference to its activity in cells maintained in the
presence of 10% serum, which was equal to 1. The average of two
independent experiments is shown. C, the activity of
ectopically expressed ASK1 is decreased in the presence of
kinase-competent IGF-IR. ASK1-HA was transiently expressed in the
presence of full-length IGF-IR (IGF-IR) or a kinase-competent
(IGF-IRIC) or kinase-mutant (IGF-IRKM)
myristylated form of the intracellular domain of the receptor's
-chain in 293 cells. ASK1-HA was immunoprecipitated and used in
kinase assays in the presence of [
-32P]ATP and myelin
basic protein (MBP) as a substrate. The products of the
reaction were resolved in gels and detected by phosphorimager analysis
of 32P incorporation in ASK1 (upper panel) and
MBP (middle panel). Immunoprecipitated HA-ASK1 was analyzed
by immunoblot (lower panel). D, the histogram
shows the mean ± S.E. for densitometric ratios of
32P-labeled/protein determined by phosphorimager analysis.
Values of 32P-labeled protein were normalized to ASK1
protein levels by immunoblotting as described in C. p values were calculated by Student's paired t
test.
-chain (IGF-IRIC), or a kinase-inactive form of the myristylated IGF-IR intracellular domain (IGF-IRK) in
293 cells. As reported previously (3), ectopically expressed ASK1-HA showed high constitutive activity in in vitro kinase assays
(Fig. 1, panel C). We observed variability in ASK1-HA
protein levels when it was expressed in cells in the presence of
IGF-IR; therefore, to determine whether ASK1-HA activity was affected
by IGF-IR signaling independent of variations in ASK1-HA protein
levels, we determined the ratio between ASK1-HA kinase activity by
radionuclide labeling and phosphorimager analysis and the densitometric
values of ASK1-HA protein by immunoblot in cells expressing ASK1-HA in
the presence of different forms of IGF-IR. ASK1 specific activity was
decreased in the presence of full-length IGF-IR (Fig. 1, panel
D). IGF-IRIC, which showed high constitutive kinase
activity (Fig. 2, panel D and
data not shown), further inhibited both ASK1 autophosphorylation and
the phosphorylation of myelin basic protein by ASK1 (Fig. 1,
panels C and D). This effect was partially
abolished by mutation of the ATP binding site in the IGF-IR kinase
domain. Mutation of lysine 1003 to arginine in the ATP binding site of
the receptor has been shown to abolish its kinase activity (29). The
partial (rather than complete) restoration of ASK1 kinase activity
observed in the presence of the kinase-inactive form of the IGF-IR
intracellular domain suggests that a kinase-independent mechanism
for ASK1 inhibition by IGF-IR may exist.
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Fig. 2.
ASK1 is phosphorylated on Tyr in the presence
of kinase-active IGF-IR. A, ASK1-HA was transiently
expressed in 293 cells in the presence of kinase-competent or
kinase-mutant forms of IGF-IR and immunoprecipitated using anti-HA
antibodies. Complexes were separated on 3-8% Tris acetate gels and
immunoblotted with anti-HA antibodies. B, lysates from 293 cells transiently expressing the indicated proteins were subject to
immunoprecipitation using anti-ASK1 antibodies, resolved in gels and
immunoblotted with antiphospho-Tyr antibodies (upper panel)
or anti-ASK1 antibodies (lower panel). C, the
indicated IGF-IR forms and either ASK1-HA or ASK1KM-HA were
separately immunoprecipitated from lysates of 293 cells and used alone
or combined in in vitro kinase assay reactions in the
presence of [ -32P]ATP as indicated. The
32P-labeled products were separated in gradient gels and
detected by phosphorimager analysis (left panel). The
activity of ASK1-HA or ASK1KM-HA immunoprecipitated from
293 cells was determined in in vitro kinase assays by
incorporation of [
-32P]ATP (right
panel).
chain intracellular domain (Fig. 2, panel B).
IGF-I stimulation of cells expressing full-length IGF-IR did not
significantly increase ASK1-HA phosphotyrosine immunoreactivity,
suggesting that transiently expressed IGF-IR may signal in the absence
of exogenous IGF-I stimulation. To determine whether transiently expressed IGF-IR may become active in the absence of exogenously added
serum or IGF-I, we assayed IGF-IR complexes immunoprecipitated from 293 cells in in vitro kinase assays and found that these complexes were able to transphosphorylate in vitro (data not
shown). These observations suggest that IGF-IR may form kinase-active complexes when transiently expressed in 293 cells in the absence of
ligand binding. Even though survival of 293 cell cultures requires the
addition of serum or IGF-I to the media, we cannot rule out the
possibility that suboptimal amounts of IGF-I may be produced by 293 cells maintained in serum-free conditions for the duration of the
transient transfection experiments described in these studies, ~12 h.
We concluded that ASK1 is phosphorylated on a tyrosine residue in a
manner dependent on IGF-IR activity and independent of exogenously
added IGF-I.
chain and a kinase-mutant form of ASK1-HA from
293 cells and performed in vitro kinase assays in the
presence of 32P-labeled
ATP. A kinase-mutant form of
ASK1-HA (ASK1KM-HA), which possesses no detectable kinase
activity (Fig. 2C, right panel), was labeled with
32P when IGF-IR or the kinase-competent intracellular
domain of the receptor's
-chain were present in the reaction (Fig.
2C, left panel), suggesting that IGF-IR can
phosphorylate ASK1 in vitro. Kinase-active ASK1-HA was
included in the experiments as a control for the kinase reaction and to
indicate the position of migration of 32P-labeled ASK1-HA
protein bands in gels.
-chain of
IGF-IR was present in complexes immunoprecipitated with an anti-ASK1
antibody in extracts from L929 cells maintained both in the presence
and in the absence of serum (Fig. 3,
panel A). A relative increase in the amount of IGF-IR
-chain present in complexes immunoprecipitated with anti-ASK1 was
observed in lysates from serum-starved L929 cells.
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Fig. 3.
ASK1 forms a complex with IGF-IR.
A, ASK1 was immunoprecipitated from L929 cells maintained in
the presence of 10% serum or deprived of serum and left untreated or
treated with IGF-I as described under "Experimental Procedures."
Complexes were resolved in gels and immunoblotted with anti-IGF-IR
antibodies (upper panel) or anti-ASK1 antibodies
(lower panel). B, lysates from 293 cells
transiently expressing the indicated proteins were subject to
immunoprecipitation with anti-HA antibodies. Complexes were resolved in
gels and immunoblotted with anti-IGF-IR antibodies (upper
panel) or anti-HA antibodies (lower panel).
C, lysates from 293 cells transiently expressing the
indicated proteins were subject to immunoprecipitation with anti-IGF-IR
antibodies. Complexes were resolved in gels and immunoblotted with
anti-HA or a-IGF-IR antibodies. The arrows indicate the
protein bands corresponding to both forms of the IGF-IR intracellular
domain, which comigrate with the IgG light chain. D, 293 cells transiently expressing the indicated proteins were fixed and
incubated with anti-ASK1 and anti-IGF-IR primary antibodies followed by
FITC- and Texas Red-conjugated secondary antibodies, respectively.
Samples were processed as described under "Experimental
Procedures." Images were acquired with a Nikon Eclipse-800
microscope, analyzed using Compix Simple PCI software and
deconvolved using a MLE algorithm as described under
"Experimental Procedures." Magnification is ×1000.
-chain, which encompasses its kinase domain. ASK1-HA was
transiently expressed in 293 cells together with IGF-IR or with
kinase-active and kinase-inactive forms of the IGF-IR
-chain
intracellular domain and reciprocal immunoprecipitations were
performed. Both a kinase-active and a kinase-inactive form of the
IGF-IR
-chain, as well as the full-length receptor, were found in
complexes immunoprecipitated with anti-HA antibodies (Fig. 3,
panel B). Conversely, complexes immunoprecipitated with anti-IGF-IR antibodies contained ASK1-HA (Fig. 3, panel C).
These results indicate that the interaction between ASK1 and the IGF-IR involves the cytoplasmic portion of the IGF-IR
-chain. Consistent with the constitutive nature of the interaction between the endogenous ASK1 and IGF-IR in L929 cells, the formation of IGF-IR/ASK1 complexes was not affected by mutation of the ATP-binding site in the IGF-IR kinase domain. It has been shown that the mechanism of ASK1 activation involves, in part, stimulus-dependent homo-oligomerization
(30). Thus, it is possible that the interaction with IGF-IR blocks ASK1 homo-oligomerization and activation, and may explain why kinase-mutant forms of IGF-IR partially retained the ability to inhibit ASK1 (Fig. 1,
panels C and D).
N-HA) (15) together with IGF-IR in 293 cells maintained in the
absence of serum. While full-length ASK1-HA became
phosphotyrosine immunoreactive in the presence of IGF-IR, no detectable
phosphorylated tyrosine residues were found in ASK1
N-HA,
irrespective of IGF-I stimulation (Fig. 4, panel A). Therefore, the
Tyr residue(s) that are phosphorylated in ASK1 by IGF-IR map to the
N-terminal 648 amino acids of the kinase. Both the IGF-IR and the
intracellular portion of its
-chain were detected in complexes
immunoprecipitated with ASK1
N-HA, suggesting that the N-terminal
domain of ASK1 is not required for its interaction with IGF-IR (data
not shown).
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Fig. 4.
The N-terminal regulatory domain of ASK1 is
Tyr-phosphorylated. Lysates from 293 cells transiently expressing
the indicated proteins were subject to immunoprecipitation using
anti-HA antibodies. The complexes were resolved in gels and
immunoblotted with antiphospho-Tyr (upper panel) or anti-HA
antibodies (lower panels).
and (b) activation of JNK1 by ASK1 in 293 cells
maintained in the absence of serum. IGF-IR-dependent inhibition of ASK1 activity was not affected by TNF
stimulation (25 versus 19% reduction in ASK1 activity, respectively). These results suggest that IGF-IR signaling can inhibit ASK1 irrespective of
TNF
stimulation in 293 cells (Fig. 5,
panel A). Whether IGF-IR blocks the
TRAF2-dependent activation of ASK1 or inhibits ASK1 downstream of its activation by TNF-R cannot be deduced from our data.
Consistent with the inhibition of ASK1 activity by IGF-IR, the
ASK1-dependent activation of JNK1 in 293 cells was
decreased in the presence of IGF-IR (Fig. 5, panel B).
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Fig. 5.
IGF-IR inhibits ASK1 irrespective of
TNF stimulation and decreases ASK1 signaling
to JNK1 in 293 cells. A, the activity of ASK1-HA
immunoprecipitated from lysates of 293 cells transiently expressing the
indicated proteins was determined in in vitro kinase assays
using [
-32P]ATP (upper panel). ASK1-HA
complexes were resolved in gels and immunoblotted as indicated
(lower panel). The specific activity of ASK1 (Sp
activity) was calculated as the ratio between 32P
incorporation and the densitometric values of ASK1 protein by
phosphorimager analysis of kinase assays and densitometry as described
under "Experimental Procedures" and multiplied by 100. B, JNK1 was immunoprecipitated from lysates of 293 cells
transiently expressing the indicated proteins. Complexes were resolved
in gels and immunoblotted using an antiphospho-JNK1 antibody
(upper panel) or an anti-JNK1 antibody (lower
panel). The specific activity of JNK1 was calculated as the ratio
between the densitometric values for antiphospho-JNK1 immunoreactivity
and anti-JNK1 immunoreactivity as described under "Experimental
Procedures" and multiplied by 100.
- and
H2O2-induced apoptosis (17) and
ASK1-dependent activation of JNK, and p38 has been shown to
mediate apoptosis induced by genotoxic stress (32). To determine whether the inhibition of ASK1 activity by IGF-IR has a role in the
modulation of ASK1-induced apoptosis, we examined the effect of IGF-IR
signaling in apoptosis induced by transient expression of ASK1-HA in
293 cells in serum-free conditions, as stimulation of endogenous IGF-IR
is sufficient to inhibit ASK1 activation (Fig. 1) and ASK1-induced cell
death. An increase in the percentage of cells with less than 2N DNA
content, indicative of apoptosis, was observed in cultures of 293 cells
transiently expressing ASK1-HA with respect to those transfected with
an empty vector in the absence of serum (Fig.
6). The presence of IGF-IR in cultures of
293 cells transiently expressing ASK1-HA in serum-free conditions completely abolished ASK-1-induced apoptosis. No significant difference in the percentage of apoptotic cells present in cultures expressing ASK1 and IGF-IR was observed when the cultures were maintained in the
presence of the PI3K inhibitor wortmannin. These results indicate that
IGF-IR can suppress ASK1-induced cell death independent of PI3K
activity.
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Fig. 6.
IGF-IR suppresses ASK1-induced
apoptosis. Percentages of apoptotic cells present in cultures of
293 cells expressing the indicated proteins were determined by PI
staining and flow cytometry as described under "Experimental
Procedures." p values were determined by Student's
t test.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(24). The mechanisms by which IGF-I signaling blocks the
stress-activated MAP kinase cascade, however, have not yet been elucidated.
-chain. We found that ASK1 was present in a complex with
the IGF-IR in a compartment juxtaposed to the nucleus in 293 cells
overexpressing both kinases. Ectopically expressed ASK1 also
colocalized with kinase-active and kinase-inactive myristylated forms
of the intracellular domain of the IGF-IR at the plasma membrane of 293 cells. Consistent with our observation that endogenous IGF-IR and ASK1
were found in complexes immunoprecipitated from L929 cells irrespective
of IGF-I or serum stimulation, the two kinases colocalized in
perinuclear structures reminiscent of ER in serum-stimulated or
serum-deprived L929 cells (data not shown).
stimulation did not affect IGF-IR
inhibition of ASK1. Whether IGF-IR blocked ASK1 activation or whether
the TNF
-stimulated ASK1 activity was inhibited by IGF-IR could not
be deduced from our data. On the other hand, the activation of JNK1 and
the induction of programmed cell death downstream of ASK1 activation
were inhibited by IGF-IR even when PI3K activity was blocked by wortmannin.
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ACKNOWLEDGEMENTS |
---|
We thank Roya Khosravi-Far for the pCMV-JNK1 vector and the Bredesen Laboratory for useful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant NS33376 from the NINDS, National Institutes of Health (to D. E. B.) and DOD/DAMD17-98-1-8613 from the United States Department of Defense.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 correspondence should be addressed: Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945-1400. Tel.: 415-209-2090; Fax: 415-209-2230; E-mail: dbredesen@buckinstitute.org.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M211398200
2 V. Galvan and D. E. Bredesen, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: IGF-IR, type 1 insulin-like growth factor receptor; HA, hemagglutinin; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; JNK, Jun N-terminal kinase; PI3K, phosphoinositide 3-kinase; TNF, tumor necrosis factor; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; ASK1, apoptosis signal-regulating kinase.
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