From the Program in Molecular Pharmacology and
Chemistry and the
Department of Clinical Chemistry, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021 and the
¶ Department of Pharmacology, Weill Graduate School of
Medical Sciences of Cornell University, New York, New York,
10021
Received for publication, December 9, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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Granulocyte-macrophage
colony-stimulating factor (GM-CSF) stimulates cellular glucose uptake
by decreasing the apparent Km for substrate
transport through facilitative glucose transporters on the plasma
membrane. Little is known about this signal transduction pathway and
the role of the Granulocyte-macrophage colony-stimulating factor
(GM-CSF)1 is an important
cytokine involved in the growth and maturation of hematopoietic cells
and in regulating host defense functions (1). The receptor for GM-CSF
is composed of two subunits, It has long been known that cytokines like IL-3 and GM-CSF modulate
glucose uptake in hematopoietic cells (19-24). Stimulation of cellular
glucose uptake by cytokines is believed to be part of their function as
survival factors by enhancing the availability of substrate for energy
generation required for cell metabolism (25). Non-hematopoietic cells
expressing high affinity GMR and melanoma cells expressing an isolated
Neoplastic transformation and viral infection increase cellular glucose
uptake through augmented facilitative glucose transporter (GLUT)
expression on the cell surface (30-32). Similarly insulin and certain
growth factors cause increased GLUT translocation to the cell membrane
that is dependent on activation of PI 3-kinase and its downstream
targets (33, 34). Up-regulation of glucose transport in a cell
model for chronic myeloid leukemia is also mediated via activation of
PI 3-kinase (35). The ability of GM-CSF to decrease the apparent
Km of GLUT1 without changing the
Vmax suggests that initial signaling from the
GMR results in modulation of the intrinsic properties of the
transporters (26, 36, 37). GM-CSF also signals for increased transport of the oxidized form of vitamin C, as dehydroascorbic acid, through GLUT1 by a similar mechanism (37). To elucidate this signaling process
and the role of the isolated Cell Lines and Culture Methods--
293T cells (38) were
maintained in high glucose Dulbecco's modified Eagle's medium
supplemented with 10% bovine calf serum, 1% sodium pyruvate, 1%
L-glutamine, and antibiotics. Neutrophilic and
promyelocytic HL-60 cells were maintained in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and antibiotics. Leukopacks from normal donor
blood were provided by the New York Blood Center, and the lymphocytes were purified by Ficoll-Hypaque density centrifugation followed by
plastic adherence depletion of monocytes/macrophages. U937 cells were
maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum.
In Vitro Transcription and RNA Injection--
Plasmid containing
human 2-Deoxy-D-glucose Uptake--
The uptake of
2-deoxy-D-glucose (DOG) by Xenopus oocytes was
measured as described previously (27). Briefly, oocytes (15 per group)
were incubated with GM-CSF (1 nM) (R&D Systems,
Minneapolis, MN) or the antibodies S20 (0.4 µg/ml) (recognizing an
extracellular epitope of Immunoprecipitation of Detection of p85 in HL-60--
Lysates from HL-60 cells with or
without treatment with GM-CSF were incubated with 1 µg of anti- Construction of Mutant Plasmids--
Human
Site-specific mutants were generated in the plasmid GM-CSF-stimulated Glucose Transport Depends on PI 3-Kinase--
We
investigated the role of PI 3-kinase in GM-CSF-stimulated transport of
glucose in Xenopus oocytes expressing the human
GM-CSF induces glucose uptake in human cells expressing the high
affinity receptors The p85 Regulatory Subunit of PI 3-Kinase Associates with
Identification of a p85-binding Motif in the Cytoplasmic Domain of
The Cytoplasmic Domain of
We addressed the question whether the residual glucose uptake induced
by GM-CSF through GM-CSF-induced Activation of PI 3-Kinase Involves Generation of
H2O2--
It is known that GM-CSF uses
reactive oxygen species (ROS) as second messenger molecules in its
signaling pathway (41, 42) and that ROS in the form of hydrogen
peroxide has a role in stimulating glucose uptake (43, 44). We
hypothesized that GM-CSF binding to the low affinity receptor generates
H2O2 extracellularly, and H2O2 diffuses through the plasma membrane to
activate PI 3-kinase. Oocytes expressing
We further tested the effect of wortmannin on
H2O2-induced glucose uptake in oocytes injected
with Monoclonal Antibodies to Antibody to
We propose a signaling pathway for GM-CSF-induced glucose uptake
whereby GM-CSF receptor interaction with ligand or antibody leads to
local generation of H2O2 that permeates the
cell membrane and activates PI 3-kinase signaling. This pathway is
illustrated in Fig. 8.
Hematopoietic cytokines stimulate the proliferation of precursor
cells and promote the survival and function of mature cells. These
processes require energy, and cytokines such as IL-3, lL-1, and GM-CSF
are known to enhance glucose transport in target cells (19, 20, 27,
46-48). Transport of glucose across the plasma membrane occurs
predominantly through facilitative GLUTs, and cellular glucose uptake
can be modulated by regulating the number of transporters on the cell
surface and/or by altering their transport efficiency (49-52). Both
GM-CSF and IL-3 enhance the intrinsic efficiency of GLUT1 as reflected
by a decrease in the Km without a change in maximum
velocity (Vmax) (36, 37). GM-CSF stimulates
glucose transport in Xenopus oocytes expressing the isolated
human In oocytes, as in mammalian cells, PI 3-kinase activation has been
implicated as an important signaling intermediate for
insulin-stimulated glucose uptake (53, 54). We hypothesized that PI
3-kinase was involved in the stimulation of glucose uptake by GM-CSF.
In oocytes expressing In several cell types insulin elicits rapid production of hydrogen
peroxide (44), and one of the subsequent cell responses to the
H2O2 production by insulin is increase in
glucose uptake (58). Generation of extracellular peroxide has been
demonstrated previously in fibroblasts and endothelial cells treated
with transforming growth factor- Lerner and colleagues (45) have provided strong evidence for
antigen-antibody interaction leading to generation of
H2O2 through catalysis of molecular oxygen and
H2O. We found that anti- The role of the subunit of the GM-CSF receptor (
GMR) in
modulating transporter activity. We examined the function of phosphatidylinositol 3-kinase (PI 3-kinase) in GM-CSF-stimulated glucose uptake and found that PI 3-kinase inhibitors, wortmannin and
LY294002, completely blocked the GM-CSF-dependent increase of glucose uptake in Xenopus oocytes expressing the low
affinity
GMR and in human cells expressing the high affinity
GMR complex. We identified a Src homology 3 domain-binding
motif in
GMR at residues 358-361 as a potential interaction
site for the PI 3-kinase regulatory subunit, p85. Physical evidence for
p85 binding to
GMR was obtained by co-immunoprecipitation with
antibodies to
GMR and p85, and an
GMR mutant with alteration of
the Src homology 3 binding domain lost the ability to bind p85.
Experiments with a construct eliminating most of the intracellular
portion of
GMR showed a 50% reduction in GM-CSF-stimulated glucose
uptake with residual activity blocked by wortmannin. Searching for a
proximally generated diffusible factor capable of activating PI
3-kinase, we identified hydrogen peroxide
(H2O2), generated by ligand or antibody binding
to
GMR, as the initiating factor. Catalase treatment abrogated
GM-CSF- or anti-
GMR antibody-stimulated glucose uptake in
GMR-expressing oocytes, and H2O2 activated
PI 3-kinase and led to some stimulation of glucose uptake in uninjected
oocytes. Human myeloid cell lines and primary explant human lymphocytes expressing high affinity GM-CSF receptors responded to
GMR antibody with increased glucose uptake. These results identify the early events
in the stimulation of glucose uptake by GM-CSF as involving local
H2O2 generation and requiring PI 3-kinase
activation. Our findings also provide a mechanistic explanation for
signaling through the isolated
subunit of the GM-CSF receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(
GMR) and
(
GMR), and
is found on hematopoietic cells, myeloid progenitors, mature
granulocytes, and mononuclear phagocytes (2, 3). The GM-CSF receptor is
also expressed on non-hematopoietic tissues including endothelial
cells, oligodendrocytes, placenta, spermatozoa, prostate, and various
immortalized cell lines (4-12). The isolated
GMR binds GM-CSF with
low affinity (Kd 2-5 nM) and forms a
complex with
GMR to create the high affinity receptor
(Kd 30 pM). While the GM-CSF, IL-5, and
IL-3 receptors share a common
chain (
GMR), the
subunit is
unique to each receptor and determines the binding specificity and the
distinct responses mediated by each ligand (13, 14). The
GMR subunit
plays a central role in GM-CSF cell signaling (15); however, the
subunit is also required for signaling mediated by the high affinity
receptor beyond its role in ligand binding (16, 17). A direct signaling function for an isolated
subunit was recently elucidated by the
finding that the
IL-5 receptor interacts with syntenin, inducing
IL-5-mediated transcriptional responses (18).
GMR also respond to GM-CSF by increasing glucose uptake (12, 26). We
previously found that Xenopus oocytes expressing only the
isolated
GMR respond to GM-CSF with increased glucose uptake (27).
The only physiological signaling role tentatively assigned to the low
affinity GM-CSF receptor, independent of the
GMR, is the regulation
of glucose uptake (22, 27). The in vivo relevance of GM-CSF
in mediating glucose uptake was demonstrated in the development of
preimplantation embryos in mice where an isolated
GMR is expressed
without
GMR. Signaling through this low affinity receptor is
associated with increased glucose uptake and enhanced proliferation and
viability of blastomeres (28). Studies with bone marrow from
GMR-null mice, however, have suggested that
GMR may be required
for GM-CSF signaling for glucose transport in mouse bone marrow cells
(29).
GMR, we undertook experiments in frog
oocytes expressing
GMR and human cells expressing both the
GMR
and
GMR subunits. The results reported here point to a central role
for PI 3-kinase in signaling for increased glucose uptake and indicate
that hydrogen peroxide generated proximally at the receptor is a key
initial signaling event.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GMR cDNA cloned in pBluescript (Stratagene) was linearized
with NotI. The digested DNA was extracted with phenol and
precipitated with ethanol, and RNA was transcribed in vitro
(Ambion, Austin, TX). Stage V and VI oocytes were harvested from the
ovaries of adult female Xenopus laevis frogs
(Nasco, Ft. Atkinson, WI) and cultured in OR2 (Bufferad Inc.) as
described previously (27). After 48 h of culture, 20 ng of capped
mRNA in 50 µl of diethyl pyrocarbonate water was
microinjected into the vegetal pole of the oocytes as described
previously (27). Glucose uptake experiments were performed 48 h
postinjection.
GMR mutants were generated in pcDNA4/His-V5c
(Invitrogen). RNA was generated by in vitro transcription of
these plasmids, linearized with XhoI, and injected as described.
GMR, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), 17A (0.4 µg/ml) (recognizing an extracellular epitope of
GMR, Sigma), or N20 (0.4 µg/ml) (recognizing the extracellular
portion of
GMR, Santa Cruz Biotechnology, Inc.) at room temperature
for 1 h. Oocytes were incubated with
2-deoxy-D-[1,2-3H]glucose (PerkinElmer Life
Sciences) and DOG to a final concentration of 0.1-0.4 µCi/ml
and 0.2 mM, respectively. After 10 min at room temperature,
DOG uptake was terminated by dilution with OR2, and the oocytes were
washed with cold OR2 medium four times. Oocytes were then lysed in 20 mM Tris-HCl (pH 8.0) containing 0.2% SDS, and the amount
of radioactive DOG trapped intracellularly was measured by
scintillation counting. The uptake of DOG in HL-60, U937 cells, and
human lymphocytes was measured as described above. Catalase
(Sigma) was added to a final concentration of 1900 or 3800 units/ml for
5 min before GM-CSF treatment.
GMR and
GMR from Transfected 293T
Cells--
293T cells were transfected using Superfect (Qiagen) in
100-mm plates at 50% confluence with 5 µg of plasmids for
GMR,
GMR-PA,
GMR-A351, or
GMR-YA according to the manufacturer's
instructions. Forty-eight hours after transfection the cells were
harvested and lysed by sonication in lysis buffer (20 mM
Tris, 150 mM NaCl, 0.1% Triton X-100) containing protease
and phosphatase inhibitor mixtures (Sigma). For immunoprecipitation the
samples were centrifuged at 12,000 × g for 10 min at
4 °C, and the cleared lysates were further treated with protein
A-Sepharose beads (Sigma). Anti-
GMR antibody S20 was added at 1 µg
per sample for 2 h, and the antibodies were captured on protein
A-Sepharose beads for 2 h at 4 °C. The beads were washed with
lysis buffer four times, and the proteins were analyzed by
immunoblotting. Anti-
GMR antibodies C18 and S50 and anti-p85
antibody were used for detection of
GMR and p85, respectively.
GMR
antibody (S20) for 16 h at 4 °C on an oscillating platform.
Protein G-agarose (20 µg) was then added and left for 1 h at
4 °C. The protein G beads were collected by centrifugation and
washed three times with lysis buffer. The supernatants were removed
carefully, and the beads were resuspended with 1× sample loading
buffer and heated for 5 min at 95 °C before separation by 7.5%
SDS-PAGE. Western blotting to analyze the PI 3-kinase (p85) was
performed in the following manner. Nonspecific sites were blocked using
5% nonfat milk in Tris-buffered saline before incubating the blot with
anti-p85 antibody (Santa Cruz Biotechnology, Inc.) for 16 h at
4 °C at a final dilution of 1:1000 in fresh blocking buffer. The
membrane was washed three times in Tris-buffered saline, incubated for 1 h at room temperature with anti-mouse IgG, horseradish
peroxidase-linked whole antibody (Amersham Biosciences), diluted 1:4000
in blocking buffer, and washed with Tris-buffered saline and 0.1%
Tween 20, and proteins were revealed using the ECL detection
system (Amersham Biosciences).
GMR was subcloned
into HindIII and NotI sites of pcDNA4/His-V5c
(Invitrogen) using the following 5' primer:
GCCGCAAGCTTAGCACCATGCTTCTCCTGGT GACA-3'. The reverse primer for
full-length
GMR was GATAGTTTAGCGGCCGCGGTAATTTCCTTCACGGT to generate
plasmid
GMR. The reverse primer for the carboxyl-terminal deletion
GMR-A351 was 5'-TTTAAAAGGTTCCTTAGGGCGGCCGCTAAACTATC-3'.
GMR using a
mutagenesis kit (Stratagene). Briefly,
GMR-containing plasmid was
denatured and annealed with oligonucleotide primers containing the
desired mutation. The following primers were used for proline substitution by alanine: forward primer,
5'-CTTAGGATACAGCGGCTGTTCGCAGCAGTTGCACAGATCAAAGACAACTGAAT-3'; reverse primer, 5'-ATTCAGTTTGTCTTTGATCTGTGCAACTGCTGCGAACA GCCGCTGTA TCCTAAG-3'. For tyrosine substitution by alanine the forward primer was
5'-ATCATCTGGGAGGAATTCACCCCAGAGGAAGGGAAAGGCGCACGCGAAGAGGT-3', and the reverse primer was 5'-GACCTCTTCGCGTGCGCCTTTCCCTTCCTCTGGGGTGAAT TCCTCCCAGATGAT-3'. The mutagenic primers were incorporated using Pfu Turbo DNA polymerase. The non-mutagenic methylated
parental DNA template was digested with DpnI. The resultant
circular nicked double-stranded DNA was transformed into XL10-Gold
ultra competent cells (Stratagene). The mutation was confirmed
by sequencing of the resultant plasmids.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GMR.
Oocytes injected with
GMR RNA expressed
GMR as detected by
immunoblotting using an anti-
GMR antibody, C18 (Fig.
1A). Reverse transcription PCR
analysis indicated that Xenopus oocytes do not
express a
GMR transcript detectable by primers specific for human
GMR (data not shown). Also
GMR protein was not detectable by
immunoblotting using anti-human
subunit antibody. These results are
consistent with our previous finding that oocytes expressing the
GMR
had only low affinity GM-CSF binding. Oocytes expressing the
GMR and
incubated with 1 nM GM-CSF for 1 h showed a 2-fold increase in glucose uptake (Fig. 1B) (27). The increase in
glucose uptake was completely inhibited by incubation with 100 nM wortmannin for 15 min prior to GM-CSF treatment, while
the vehicle control buffer had no effect (Fig. 1B).
Wortmannin is a highly specific inhibitor of PI 3-kinase (39), and the
inhibitory effect of wortmannin was dose-dependent and did
not affect the basal level of glucose transport. The IC50
for inhibition of GM-CSF-stimulated glucose uptake by wortmannin was 2 nM (Fig. 1C). LY294002, a structurally distinct
synthetic inhibitor of PI 3-kinase, also suppressed GM-CSF-stimulated glucose uptake. At 50 µM, LY294002 completely inhibited
GM-CSF-induced glucose uptake with no effect on basal glucose transport
(Fig. 1D). In these experiments the vehicle control
(ethanol) slightly inhibited the stimulation by GM-CSF, but the effect
of LY294002 was dose-dependent with IC50 of 2 µM. These results suggest that PI 3-kinase is involved in
GMR-dependent signaling for glucose transport.
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Fig. 1.
PI 3-kinase inhibitors block
GM-CSF-dependent glucose uptake in Xenopus
oocytes. A, expression of GMR in oocytes
injected with RNA encoding
GMR. Extracts from oocytes were subjected
to SDS-PAGE, and
GMR was detected by immunoblotting with anti-
GMR
antibody C18. B, wortmannin inhibits glucose uptake induced
by GM-CSF. Oocytes expressing
GMR were incubated with 100 nM wortmannin or Me2SO (DMSO)
for 15 min prior to GM-CSF treatment. Uptake of DOG by oocytes was
quantified after 10 min. The closed bars indicate
oocytes incubated with 1 nM GM-CSF. C,
dose-dependent inhibition of [3H]DOG uptake
by wortmannin. DOG uptake is expressed as percentage of increase of
basal uptake. Data represent the average of triplicate experiments, and
standard deviation is indicated. D, LY294002 inhibits
glucose uptake induced by GM-CSF. Oocytes expressing
GMR were
preincubated with LY294002 or vehicle (ethanol) prior to GM-CSF
treatment. DOG uptake was quantitated 10 min after and is expressed as
pmol/oocytes. The closed bars indicate oocytes incubated
with 1 nM GM-CSF. Standard deviation of triplicate
experiments is indicated. E, dose-dependent
inhibition of [3DOG] uptake by LY294002. DOG uptake is
expressed as percentage of increase of basal uptake. Data represent the
average of triplicate experiments, and standard deviation is indicated.
F, wortmannin inhibits GM-CSF-dependent glucose
uptake in neutrophilic HL-60 cells. HL-60 cells were treated with 50 nM wortmannin prior to GM-CSF treatment for 1 h. DOG
uptake was quantitated 10 min after GM-CSF stimulation. Data are
expressed as -fold induction. Data represent the average of
experiments in triplicate, and standard deviation is indicated.
GMR and
GMR (27). We evaluated the effect of the PI 3-kinase inhibitor wortmannin on GM-CSF-induced glucose uptake in neutrophilic HL-60 cells (40). Cells treated with 1 nM GM-CSF showed a 2.1-fold increase in glucose uptake that
was completely inhibited by 50 nM wortmannin (Fig.
1F). These results suggest that signaling for
GM-CSF-stimulated glucose uptake in cells expressing the high affinity
receptor is also PI 3-kinase-dependent.
GMR--
To investigate the mechanism of activation of PI
3-kinase pathway by
GMR we performed experiments to determine
whether the regulatory subunit of PI 3-kinase (p85) interacts with
GMR. The
GMR monoclonal antibody S20 was used to
immunoprecipitate cellular extracts from HL-60 cells treated or
untreated with GM-CSF. The immunoprecipitates were analyzed by SDS-PAGE
and immunoblotted with anti-p85 antibody. As shown in Fig.
2A, there is a
ligand-independent interaction between
GMR and p85. The interaction
between
GMR and p85 was confirmed by co-immunoprecipitation with
an anti-p85 antibody and detected by immunoblotting with an anti-
GMR
antibody (Fig. 2B). These results indicate a physical
interaction between PI 3-kinase and
GMR.
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Fig. 2.
GMR interacts with PI
3-kinase. A, cell lysates from HL-60 cells incubated
with 1 nM GM-CSF for the period of time indicated were
immunoprecipitated with anti-
GMR (S20), separated by SDS-PAGE, and
immunoblotted with anti-p85 antibody. B, cell lysates from
HL-60 cells incubated with 1 nM GM-CSF for the time
indicated were immunoprecipitated with anti-p85 antibody, separated by
SDS-PAGE, and immunoblotted with anti-
GMR antibody.
IP, immunoprecipitation; WB, Western
blot.
GMR--
p85 associates with
GMR whose cytoplasmic domain has 54 amino acid residues. We identified an SH-3 domain-binding motif
consisting of a proline-rich segment (PPVP) proximal to the
transmembrane domain of the
GMR that could potentially be an
anchoring site for p85. Distal to this region near the carboxyl
terminus is the only tyrosine residue in the carboxyl terminus. To
investigate the role of these residues, alanine substitution mutations
were introduced whereby proline-rich SH-3 domain-binding region (PPVP) was changed (AAVA) using site-directed mutagenesis to generate the
plasmid
GMR-PA, and the tyrosine residue was mutated (Y389A) to
generate the plasmid
GMR-YA. We also generated a truncation mutant,
GMR-A351, that carries a deletion of the cytoplasmic domain
retaining only four amino acid residues proximal to the transmembrane
domain. A schematic representation of the
GMR constructs is shown in
Fig. 3A. Wild type or mutant
plasmid cDNA was transfected into 293T cells, total cell lysates
were subjected to immunoprecipitation with anti-
GMR antibody (S20),
and the immunoprecipitated proteins were analyzed by SDS-PAGE and
immunoblotted using anti-p85 antibody. While the wild type receptor
showed binding to p85, there was loss of binding to the truncation
mutant
GMR-A351 and to the
GMR-PA (Fig. 3B, top
panel). The mutant
GMR-YA, however, retained binding,
suggesting that the proline motif is critical for binding to p85 in
quiescent cells. The membrane was stripped and reprobed with
anti-
GMR antibodies. C18 antibody recognizes an intracellular epitope and therefore does not recognize the truncation mutant
GMR-A351. Antibody S50 against an extracellular epitope was used to
detect the expression of all the constructs (Fig. 3B,
lower panel). These results point to the proline-rich motif
as the site of p85 binding to
GMR.
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Fig. 3.
Identification of p85 binding domains in
GMR. A, schematic representation of
GMR mutants. B, cell lysates from 293T cells transfected
with wild type and mutant
GMR were immunoprecipitated with S20
antibody, and the immunoprecipitates were analyzed by Western blotting
using anti-p85 antibody (upper panel). The blot was stripped
and reprobed with anti-
GMR antibody S50 (lower panel) and
with anti-
GMR antibody C18 (middle panel). IP,
immunoprecipitation; WB, Western blot.
GMR Is Important but Not Essential
for GM-CSF-stimulated Glucose Uptake in Oocytes--
RNA from the wild
type (
GMR) and mutant plasmids
GMR-A351,
GMR-PA, and
GMR-YA
was injected into oocytes and tested for the ability to mediate
increased glucose uptake in response to GM-CSF (Fig.
4A). GM-CSF-treated oocytes
expressing wild type
GMR showed a 2.5-fold increase in glucose
uptake; however, oocytes injected with
GMR RNA alone did not respond
to GM-CSF. GM-CSF-stimulated glucose uptake in oocytes expressing the
GMR mutant
GMR-PA was decreased but not eliminated (Fig.
4A). When the truncation mutant of the cytoplasmic domain of
GMR-A351 was used in the same experiment there was approximately a
50% decrease in the stimulated glucose uptake compared with the wild
type receptor. The
GMR-YA mutant mediated a moderately attenuated
glucose uptake compared with the wild type
GMR (Fig. 4A).
The expression levels of the different
GMR constructs were
comparable (data not shown).
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Fig. 4.
Disruption of GMR
function by mutations in the cytoplasmic domain. A,
oocytes injected with wild type
GMR or mutants
GMR-A351,
GMR-PA, and
GMR-YA were treated with GM-CSF, and glucose uptake
was measured as explained under "Experimental Procedures."
B, oocytes injected with wild type
GMR or mutants
GMR-A351 were treated with 0.1, 1, and 10 nM wortmannin
15 min before GM-CSF stimulation. DOG uptake is shown as pmol/oocyte.
wt, wild type.
GMR-A351 is dependent on PI 3-kinase. Oocytes
injected with
GMR-A351 and wild type
GMR were preincubated with
wortmannin and treated with GM-CSF. As shown in Fig. 4B
wortmannin inhibited the stimulation of glucose uptake in the
truncation mutant. These results indicate that GM-CSF-stimulated
glucose uptake is attenuated by deletion of the intracellular domain of
GMR but that the residual activity is also dependent on PI 3-kinase. These results imply that there is PI 3-kinase activation by GM-CSF even
when p85 cannot bind to
GMR.
GMR or the truncation
mutant receptor
GMR-A351 were incubated with different
concentrations of catalase for 5 min before treatment with GM-CSF. As
shown in the Fig. 5A catalase markedly inhibited GM-CSF-stimulated glucose uptake indicating a key
role for H2O2 in stimulating glucose transport.
To further establish the role of H2O2 in
stimulated glucose uptake we directly tested the effect of peroxide on
glucose uptake in control oocytes and oocytes expressing
GMR
constructs. Exposure to 50 µM
H2O2 for 30 min caused about 2-fold stimulation
of glucose uptake in oocytes expressing
GMR (Fig. 5B).
Oocytes injected with
GMR-A351 showed only a 1.3-fold activation in
response to H2O2, a result similar to that in
uninjected oocytes (Fig. 5B). These results indicate a role
for H2O2 in stimulating glucose transport and show that
GMR plays an important but not essential role in mediating the response. The activation of glucose transport in the truncation mutant is similar to uninjected oocytes indicating that PI 3-kinase can
be activated by H2O2 in oocytes.
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Fig. 5.
Role of peroxide in GM-CS-induced glucose
uptake. A, oocytes injected with wild type GMR or
mutant
GMR-A351 were pretreated with 1900 units of catalase
(1×) or 3800 units of catalase (2×) prior to
GM-CSF stimulation. Experiments were done in triplicate, and the
standard deviation is indicated. B, oocytes injected with
wild type
GMR or mutant
GMR-A351 or not injected were stimulated
with 50 µM H2O2 and then assayed
for DOG uptake as described under "Experimental Procedures."
C, oocytes injected with wild type
GMR or mutants
GMR-A351 or not injected were stimulated with 50 µM
H2O2 in the presence or absence of 25 nM wortmannin and then assayed for DOG uptake as described
under "Experimental Procedures." wt, wild type.
GMR or the truncation mutant
GMR-A351. As in
GM-CSF-stimulated oocytes, the peroxide-induced stimulation of glucose
uptake in oocytes expressing wild type or mutant
GMR was inhibited
by 25 nM wortmannin (Fig. 5C). These findings
link peroxide generation to PI 3-kinase activation and indicate the
role of p85 binding site in
GMR in the stimulation of glucose uptake.
GMR Stimulate Glucose
Transport--
It has been shown that antibody-antigen interaction can
lead to the generation of hydrogen peroxide (45). We reasoned that, similar to ligand, antibody to
GMR could lead to the generation of
H2O2 and stimulation of glucose uptake. We
investigated whether the commercially available anti-
GMR antibodies
S20 and 17A, which recognize the extracellular region of
GMR, could
stimulate glucose uptake. Fig.
6A shows that anti-
GMR
monoclonal antibodies S20 and 17A stimulated glucose transport about
2-fold in oocytes injected with
GMR. In contrast, the polyclonal
antibody N20 that recognizes an extracellular epitope of
GMR had no
effect. Oocytes injected with H2O and incubated with
GM-CSF, S20, 17A, and N20 antibodies showed no stimulation of glucose
uptake. Oocytes expressing
GMR and incubated with the antibodies 17A
and S20 for 15 min showed an increase in glucose uptake of about
2-fold. After 60 min of incubation, similar levels of stimulation of
glucose uptake were induced by the antibodies and GM-CSF (Fig.
6B). The stimulation of glucose uptake by S20 antibody was
completely inhibited by wortmannin (Fig. 6C), indicating
that PI 3-kinase is required for antibody- as well as GM-CSF-induced
glucose transport. Treatment of
GMR-injected oocyte with catalase
also abrogated antibody-induced increase in glucose uptake (Fig.
6C). These results indicate that antibody or ligand
interaction with
GMR on oocytes leads to increased glucose uptake
that is dependent on the generation of peroxide and subsequent
activation of PI 3-kinase.
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Fig. 6.
Antibodies against the extracellular portion
of the GMR stimulate glucose transport.
A, uptake of DOG by oocytes. Oocytes were injected with
either RNA encoding the
GMR or water (control). After 48 h at
room temperature the oocytes were incubated for 1 h with 1 nM GM-CSF (GM) or 0.4 µg/ml antibodies as
indicated. Uptake of DOG was quantified after 10 min. B,
time course effect of GM-CSF and antibodies on DOG uptake in oocytes
expressing
GMR. Oocytes injected with RNA encoding the
GMR were
incubated with the antibodies indicated for 15, 30, and 60 min. The DOG
uptake was determined over a 10-min period. C, effect of
wortmannin and catalase on monoclonal antibody S20-induced glucose
uptake. Oocytes injected with wild type
GMR were pretreated with
3800 units of catalase (Cat) or 50 nM wortmannin
(W) before treatment with GM-CSF or monoclonal antibody
S20.
GMR Stimulates Glucose Uptake in Mammalian
Cells--
To extend the oocyte experiments we investigated the effect
of anti-
GMR antibodies on human cells expressing the high affinity receptor. Similar to the results in oocytes, we found that antibodies to the
subunit (S20 and 17A) stimulated glucose uptake in the promyelocytic HL-60 cell line and in the monocytic cell line U937 (Fig.
7, A and B). The
antibody N20, which reacts with the
subunit, had no effect on
glucose uptake. HL-60 cells treated with anti-
GMR antibodies showed
a 20% increase in glucose uptake. In U937 cells, GM-CSF and the
anti-
GMR antibodies stimulated glucose uptake to comparable levels.
We also tested the effect of the antibodies on freshly isolated
peripheral blood lymphocytes and observed an increase in glucose uptake
in response to GM-CSF and antibodies S20 and 17A but not with antibody
N20 (Fig. 7C).
View larger version (16K):
[in a new window]
Fig. 7.
Antibodies against the
GMR signal for increased DOG uptake in human
cells. Cells were incubated with GM-CSF (GM) and the
antibodies for 1 h, and uptake of DOG was measured with 2-min
uptake time. A, promyelocytic HL-60 cell line. B,
monocytic U937 cell line. C, normal blood lymphocytes. DOG
uptake is expressed as percentage of increase over basal uptake.
View larger version (23K):
[in a new window]
Fig. 8.
Schematic representation of the mechanisms of
GM-CSF-dependent glucose uptake. GM-CSF binding to
GMR induces glucose uptake by changing the affinity of facilitative
glucose transporters. The initial step for glucose signaling involves
H2O2 production followed by activation of the
PI 3-kinase signaling pathway, which is inhibited by wortmannin
and LY294002. TM, transmembrane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GMR, HL-60 neutrophilic cells expressing the
GMR heterodimer, and certain other cell types responsive to GM-CSF (12, 26,
27, 36). Little is known, however, about how GM-CSF signals for
increased glucose uptake, and there is controversy regarding the
requirement for
GMR (29). Signaling from the isolated
GMR for
glucose uptake in oocytes does not involve activation of the
mitogen-activated protein kinase pathway, and GM-CSF stimulation of glucose uptake in HL-60 neutrophilic cells does not appear to
involve activation of kinase pathways that are inhibitable by
staurosporine (27). More recently the in vivo relevance of GM-CSF-stimulated glucose uptake through the low affinity
GMR has
been described in the development of preimplantation embryos in mice.
These embryos express an isolated
GMR, and there is increased
glucose uptake and enhanced proliferation and viability of blastomeres
in response to GM-CSF (28). We investigated the initial signaling
events of GM-CSF-stimulated glucose uptake and sought to
mechanistically define a role for the isolated
GMR.
GMR, GM-CSF-stimulated glucose uptake was inhibited by wortmannin and a structurally distinct inhibitor of PI
3-kinase, LY294002, in a dose-dependent manner.
GM-CSF-stimulated glucose uptake in HL-60 cells expressing the high
affinity receptor was also blocked by wortmannin. The inhibitor results
therefore implicate the involvement of PI 3-kinase in the stimulation
of glucose uptake signaled through
GMR and the
GMR high
affinity receptor. We also found clear evidence for a physical
interaction between
GMR and p85. In searching for potential domains
of
GMR that interact with p85, we examined an SH-3 domain-binding
motif, QRLFPPVP, that is conserved among the GM-CSF, IL-3, and IL-5
receptors in the intracellular membrane proximal region (17, 55). A tyrosine residue toward the carboxyl end of
GMR (amino acid residue 389) is not in the context of a consensus motif used for p85 binding by
certain receptors (56, 57). Mutation of the SH-3 domain-binding site
abrogated the interaction of p85 to
GMR as did a mutation deleting most of the cytoplasmic region of the
GMR. Oocytes
expressing these mutants showed GM-CSF-stimulated glucose uptake of
about 50% of wild type
GMR. This result suggested that interaction of p85 with the SH-3 domain-binding motif, while required for PI
3-kinase binding, was only partly responsible for GM-CSF-induced glucose uptake. The residual activity of the mutants was dependent on
PI 3-kinase activation as it was completely blocked by wortmannin.
and ROS-generating systems such as
glucose/glucose oxidase or xanthine/xanthine oxidase can increase
glucose uptake in 3T3-L1 adipocytes and L6 myotubes (43). Treatment
with peroxidase in these cases inhibited the increase in glucose uptake
indicating that extracellular H2O2 was
connected to stimulation of glucose transport (43). In addition, GM-CSF
is known to use ROS for signaling (41, 42), and ROS can act as a second
messenger in cellular signaling by modifying the activity of
redox-sensitive enzymes including kinases and phosphatases (59-61). We
found that catalase and PI 3-kinase inhibitors markedly decreased
GM-CSF-stimulated glucose uptake in oocytes expressing wild type or
truncated
GMR. Hydrogen peroxide also induced PI
3-kinase-dependent glucose uptake in oocytes expressing
wild type and to a lesser extent the truncated
GMR.
H2O2 had some activity in stimulating glucose
uptake in oocytes not expressing
GMR. The experiments in oocytes
expressing
GMR yielded results consistent with the thesis that
GM-CSF binding to the low affinity receptor generates
H2O2, which then stimulates glucose uptake via
the activation of PI 3-kinase.
GMR antibody stimulated glucose
transport in Xenopus oocytes expressing
GMR and also in
HL-60 cells, U937, and peripheral blood lymphocytes in a
wortmannin-inhibitable manner. Antibody to
GMR induced glucose uptake that was blocked by catalase, suggesting that peroxide production was mediating induction of glucose uptake.
subunit of the GM-CSF receptor in signal
transduction through the high affinity receptor has been illuminated by
mutational and structural studies (55, 62-64); however, there has been
controversy regarding the ability of the isolated
GMR to signal
(29). The results reported here define a signaling system for enhanced
glucose uptake dependent on H2O2 generation leading to the activation of PI 3-kinase. The most proximal initiation appears to be peroxide generated from ligand or antibody interaction with the extracellular domain of
GMR. Permeation of peroxide leads
to PI 3-kinase activation, which is facilitated by an SH-3 domain-binding motif in the cytoplasmic portion of the
GMR. Since most of the intracellular portion of
GMR is not absolutely required for PI 3-kinase activation, we envision that such activation could occur at a proximal cell membrane location. PI 3-kinase initiates well
known signaling pathways; however, it is not known how it causes
increased transport by reducing Km. Since vitamin C
is transported as dehydroascorbic acid through GLUT1 and its uptake is
stimulated by GM-CSF (37), the GM-CSF-stimulated PI 3-kinase pathways
can operate to enhance cellular antioxidant function as well as provide
increased metabolic substrate for cellular function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Isabel Perez-Cruz for isolation of lymphocytes from blood and Oriana Bórquez-Ojeda and Alicia Pedraza for technical help.
![]() |
FOOTNOTES |
---|
* This study was supported by National Institutes of Health Grants CA30388 and 2P30 CA08748-28, New York State Department of Health Grant M010283, the Schultz Foundation, and the Lebensfeld Foundation.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.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 212-639-8483; Fax: 212-772-8589; E-mail: d-golde@ski.mskcc.org.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M212541200
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ABBREVIATIONS |
---|
The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; GMR, GM-CSF receptor; PI, phosphatidylinositol; SH-3, Src homology 3; GLUT, glucose transporter; DOG, 2-deoxy-D-glucose; ROS, reactive oxygen species; IL, interleukin.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gasson, J. C. (1991) Blood 77, 1131-1145[Medline] [Order article via Infotrieve] |
2. |
McKinstry, W. J.,
Li, C. L.,
Rasko, J. E.,
Nicola, N. A.,
Johnson, G. R.,
and Metcalf, D.
(1997)
Blood
89,
65-71 |
3. | Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K., Yokota, T., and Miyajima, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9655-9659[Abstract] |
4. | Bussolino, F., Wang, J. M., Defilippi, P., Turrini, F., Sanavio, F., Edgell, C. J., Aglietta, M., Arese, P., and Mantovani, A. (1989) Nature 337, 471-473[CrossRef][Medline] [Order article via Infotrieve] |
5. | Avalos, B. R., Gasson, J. C., Hedvat, C., Quan, S. G., Baldwin, G. C., Weisbart, R. H., Williams, R. E., Golde, D. W., and DiPersio, J. F. (1990) Blood 75, 851-857[Abstract] |
6. | Baldwin, G. C., Gasson, J. C., Kaufman, S. E., Quan, S. G., Williams, R. E., Avalos, B. R., Gazdar, A. F., Golde, D. W., and DiPersio, J. F. (1989) Blood 73, 1033-1037[Abstract] |
7. | Baldwin, G. C., Golde, D. W., Widhopf, G. F., Economou, J., and Gasson, J. C. (1991) Blood 78, 609-615[Abstract] |
8. | Taketazu, F., Chiba, S., Shibuya, K., Kuwaki, T., Tsumura, H., Miyazono, K., Miyagawa, K., and Takaku, F. (1991) J. Cell. Physiol. 146, 251-257[Medline] [Order article via Infotrieve] |
9. |
Lee, S. C.,
Liu, W.,
Roth, P.,
Dickson, D. W.,
Berman, J. W.,
and Brosnan, C. F.
(1993)
J. Immunol.
150,
594-604 |
10. |
Rivas, C. I.,
Vera, J. C.,
Delgado-Lopez, F.,
Heaney, M. L.,
Guaiquil, V. H.,
Zhang, R. H.,
Scher, H. I.,
Concha, I. I.,
Nualart, F.,
Cordon-Cardo, C.,
and Golde, D. W.
(1998)
Blood
91,
1037-1043 |
11. | Hirsch, T., Eggstein, S., Frank, S., Farthmann, E. H., and von Specht, B. U. (1995) Biochem. Biophys. Res. Commun. 217, 138-143[CrossRef][Medline] [Order article via Infotrieve] |
12. | Zambrano, A., Noli, C., Rauch, M. C., Werner, E., Brito, M., Amthauer, R., Slebe, J. C., Vera, J. C., and Concha, I. I. (2001) J. Cell. Biochem. 80, 625-634[CrossRef][Medline] [Order article via Infotrieve] |
13. | Scheid, M. P., Lauener, R. W., and Duronio, V. (1995) Biochem. J. 312, 159-162[Medline] [Order article via Infotrieve] |
14. | Geijsen, N., Koenderman, L., and Coffer, P. J. (2001) Cytokine Growth Factor Rev. 12, 19-25[CrossRef][Medline] [Order article via Infotrieve] |
15. | Miyajima, A., Mui, A. L., Ogorochi, T., and Sakamaki, K. (1993) Blood 82, 1960-1974[Medline] [Order article via Infotrieve] |
16. | Polotskaya, A., Zhao, Y., Lilly, M. L., and Kraft, A. S. (1993) Cell Growth & Differ. 4, 523-531[Abstract] |
17. | Moon, B. G., Yoshida, T., Shiiba, M., Nakao, K., Katsuki, M., Takaki, S., and Takatsu, K. (2001) Immunology 102, 289-300[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Geijsen, N.,
Uings, I. J.,
Pals, C.,
Armstrong, J.,
McKinnon, M.,
Raaijmakers, J. A.,
Lammers, J. W.,
Koenderman, L.,
and Coffer, P. J.
(2001)
Science
293,
1136-1138 |
19. | Whetton, A. D., Bazill, G. W., and Dexter, T. M. (1985) J. Cell. Physiol. 123, 73-78[Medline] [Order article via Infotrieve] |
20. | Nefesh, I., Bauskin, A. R., Alkalay, I., Golembo, M., and Ben-Neriah, Y. (1991) Int. Immunol. 3, 827-831[Abstract] |
21. | Dexter, T. M., Whetton, A. D., and Bazill, G. W. (1984) Differentiation 27, 163-167[Medline] [Order article via Infotrieve] |
22. |
Tan, A. S.,
Ahmed, N.,
and Berridge, M. V.
(1998)
Blood
91,
649-655 |
23. | Berridge, M. V., and Tan, A. S. (1995) Biochem. J. 305, 843-851[Medline] [Order article via Infotrieve] |
24. |
McCoy, K. D.,
Ahmed, N.,
Tan, A. S.,
and Berridge, M. V.
(1997)
J. Biol. Chem.
272,
17276-17282 |
25. |
Vander Heiden, M. G.,
Plas, D. R.,
Rathmell, J. C.,
Fox, C. J.,
Harris, M. H.,
and Thompson, C. B.
(2001)
Mol. Cell. Biol.
21,
5899-5912 |
26. |
Spielholz, C.,
Heaney, M. L.,
Morrison, M. E.,
Houghton, A. N.,
Vera, J. C.,
and Golde, D. W.
(1995)
Blood
85,
973-980 |
27. | Ding, D. X., Rivas, C. I., Heaney, M. L., Raines, M. A., Vera, J. C., and Golde, D. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2537-2541[Abstract] |
28. |
Robertson, S. A.,
Sjoblom, C.,
Jasper, M. J.,
Norman, R. J.,
and Seamark, R. F.
(2001)
Biol. Reprod.
64,
1206-1215 |
29. |
Scott, C. L.,
Hughes, D. A.,
Cary, D.,
Nicola, N. A.,
Begley, C. G.,
and Robb, L.
(1998)
Blood
92,
4119-4127 |
30. | Medina, R. A., and Owen, G. I. (2002) Biol. Res. 35, 9-26[Medline] [Order article via Infotrieve] |
31. | Siegert, W., and Monch, T. (1981) Blut 43, 297-305[Medline] [Order article via Infotrieve] |
32. | Corkey, R. F., Corkey, B. E., and Gimbrone, M. A., Jr. (1981) J. Cell. Physiol. 106, 425-434[Medline] [Order article via Infotrieve] |
33. |
Ishizuka, T.,
Kajita, K.,
Miura, A.,
Ishizawa, M.,
Kanoh, Y.,
Itaya, S.,
Kimura, M.,
Muto, N.,
Mune, T.,
Morita, H.,
and Yasuda, K.
(1999)
Am. J. Physiol.
276,
E196-E204 |
34. |
Egawa, K.,
Sharma, P. M.,
Nakashima, N.,
Huang, Y.,
Huver, E.,
Boss, G. R.,
and Olefsky, J. M.
(1999)
J. Biol. Chem.
274,
14306-14314 |
35. | Bentley, J., Walker, I., McIntosh, E., Whetton, A. D., Owen-Lynch, P. J., and Baldwin, S. A. (2001) Br. J. Haematol. 112, 212-215[CrossRef][Medline] [Order article via Infotrieve] |
36. | Ahmed, N., Kansara, M., and Berridge, M. V. (1997) Biochem. J. 327, 369-375[Medline] [Order article via Infotrieve] |
37. |
Vera, J. C.,
Rivas, C. I.,
Zhang, R. H.,
and Golde, D. W.
(1998)
Blood
91,
2536-2546 |
38. |
Pear, W. S.,
Nolan, G. P.,
Scott, M. L.,
and Baltimore, D.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8392-8396 |
39. | Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Biochem. J. 351, 95-105[CrossRef][Medline] [Order article via Infotrieve] |
40. | Tomonaga, M., Gasson, J. C., Quan, S. G., and Golde, D. W. (1986) Blood 67, 1433-1441[Abstract] |
41. |
Carcamo, J. M.,
Borquez-Ojeda, O.,
and Golde, D. W.
(2002)
Blood
99,
3205-3212 |
42. |
Sattler, M.,
Winkler, T.,
Verma, S.,
Byrne, C. H.,
Shrikhande, G.,
Salgia, R.,
and Griffin, J. D.
(1999)
Blood
93,
2928-2935 |
43. | Kozlovsky, N., Rudich, A., Potashnik, R., and Bashan, N. (1997) Free Radic. Biol. Med. 23, 859-869[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Mahadev, K.,
Zilbering, A.,
Zhu, L.,
and Goldstein, B. J.
(2001)
J. Biol. Chem.
276,
21938-21942 |
45. |
Wentworth, P., Jr.,
Jones, L. H.,
Wentworth, A. D.,
Zhu, X.,
Larsen, N. A.,
Wilson, I. A.,
Xu, X.,
Goddard, W. A., III,
Janda, K. D.,
Eschenmoser, A.,
and Lerner, R. A.
(2001)
Science
293,
1806-1811 |
46. | Whetton, A. D., Bazill, G. W., and Dexter, T. M. (1984) EMBO J. 3, 409-413[Abstract] |
47. | Hamilton, J. A., Vairo, G., and Lingelbach, S. R. (1988) J. Cell. Physiol. 134, 405-412[Medline] [Order article via Infotrieve] |
48. | Freedman, M. H., Grunberger, T., Correa, P., Axelrad, A. A., Dube, I. D., and Cohen, A. (1993) Blood 81, 3068-3075[Abstract] |
49. | Gould, G. W., and Holman, G. D. (1993) Biochem. J. 295, 329-341[Medline] [Order article via Infotrieve] |
50. |
Cushman, S. W.,
and Wardzala, L. J.
(1980)
J. Biol. Chem.
255,
4758-4762 |
51. | Cushman, S. W., Goodyear, L. J., Pilch, P. F., Ralston, E., Galbo, H., Ploug, T., Kristiansen, S., and Klip, A. (1998) Adv. Exp. Med. Biol. 441, 63-71[Medline] [Order article via Infotrieve] |
52. |
Bird, T. A.,
Davies, A.,
Baldwin, S. A.,
and Saklatvala, J.
(1990)
J. Biol. Chem.
265,
13578-13583 |
53. | Egert, S., Nguyen, N., Brosius, F. C., and Schwaiger, M. (1997) Cardiovasc. Res. 35, 283-293[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Gould, G. W.,
Jess, T. J.,
Andrews, G. C.,
Herbst, J. J.,
Plevin, R. J.,
and Gibbs, E. M.
(1994)
J. Biol. Chem.
269,
26622-26625 |
55. |
Matsuguchi, T.,
Zhao, Y.,
Lilly, M. B.,
and Kraft, A. S.
(1997)
J. Biol. Chem.
272,
17450-17459 |
56. |
Thakker, G. D.,
Hajjar, D. P.,
Muller, W. A.,
and Rosengart, T. K.
(1999)
J. Biol. Chem.
274,
10002-10007 |
57. |
Kontos, C. D.,
Stauffer, T. P.,
Yang, W. P.,
York, J. D.,
Huang, L.,
Blanar, M. A.,
Meyer, T.,
and Peters, K. G.
(1998)
Mol. Cell. Biol.
18,
4131-4140 |
58. |
Mahadev, K.,
Wu, X.,
Zilbering, A.,
Zhu, L.,
Lawrence, J. T.,
and Goldstein, B. J.
(2001)
J. Biol. Chem.
276,
48662-48669 |
59. | Finkel, T. (2000) FEBS Lett. 476, 52-54[CrossRef][Medline] [Order article via Infotrieve] |
60. | Kamata, H., and Hirata, H. (1999) Cell. Signal. 11, 1-14[CrossRef][Medline] [Order article via Infotrieve] |
61. | Suzuki, Y. J., Forman, H. J., and Sevanian, A. (1997) Free Radic. Biol. Med. 22, 269-285[CrossRef][Medline] [Order article via Infotrieve] |
62. | DiPersio, J. F., Golde, D. W., and Gasson, J. D. (1990) Int. J. Cell Cloning 8 Suppl. 1, 63-75[Abstract] |
63. |
Lilly, M. B.,
Zemskova, M.,
Frankel, A. E.,
Salo, J.,
and Kraft, A. S.
(2001)
Blood
97,
1662-1670 |
64. |
Evans, C. A.,
Ariffin, S.,
Pierce, A.,
and Whetton, A. D.
(2002)
Blood
100,
3164-3174 |