From the Department of Medicine, Division of
Endocrinology, the ** Cancer Biology Program, Division of
Hematology-Oncology Beth Israel Deaconess Medical Center, and the
§ Joslin Diabetes Center and Department of Medicine, Harvard
Medical School, Boston, Massachusetts 02215
Received for publication, August 15, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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The protein tyrosine phosphatase SHP-2 has
been proposed to serve as a regulator of leptin signaling, but its
specific roles are not fully examined. To directly investigate the role
of SHP-2, we employed dominant negative strategies in transfected
cells. We show that a catalytically inactive mutant of SHP-2 blocks
leptin-stimulated ERK phosphorylation by the long leptin receptor,
ObRb. SHP-2, lacking two C-terminal tyrosine residues, partially
inhibits ERK phosphorylation. We find similar effects of the SHP-2
mutants after examining stimulation of an ERK-dependent
egr-1 promoter-construct by leptin. We also demonstrate ERK
phosphorylation and egr-1 mRNA expression in the
hypothalamus by leptin. Analysis of signaling by ObRb lacking
intracellular tyrosine residues or by the short leptin receptor, ObRa,
enabled us to conclude that two pathways are critical for ERK
activation. One pathway does not require the intracellular domain of
ObRb, whereas the other pathway requires tyrosine residue 985 of ObRb.
The phosphatase activity of SHP-2 is required for both pathways,
whereas activation of ERK via Tyr-985 of ObRb also requires
tyrosine phosphorylation of SHP-2. SHP-2 is thus a positive regulator
of ERK by leptin receptors, and both the adaptor function and the
phosphatase activity of SHP-2 are critical for this regulation.
Leptin is a 16-kDa hormone derived from adipose tissue that acts
on specific regions of the brain to regulate food intake, energy
expenditure, and neuroendocrine function in response to nutritional
perturbations (1-5). Lack of functional leptin or of leptin receptors
produces severe obesity in mice and, in rare cases, in humans (1,
6-10). The common form of human obesity is characterized by
hyperleptinemia and leptin resistance that yet has to be explained
(11).
Leptin regulates the expression of several genes in the hypothalamus
via direct actions on neurons expressing leptin receptors. These
include socs-3
(suppressor-of-cytokine-signaling)
(12), c-fos (13), and genes encoding neuropeptides,
which are involved in the regulation of appetite and body weight (14),
including NPY (15, 16), AgRP (17), CART
(18), POMC (19, 20), and TRH (21).
The intracellular signaling pathways by which leptin regulates these
genes are unknown, and further studies in this area are, therefore,
likely to be critical for the understanding of the mechanism(s) leading
to the leptin resistance characterizing human obesity.
Leptin is structurally related to cytokines and acts on receptors that
belong to the cytokine-receptor superfamily (6). Several different
leptin receptor isoforms exists, including a long form
(ObRb),1 which is highly
expressed in regions of the hypothalamus, and a short form (ObRa),
which is highly expressed within microvessels of the blood-brain
barrier (6, 7, 22-24). Although ObRb has a 302-amino acid long
intracellular domain, ObRa is predicted to encompass only a 34-residue
cytoplasmic domain. Leptin receptors lack intrinsic catalytic activity,
and analogous with other cytokine receptor systems, leptin stimulation
results in activation of intracellular Janus tyrosine kinases (JAKs)
that are associated with conserved JAK-binding motifs present in the
membrane proximal region of leptin receptors (25, 26). Activated JAKs
then phosphorylate phosphotyrosine residues in the intracellular domain
of ObRb (26). ObRa does not contain any intracellular tyrosine residues.
The murine ObRb receptor contains three intracellular tyrosine
residues, located at positions 985, 1077, and 1138. These amino acids
are conserved among known species of long form leptin receptors. Tyrosine phosphorylation sites provide binding motifs for src homology
2 (SH2)-domain containing proteins, such as STATs (signal transducer
and activator of transcription) (27, 28). Tyrosine 1138 is located 3 residues N-terminal to a glutamine residue (YXXQ), generating a consensus STAT3-binding motif (29). STATs then become
tyrosine-phosphorylated in response to JAK activation and translocate
to the nucleus to regulate gene transcription. Consistently, removal of
Tyr-1138 severely reduces leptin-induced STAT3 activation (30-33).
Although several different STAT isoforms have been shown to be
activated by leptin in cell systems (22, 30), only STAT3 has been found
to be activated in vivo in the hypothalamus (34). The
severely obese db/db mice lack the intracellular domain of ObRb (7, 8), and therefore cannot mediate activation of STAT3 in the
hypothalamus (34). It therefore seems likely, but yet unproven, that
STAT3 activation is a crucial component in leptin's pathway(s) to
regulate body weight.
In addition to STAT3 activation, other key signaling components are
likely to be regulated by leptin receptors. In vitro or in vivo studies show that leptin can stimulate insulin
receptor substrate phosphorylation (26), phosphatidylinositol 3-kinase activity (35-37), and MAPK (ERK) activity (26, 37, 38). Recent data
suggest that tyrosine 985 is required for maximal activation of the ERK
pathway by leptin (33). Furthermore, the SH2-domain containing protein
tyrosine phosphatase, SHP-2 (for review, see Ref. 39), binds to Tyr-985
and this site is required for tyrosine phosphorylation of SHP-2
following leptin treatment (32, 33, 40). SHP-2 has been shown to play a
positive role in mediating ERK activation by cytokine receptors and by
receptor tyrosine kinases (41-45). Studies also suggest that SHP-2
acts as both a negative and positive regulator of STAT signaling via
different cytokine receptors (45-48). The roles of the phosphatase
activity and tyrosine phosphorylation of SHP-2 in regulation of
signaling pathways are not well characterized and may differ in
different signaling systems.
Data suggest that Tyr-985 of ObRb mediates negative regulation of the
STAT pathway (32). We have recently provided a mechanism for this
observation by demonstrating that SOCS-3, a leptin-inducible SH2-domain
containing protein, requires binding to phosphorylated Tyr-985 to
efficiently inhibit ObRb signaling (49). We have previously shown that
both ObRa and ObRb have the ability to stimulate the ERK pathway (26)
and that SHP-2 is likely to play a positive role in ERK activation via
Tyr-985 of ObRb (33). However, because the exact requirements for
different leptin receptor domains and SHP-2 functions are unclear in
leptin signaling to the ERK pathway, we decided to more carefully
examine this by applying dominant-negative SHP-2 strategies. Our data
show that SHP-2 is a positive regulator of the ERK pathway by both
short and long leptin receptors. We conclude that SHP-2 affects ERK
activation via two pathways. One pathway does not require the
intracellular domain unique for ObRb, whereas the other requires
tyrosine residue 985 of ObRb. The phosphatase activity of SHP-2 is
required for both pathways, whereas activation of ERK via Tyr-985 of
ObRb also requires tyrosine phosphorylation of SHP-2. We speculate that
SHP-2 is also an indirect positive regulator of STAT signaling by
competitive inhibition of SOCS-3 binding to Tyr-985.
Materials--
Recombinant mouse leptin was obtained from Eli
Lilly (Indianapolis, IN). 125I-leptin was purchased from
PerkinElmer Life Sciences (Boston, MA). Murine erythropoietin (EPO) was
purchased from PharMingen (San Diego, CA). The expression vectors
encoding long (ObRb) and short (ObRa) murine leptin receptors were
generated as described earlier (26). A chimeric receptor (ELR) (and
tyrosine mutants), encompassing the extracellular domain of the murine
EPO receptor and the intracellular domain of murine ObRb, was made as
described earlier (33). Vectors encoding SHP-2, SHP-2 (C459S), and
SHP-2 (Y2F) were described previously by Bennett et al.
(42). JAK1 and JAK2 plasmids were from Dr. R. Kukunaga (Osaka
University, Japan). The HA epitope-tagged ERK1 and STAT3 expression
vectors were provided by Dr. J. Chernoff (Fox Chase Cancer Center,
Philadelphia) and Dr. J. Blenis (Harvard Medical School, Boston),
respectively. cDNA expression vectors encoding constitutively
active and dominant negative MEK1 mutants were gifts from Dr. C. Marshall (Institute of Cancer Research, London). The socs-3
promoter-luciferase construct was kindly given by Dr. S. Melmed (UCLA,
Los Angeles, CA), whereas the STAT-responsive-luciferase (GAS)
reporter plasmid was from Dr. L. Stancato and Dr. R. Pine (Sphinx
Pharmaceuticals, Durham, NC). Dr. G. Walz (Beth Israel Deaconess
Medical Center, Boston) kindly provided the egr1-luc
construct. All reagents for transfection were from Life Technologies,
Inc. (Gaithersburg, MD). Anti-phosphotyrosine (pY, 4G10) and
phospho-specific STAT3 (Y705) antibodies were from Upstate
Biotechnologies (Lake Placid, NY). JAK1, JAK2, and monoclonal SHP-2
antibodies were purchased from Santa Cruz (Santa Cruz, CA). The
phospho-specific MAPK antibody was from Promega (Madison, WI). The
leptin receptor antibody was generated as described by Bjørbæk
et al. (26), and the monoclonal HA antibody (12CA5) was from
Roche Molecular Biochemicals (Indianapolis, IN). For experiments shown
in Fig. 6A (see below) we used an antigen affinity-purified rabbit phospho-specific JAK2 antibody generated against a
dual-tyrosine-phosphorylated peptide corresponding to tyrosine
residues (Tyr-1007, Tyr-1008) in the activation loop of murine JAK2. A
rabbit SHP-2 antibody generated against a glutathione
S-transferase fusion protein containing the entire SHP-2
molecule (50) was also used in Fig. 6A. Cells stably
expressing mutant or wild-type chimeric ELR isoforms were generated as
described earlier (33).
Cell Culture and Transient Transfection--
CHO cells were
grown in Ham's F-12 medium supplemented with 10% fetal calf serum,
100 units/ml penicillin, and 10 µg/ml streptomycin at 37 °C in 5%
CO2. COS-1 and 293 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml
penicillin, and 10 µg/ml streptomycin at 37 °C in 5%
CO2. All cells were serum-deprived for 12-15 h before
stimulation with hormones. Cells were transfected with LipofectAMINE
according to the recommendations by the manufacturer (Life
Technologies, Inc.). Cells were harvested by rinsing in ice-cold
phosphate-buffered saline and scraping into ice-cold lysis buffer
(radioimmune precipitation buffer supplemented with 2 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). Lysates were clarified by centrifugation, and supernatants were immunoprecipitated as described below.
Immunoprecipitation and Immunoblotting--
Immunoprecipitations
were performed as described earlier by Bjørbæk et al.
(26). Briefly, clarified lysates were incubated at 4 °C with
antibodies together with protein A-agarose beads. After three washes in
ice-cold radioimmune precipitation buffer, the samples were subjected
to SDS-PAGE. Proteins were then transferred to nitrocellulose membranes
and blocked in nonfat dry milk or bovine serum albumin. After
incubation with antibodies, nitrocellulose membranes were washed and
targeted proteins were detected using either enhanced chemiluminescence
(ECL), as described by the manufacturer (Amersham Pharmacia
International, Buckinghamshire, UK), or by using
125I-protein A (ICN).
Luciferase and 125I-Leptin Binding Assays--
COS-1 cells were
transfected as described above, serum-deprived for 12-15 h, and
incubated with 100,000 cpm of 125I-leptin in Dulbecco's
modified Eagle's medium containing 0.1% of bovine serum albumin at
4 °C for 4 h, in the presence or absence of 200 nM
unlabeled leptin. Cells were then washed four times with ice-cold
binding medium, and the radioactivity associated with the cells was
measured in a gamma counter.
ERK Phosphorylation and Quantification of socs-3 and egr-1
mRNAs in Hypothalamus--
Male C57Bl/6J and ob/ob mice
were purchased from Jackson Laboratories (Bar Harbor, ME). The animals
and procedures used were in accordance with the guidelines and approval
of the Harvard Medical School Animal Care and Use Committees. For
phospho-MAPK Western blotting, ob/ob mice were injected
intraperitoneally with recombinant leptin or vehicle and sacrificed 20 min later. Hypothalami were isolated and lysed. After normalization of
protein, lysates were resolved by SDS-PAGE and membranes were
immunoblotted with anti-phospho-MAPK antibodies. For mRNA
quantification, overnight food-deprived C57 mice were injected
intraperitoneally with 100 µg of recombinant leptin or vehicle. At
different times following injection, mice were deeply anesthetized by
inhalation of Metofane (Mallinckrodt Veterinary, Inc., Mundelein, IL)
and then decapitated. The skull was reflected from the brain, and the
hypothalamus was isolated and snap-frozen in liquid nitrogen. Total RNA
purification, cDNA synthesis, and quantitative 32P-PCR
was done as described earlier (12). The following primers were used for
specific amplification of socs-3, egr-1, and
Dominant Negative SHP-2 Proteins Attenuate Leptin-induced ERK
Phosphorylation--
We have demonstrated previously that leptin has
the ability to activate MAPK (ERK1) kinase activity via both long and
short leptin receptors in transfected COS and CHO cells (26).
Furthermore, recent data show that maximal phosphorylation of ERK by
leptin requires tyrosine 985 of ObRb (33). This residue is also
required for binding of SHP-2 to ObRb and for tyrosine phosphorylation of SHP-2 by leptin (32, 33, 40). Although these data are suggestive,
the role of tyrosine phosphorylation of SHP-2, and the importance of
the phosphatase activity of SHP-2, in the regulation of the ERK pathway
by leptin is unclear. We therefore directly examined effects of SHP-2
on ERK activation by leptin, by applying dominant negative SHP-2
strategies. CHO cells were transiently transfected with ObRb, JAK2, and
HA-ERK1 expression vectors, together with vectors encoding either
wild-type SHP-2 (SHP-2 WT), catalytically inactive SHP-2 (SHP-2 C-S),
or a SHP-2 variant with two C-terminal GRB-2 tyrosine-binding sites
mutated into phenylalanine (SHP-2 Y2F). After serum deprivation, cells
were treated with 20 nM leptin for various periods of time
followed by SDS-PAGE and Western blotting for phosphorylated ERK1 in
clarified lysates. As shown in Fig. 1A, SHP-2 C-S strongly
inhibited leptin-induced HA-ERK1 phosphorylation, whereas expression of
SHP-2 Y2F appeared to result in a partial inhibition. Effects of SHP-2
mutants on ERK1 phosphorylation was then more carefully studied at the
maximal time point (Fig. 1B). The top panel shows
phosphorylation after 10 min of stimulation, and the lower
panels show equal expression of HA-ERK1 as well as transfected
SHP-2 proteins. Significant levels of endogenous SHP-2 proteins could
also be detected in these cells (left two lanes in
lowest panel), showing that the transiently expressed SHP-2
constructs are not highly overexpressed. Western blots from several
independent experiments were quantified by laser-scanning densitometry
(not shown), demonstrating a 40% reduction in samples from
leptin-treated cells expressing SHP-2 Y2F as compared with cells
expressing wild-type SHP-2. These results show that SHP-2 is a positive
mediator of ERK activation by ObRb and that this effect requires an
intact phosphatase activity of SHP-2. Furthermore, the data also
suggest that phosphorylation of the C-terminal tyrosine residues of
SHP-2 are required for maximal ERK activation.
Leptin Stimulates egr-1 Promoter Activity via SHP-2 and the ERK
Pathway--
We next investigated whether SHP-2 might influence
transcriptional regulation of egr-1 by leptin. The immediate
early gene egr-1 encodes a zinc finger transcription factor
that is induced by a variety of signals that initiate growth and
differentiation. For example, growth hormone, interleukin-6, and
granulocyte-colony stimulating factor stimulate egr-1
gene transcription via the ERK pathway (45, 51, 52). We therefore
transiently transfected CHO cells with vectors encoding ObRb and an
egr-1 luciferase reporter construct, together with empty
vector, SHP-2 WT, SHP-2 C-S, or SHP-2 Y2F expression plasmids. Cells
were then stimulated with leptin for 6 h, and luciferase
activities were measured in cell lysates. As shown in Fig.
2A, leptin stimulates
egr-1 promoter activity in this heterologous transfection
system. Furthermore, SHP-2 C-S markedly inhibited activation of the
egr-1 promoter by leptin (Fig. 2A). Expression of
SHP-2 Y2F resulted in a 50% attenuation of egr-1 promoter
activity. To determine whether the ERK pathway mediates activation of
the egr-1 promoter by leptin, we cotransfected
constitutively active (CA) or dominant negative (DN) MEK1 expression
vectors together with ObRb and the egr-1-luc plasmid into
CHO cells. As shown in Fig. 2B, CA MEK1 greatly enhanced egr-1 promoter activities in a leptin-independent manner,
whereas DN MEK1 blocked the ability of leptin to stimulate
egr-1 transcription, demonstrating that the ERK pathway is
required, and possibly sufficient, for the regulation of
egr-1 by leptin. Finally, we demonstrate that neither SHP-2
C-S nor SHP-2 Y2F affects the activation of the egr-1
promoter by constitutively active MEK1 (Fig. 2C). Combined, our data show that leptin regulates the egr-1 promoter via
the ERK pathway. Furthermore, SHP-2 is a positive regulator of the egr-1 promoter by ObRb, and this effect of SHP-2 requires
both phosphorylation of SHP-2 and the phosphatase activity of SHP-2. Finally, SHP-2 regulates the ERK pathway at signaling steps upstream of
MEK1.
Leptin Stimulates ERK Phosphorylation and egr-1 mRNA Expression
in the Hypothalamus--
Because leptin readily stimulates ERK1
phosphorylation and egr-1 promoter activity via ObRb in
transfected cells, we examined whether leptin might also have this
ability in the hypothalamus of mice where ObRb is highly expressed.
ob/ob mice were injected intraperitoneally with 100 µg of
recombinant leptin or with vehicle and sacrificed 20 min later. Protein
lysates from hypothalami were then analyzed for ERK phosphorylation by
Western blotting. We found a modest but significant increase in
phosphorylation in the leptin-treated animals (Fig.
3A). We next measured
hypothalamic egr-1 mRNA levels after leptin treatment.
Fasted C57Bl mice were injected intraperitoneally with leptin, and
total hypothalamic RNA was isolated at different times after
administration. We then employed quantitative 32P-RT-PCR to
measure egr-1 mRNA expression as described earlier for
socs-3 mRNA (12). Radioactive PCR products from
representative samples are shown in Fig. 3B. Leptin rapidly
stimulates hypothalamic egr-1 mRNA levels, reaching
maximal levels at 30 min after injection. Expression levels returned to
baseline levels at 3 h. The results from several animals are
quantified in Fig. 3C. We have earlier demonstrated
stimulation of socs-3 mRNA levels by leptin in the hypothalamus. SOCS-3 is an intracellular inhibitor of leptin signaling and socs-3 mRNA expression is stimulated by leptin
in vivo (12) and in vitro (53). The time course
of socs-3 mRNA induction in the hypothalamus by leptin
has not been reported. We found by quantitative 32P-RT-PCR
that the increase of socs-3 mRNA levels were slower as compared with egr-1, reaching maximal levels (4-fold above
baseline) at ~2 h and returning to near baseline at 6 h (Fig. 3,
B and C). We conclude that leptin rapidly stimulates
hypothalamic egr-1 and socs-3 mRNA expression
in vivo. Leptin administration did not affect
SHP-2 Does Not Affect Leptin-induced STAT3 Tyrosine Phosphorylation
or STAT-mediated Gene Transcription--
We next examined whether
SHP-2, in addition to regulation of the MAPK pathway, would also
directly affect leptin-mediated regulation of the STAT pathway, as
suggested by Carpenter et al. (33). To first measure
leptin-induced STAT3 tyrosine phosphorylation, we transfected COS-1
cells with ObRb and HA-STAT3 expression vectors, together with SHP-2
WT, SHP-2 C-S, or SHP-2 Y2F plasmids. After stimulation of cells with
leptin for 15 min, cell lysates were blotted with phospho-specific
(Tyr-705) STAT3 antibodies. As shown in Fig.
4A, SHP-2 constructs did not
affect leptin-induced STAT3 phosphorylation. To further test whether
SHP-2 might affect STAT-mediated gene transcription, we measured
leptin-induced transcription of an artificial
STAT-dependent luciferase reporter construct
( SHP-2 Does Not Affect ObRb Cell Surface Expression or ObRb Tyrosine
Phosphorylation--
To investigate whether SHP-2 might affect leptin
receptor expression or leptin-dependent ObRb tyrosine
phosphorylation, we first measured cell surface expression of ObRb
using 125I-leptin binding assays after cotransfection of
COS-1 cells with ObRb and the various SHP-2 constructs. As shown in
Fig. 5A, none of the SHP-2
variants affected specific 125I-leptin binding,
demonstrating that ObRb cell surface expression was similar in all
samples. COS-1 cells do not express endogenous leptin receptors and
therefore do not exhibit specific 125I-leptin binding (53).
We next measured ObRb tyrosine phosphorylation by transfecting cells
with ObRb and JAK2 expression vectors together with SHP-2 WT, SHP-2
C-S, or SHP-2 Y2F cDNAs. After stimulation of the cells with leptin
for 7 min, clarified lysates were subjected to immunoprecipitation with
anti-ObR antiserum. As shown by Western blotting using
anti-phosphotyrosine (pY) antibodies of the resolved immunoprecipitated
proteins, leptin-induced ObRb phosphorylation was not significantly
affected by wild-type or dominant negative SHP-2 constructs (Fig.
5B). Altogether, these results suggest that SHP-2 does not
affect leptin-induced ERK activation by altering expression or
phosphorylation of the leptin receptor.
JAK2 Enhances ERK Phosphorylation Independently of Leptin-receptor
Tyrosine Phosphorylation--
We have earlier shown that the short
leptin receptor isoform, ObRa, has the capacity to activate ERK
activity and that JAK2 potentiates ERK activation by ObRb (26). In
addition, ObRb lacking tyrosine 985 or all tyrosine residues can
activate ERK phosphorylation, although at a reduced rate (33).
Combined, these data suggest that ERK can be activated via a
JAK2-dependent pathway that does not require tyrosine
phosphorylation of ObRb. To investigate this hypothesis further we
first studied signaling in 293 cells stably expressing chimeric
erythropoietin-leptin receptors (ELR) or chimeric receptors
lacking all three tyrosine residues (ELR-triple), with or without
transient transfection of JAK2 cDNA. Both receptors induced similar
tyrosine phosphorylation of JAK2 in JAK2-transfected cells, whereas
little phosphorylation of endogenous JAK2 could be detected (Fig.
6A, top panel). As
expected, EPO treatment of cells expressing ELR, but not ELR-triple,
induced SHP-2 and STAT3 tyrosine phosphorylation (Fig. 6A,
middle two panels). Cotransfection of JAK2 resulted in
enhanced tyrosine phosphorylation of SHP-2 but not of STAT3 tyrosine
phosphorylation. As we have shown earlier (33),
ligand-dependent phosphorylation of endogenous ERK1 and ERK2 was strongly reduced, but detectable, in cells expressing ELR-triple (Fig. 6A, second panel from
bottom). However, coexpression of JAK2 strongly enhanced
EPO-induced ERK phosphorylation by this mutant receptor. Induction of
ERK phosphorylation by WT ELR was also enhanced by coexpression of
JAK2, consistent with results we have reported earlier using
full-length ObRb (26). These data show that the ERK pathway can be
activated via JAK2 by a mechanism that does not require tyrosine
phosphorylation of the leptin receptor. To specifically investigate the
role of Tyr-985 in ERK activation via JAK2, we transiently transfected
ELR or ELR-985L into CHO cells. The cells were also cotransfected or not with low levels of JAK2 cDNA. Consistent with results that we
have reported earlier using 293 cells (33), stimulation of HA-ERK1
phosphorylation by ELR-985L was much reduced as compared with ELR in
the absence of JAK2 coexpression (Fig. 6B). However, transfection of JAK2 significantly enhanced ligand-induced ERK1 phosphorylation by both ELR and ELR-985L (Fig. 6B), although
the levels were slightly lower for ELR-985L. These data show that, in
the absence of JAK2 cotransfection, maximal ERK activation requires
Tyr-985 of ObRb. On the other hand, coexpression of JAK2 leads to
enhanced ERK phosphorylation that is largely independent of receptor
phosphorylation. Combined, the data suggest that ERK1 and ERK2 can be
activated by leptin via both receptor-tyrosine-dependent (Tyr-985) and receptor-tyrosine-independent pathways. Furthermore, we
conclude that the relative signaling via these two pathways may depend
on cellular JAK2 activity levels.
Maximal Activation of ERK via Tyr-985 of ObRb Requires Tyrosine
Phosphorylation of SHP-2 at the C Terminus--
We have shown above
that SHP-2 is a positive mediator of ERK activation by ObRb and that
this requires both the phosphatase activity and tyrosine
phosphorylation of SHP-2. Furthermore, Tyr-985 is required for maximal
ERK phosphorylation. Because SHP-2 has been shown to bind to ObRb at
tyrosine residue 985 (32, 40), we first wanted to examine whether this
amino acid was important for the regulation of the ERK pathway by
SHP-2. We therefore investigated inhibitory effects of dominant
negative SHP-2 proteins on ERK1 phosphorylation by ELR-985L. In CHO
cells stably expressing ELR-985L, we transiently transfected JAK2 and
HA-ERK1 cDNAs together with either SHP-2 WT, SHP-2 C-S, or SHP-2
Y2F expression vectors. Following treatment with EPO for 10 min,
cellular lysates were analyzed for ERK1 phosphorylation by Western
blotting as described under Fig. 1. We found that the phosphorylation
was completely blocked by the dominant negative SHP-2 C-S mutant (Fig.
7A). However, there was no
attenuation by SHP-2 Y2F, contrary to what was observed for wild-type
ObRb (Fig. 1). To further strengthen this finding, we transiently
transfected the short leptin receptor (ObRa), JAK2, HA-ERK1 together
with SHP-2 constructs into CHO cells. As shown by Western blotting in
Fig. 7B, leptin-induced HA-ERK1 phosphorylation via ObRa was
also blocked by SHP-2 C-S, but was unaffected by SHP-2 Y2F.
Autoradiograms from several independent Western blots of ERK
phosphorylation by ObRa (Fig. 7B) were quantified by
laser-scanning PhosphorImager densitometry. The results are depicted in
Fig. 7C. Combined, these data demonstrate that the
phosphatase activity of SHP-2 does not require the intracellular domain
unique to ObRb to positively regulate ERK by leptin. On the other hand,
stimulation of ERK via Tyr-985 of ObRb requires tyrosine
phosphorylation of SHP-2 at the C terminus.
We show here that leptin can activate ERK signaling in the
hypothalamus and that this stimulation is likely to occur via two pathways, both involving SHP-2. First, because the long leptin receptor
lacking tyrosine 985 exhibits a significantly reduced ability to
activate ERK phosphorylation, this residue is at least in part
mediating stimulation of the ERK pathway by ObRb. This residue binds
SHP-2 and is required for tyrosine phosphorylation of SHP-2 (32, 33,
40). Furthermore, as shown here, a tyrosine mutant of SHP-2 attenuates
activation of ERK phosphorylation and of ERK-dependent gene
transcription (egr-1 promoter) by ObRb. Consistent with a
role of phosphorylated SHP-2 in ERK regulation via Tyr-985, we find
that SHP-2 Y2F does not reduce ERK phosphorylation by neither the
Tyr-985 leptin-receptor mutant nor the short leptin receptor, ObRa.
Based on these data, we conclude that tyrosine phosphorylation of SHP-2
is a mediator of ERK activation via Tyr-985. This is likely to occur
via Grb-2 binding to SHP-2 at the C terminus followed by activation of
the Ras-Raf pathway as suggested for other signaling systems (55, 56)
and more recently for the leptin receptor (33). Altogether, these
results are strong evidence that SHP-2 acts as an adaptor in leptin
signaling to the ERK pathway. However, because the SHP-2 Y2F mutant
only partially inhibits ERK phosphorylation and
egr-1-promoter activation by ObRb, other pathways to
activate ERK are likely to be involved. Consistent with this notion is
the fact that stimulation of ERK phosphorylation does not absolutely
require tyrosine phosphorylation of ObRb, as demonstrated by the
ability of ObRa, and by long form receptors lacking all tyrosine
residues, to activate ERK. Combined, these data suggest that leptin
receptors can stimulate the ERK pathway via two mechanisms, each
requiring different intracellular receptor domains (Fig.
8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Galactosidase Assay--
After cell lysis,
aliquots were used for luciferase assay as described earlier (12).
Briefly, luciferin (Molecular Probes, Eugene, OR) and assay buffer were
injected simultaneously and measured for 20 s by a Luminometer (LB
9501, EG&G Berthold, Bad Wildbad, Germany).
-Galactosidase
activities were determined using Galacton (Tropix Inc., Bedford, MA) as
described by the manufacturer and measured for 5 s by the Luminometer.
-actin cDNAs: SOCS-3A,
5'-accagcgccacttcttcacg-3', and SOCS-3B, 5'-gtggagcatcatactgatcc-3' (450-bp product, 25 cycles); Egr-1A, 5'-tttccacaacaacagggagacc-3', and
Egr-1B, 5'-acaggcaaaaggcttctcgc-3' (425-bp product, 22 cycles); and
Actin-A, 5'-cgtaccacgggcattgtgatgg-3', and Actin-B,
5'-tttgatgtcacgcacgatttccc-3' (200-bp product, 17 cycles).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Dominant negative SHP-2 proteins attenuate
leptin-induced ERK phosphorylation. A, effects by SHP-2
mutants on the time course of ERK-1 phosphorylation in CHO cells. Cells
were transfected with ObRb, HA-ERK1, JAK2 together with vectors
encoding the WT SHP-2, C-S SHP-2, or Y2F SHP-2. Serum-deprived cells
were treated with 20 nM leptin for different periods of
time. Lysates were subjected to Western blotting (anti-P-MAPK) for
activated HA-ERK1. The lower band represents phosphorylation
of endogenous ERK1. B, attenuation of ERK1 phosphorylation
by dominant negative SHP-2 proteins. Cells were transfected as above
and treated with leptin for 10 min. Lysates were subjected to Western
blotting for activated HA-ERK1 (top panel). The same
membrane was reprobed for total HA-ERK1 expression (anti-HA,
middle panel) and for SHP-2 expression (anti-SHP-2,
lower panel). One representative blot from several
independent experiments is shown. Endogenous SHP-2 expression could
also be detected (lanes 1 and 2, bottom
panel).
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Fig. 2.
Effects of SHP-2 and of the ERK pathway on
leptin-dependent regulation of the egr-1
promoter. A, inhibition of leptin-induced
egr-1 promoter activation by SHP-2 mutants. CHO cells were
transfected with ObRb cDNA and an egr-1-luc reporter
plasmid together with either empty vector, SHP-2 WT, SHP-2 C-S, or
SHP-2 Y2F. A cmv-lacZ control plasmid was also cotransfected
into the cells to correct for differences in transfection efficiencies.
JAK2 was not coexpressed in this experiment. Serum-deprived cells were
left unstimulated or treated with 20 nM leptin for 6 h, and luciferase activities were measured in the cell lysates.
Luciferase activities were normalized with -galactosidase activities
measured in the same samples. Gray bars show results from
nontreated samples, whereas black bars show the
leptin-treated results. This experiment was performed three times, each
in triplicates. Shown are means ± S.E. of one experiment. A
significant (p = 0.003) reduction of egr-1
promoter activation was found in samples from cells transfected with
SHP-2 Y2F as compared with cells transfected with wild-type SHP-2.
SHP-2 C-S expression resulted in a near blockade of egr-1
activation. B, leptin activates egr-1 via the ERK
pathway. CHO cells were transfected with ObRb and egr-1
plasmids, together with either empty vector, constitutively active
(CA) MEK1 or dominant negative (DN) MEK1.
Leptin-dependent and -independent luciferase activities
were measured and depicted as above. This experiment was done two
times, each in triplicate. Shown are means ± S.E. from one
experiment. C, SHP-2 acts upstream of MEK1. Cells were
transfected with ObRb, egr-1-luc, together with SHP-2, SHP-2
C-S, or SHP-2 Y2F, and with either empty vector or MEK1 CA plasmids.
Leptin-dependent and -independent luciferase activities
were measured and depicted as above.
-actin mRNA levels in the same samples.
Because the time courses of the two mRNAs are different following
leptin administration, it is possible that different intracellular
signaling pathways regulate the two genes. Furthermore, based on our
transfection data, it is likely that leptin stimulates egr-1
mRNA expression in the hypothalamus via SHP-2 and the ERK pathway.
On the other hand, stimulation of socs-3 gene expression by
leptin requires activation of the STAT pathway (33, 54), and this
stimulation is not affected by the ERK pathway in transfected
cells.2
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Fig. 3.
Leptin rapidly stimulates ERK phosphorylation
and egr-1 mRNA expression in the hypothalamus of
mice. A, activation of ERK phosphorylation in the
hypothalamus by leptin. ob/ob mice were injected
intraperitoneally with vehicle or 100 µg of recombinant leptin. After
20 min, hypothalami were collected and protein lysates were analyzed
for ERK phosphorylation by Western blotting (anti-P-MAPK). Each lane
represents one animal. Shown is a representative blot of four samples
from a total of eight animals analyzed. B, leptin induces
egr-1 and socs-3 mRNA in the hypothalamus.
C57Bl mice were food-deprived overnight and injected intraperitoneally
with saline or 100 µg of recombinant leptin. Total RNA was isolated
from hypothalami at different times after injection (t = 0.5, 1, 3, and 6 h) and subjected to quantitative
32P-RT-PCR for egr-1, socs-3, and
-actin mRNA under conditions of limiting
number of PCR cycles. Radioactive PCR products were separated on
polyacrylamide gels. Shown are representative samples from
egr-1 and socs-3 mRNA measurements. Each lane
represents one animal. C, all samples were quantified by
PhosphorImager analysis. Shown are the means ± S.E.
(n = 5 animals at each time point). Results from
saline-injected animals were normalized to 100% for each gene.
-interferon-activated sequence (GAS)) in transfected COS-1 cells.
As demonstrated in Fig. 4B, leptin treatment for 6 h
induced a robust, ~40-fold increase in luciferase activities, which
were not affected by coexpression of wild-type or mutant SHP-2
proteins. Furthermore, SHP-2 constructs did not affect
leptin-dependent transcriptional activation of a
STAT3-dependent socs-3-luciferase promoter
plasmid (Fig. 4C) (54). Altogether, these results suggest
that SHP-2 does not affect leptin-dependent STAT signaling
after 15 min or after 6 h of treatment in these cells.
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Fig. 4.
SHP-2 does not affect leptin-induced STAT3
tyrosine phosphorylation or STAT3-mediated gene transcription.
A, SHP-2 does not affect rapid phosphorylation of STAT3 by
leptin. COS-1 cells were transfected with ObRb and HA-STAT3 vectors,
together with either empty vector, SHP-2 WT, SHP-2 C-S, or SHP-2 Y2F
plasmids. JAK2 was not cotransfected in these experiments.
Serum-deprived cells were left unstimulated or treated with 20 nM leptin for 15 min, and protein lysates were resolved and
blotted using phospho-specific STAT3 (Tyr-705) antibodies (top
panel). The lower panel shows equal HA-STAT3 expression
by anti-HA blotting. This experiment was done three times.
B, SHP-2 does not affect STAT-dependent gene
transcription. COS-1 cells were transfected with ObRb plasmids and
GAS-luciferase reporter plasmids, together with SHP-2 plasmids. A
cmv-lacZ control plasmid was also cotransfected into the
cells to correct for differences in transfection efficiencies. Cells
were then stimulated with 20 nM leptin for 6 h
followed by measurement of luciferase and -galactosidase activities
in the cellular lysates. Gray bars show results from
nontreated samples, whereas black bars show the
leptin-treated results. This experiment was performed two times, each
in triplicate. Shown are the means ± S.E. from one experiment.
C, SHP-2 does not affect leptin-mediated activation of the
socs-3 promoter. COS-1 cells were transfected as under
B, except that a socs3-luciferase reporter
plasmid was transfected instead of the GAS-luciferase plasmid. Cells
were treated with 20 nM leptin for 5 h. Results are
depicted as described under B.
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Fig. 5.
SHP-2 does not affect ObRb cell surface
expression or ObRb tyrosine phosphorylation. A, SHP-2
does not affect ObRb expression. COS-1 cells were transfected with ObRb
expression vectors together with the different SHP-2 plasmids.
Serum-deprived cells were then subjected to 125I-leptin
binding assay at 4 °C as described under "Experimental
Procedures." Gray bars show nonspecific binding
(competition with 200 nM unlabeled leptin). Shown are
means ± S.E. B, SHP-2 does not affect leptin-induced
ObRb tyrosine phosphorylation. Shown is a Western blot (upper
panel) using anti-pY antibodies of ObR immunoprecipitates from
cells transfected with ObRb and JAK2 together with SHP-2 constructs.
Cells were left unstimulated or treated with 20 nM leptin
for 10 min. This experiment was done three times. The lower
panel shows SHP-2 protein expression in the same samples by
Western blotting of SHP-2 immunoprecipitates.
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Fig. 6.
JAK2 enhances ERK phosphorylation
independently of leptin-receptor tyrosine phosphorylation.
A, 293 cells that stably express chimeric EPOR-leptin
receptor (ELR) or ELR with mutated tyrosines 985, 1077, and
1138 (ELR-triple) were transfected or not with JAK2
expression vectors and subsequently treated or not with erythropoietin
(EPO) for 5 min. Clarified lysates were probed for JAK2
tyrosine phosphorylation (anti-Tyr-1007, -Tyr-1008 of JAK2), JAK2
expression, STAT3 Tyr-705 phosphorylation, ERK phosphorylation, or
total ERK expression by Western blotting. SHP-2 tyrosine
phosphorylation was examined by blotting with pY antibodies after SHP-2
immunoprecipitation. Equal surface expression of chimeric receptors in
the two cell lines was determined by 125I-EPO tracer
binding studies (not shown). B, CHO cells were transiently
transfected with ELR or ELR with Tyr-985 mutated into leucine
(ELR-985L), together with HA-ERK1, with or without JAK2
plasmids. After stimulation or not with EPO for 10 min, clarified
lysates were probed for ERK1 phosphorylation (upper panel)
or for total HA-ERK1 protein expression (lower panel).
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Fig. 7.
Stimulation of ERK via Tyr-985 of ObRb
requires tyrosine phosphorylation of SHP-2. A, tyrosine
985 is required for inhibition of ERK phosphorylation by SHP-2 Y2F. CHO
cells stably expressing ELR-985L receptors were transiently transfected
with JAK2 and HA-ERK1 vectors, together with the different SHP-2
plasmids. Cells were treated with 50 nM EPO or with nothing
for 10 min. Shown is a Western blot using phospho-specific ERK
antibodies of cell lysates (top panel). The same membrane
was later probed with anti-HA antibodies (lower panel). The
same result was obtained in two other experiments. B,
expression of SHP-2 Y2F does not inhibit activation of ERK by the short
leptin receptor, ObRa. COS-1 cells were transfected with cDNAs
encoding ObRa, JAK2, and HA-ERK1, together with either SHP-2 WT, SHP-2
C-S, or SHP-2 Y2F plasmids. Cells were left unstimulated or treated
with 20 nM leptin for 10 min. Shown is a Western blot using
phospho-specific ERK antibodies of cell lysates (top panel).
The same membrane was reprobed with anti-HA antibodies (middle
panel). Total SHP-2 expression in the clarified lysates is shown
in the lower panel. C, autoradiograms from
several independent Western blots of HA-ERK1 phosphorylation via ObRa
(B) were quantified by laser-scanning PhosphorImager
densitometry. Gray and black bars depict
arbitrary units of unstimulated and stimulated HA-ERK1 phosphorylation,
respectively. Values are means ± S.E. (n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Model for the role of SHP-2 in ERK signaling
by leptin receptors. Ligand binding to the long leptin receptor,
ObRb, leads to activation of ERK via two pathways that both require
SHP-2. One pathway (A) is independent of tyrosine
phosphorylation of ObRb but requires the phosphatase activity (PTPase)
of SHP-2. The other pathway (B) requires binding of SHP-2 to
Tyr-985 followed by tyrosine phosphorylation of SHP-2. This pathway is
also dependent on the PTPase activity of SHP-2. The ERK pathway
regulates the egr-1 promoter. Tyr-1138 regulates the STAT
pathway leading to socs-3 gene activation and does not
require SHP-2. SOCS-3 proteins attenuate leptin signaling via binding
to Tyr-985 (53). The mechanism by which this occurs is unknown but may
involve inhibition of JAK activity. PM, plasma membrane;
pY, tyrosine phosphorylation. Small round objects
represents SH2 domains.
Ligand-dependent ERK phosphorylation via ObRb lacking all tyrosine residues is strongly enhanced by coexpression of JAK2, and the level is comparable to that of wild-type ObRb. This is suggestive of a direct signaling pathway from JAK2 to the ERK pathway. This alternative pathway requires the phosphatase (PTPase) activity of SHP-2, as demonstrated by the complete blockade of ERK signaling by the phosphatase inactive SHP-2 C-S. In addition, because SHP-2 C-S completely inhibits ERK activation by both the short and long leptin receptors, we conclude that the Tyr-985-dependent pathway is not sufficient to activate ERK in the absence of SHP-2 PTPase activity. Combined, our results suggest that the phosphatase activity of SHP-2 is required for both pathways leading to activation of ERK. Although our data suggest that the site of action of the SHP-2 phosphatase activity occurs at a signaling step located upstream of MEK1 and downstream of leptin receptors (Fig. 8), the substrates of SHP-2 that are important for ERK activation by leptin remain unknown. One possibility is that the SHP-2 PTPase acts at a signaling step upstream of Ras in the Ras-Raf-MEK-ERK cascade, as suggested for the role of the SHP-2 phosphatase function in ERK activation by EGF (57). Alternatively, activation of ERK by cytokine receptors, including the leptin receptor, may require a signaling step that is activated by integrin, such as the SHPS1·SHP-2 complex (58-60), but further studies are needed to investigate this possibility.
One report suggests that SHP-2 is a negative regulator of leptin-induced STAT3-transcription (32). This conclusion was based on indirect evidence showing that the Tyr-985 mutant of ObRb does not bind SHP-2 and that this receptor exhibits increased STAT-mediated gene transcription following 24 h of ligand treatment. A mechanism explaining these observations was not provided. By applying a direct approach using dominant negative SHP-2 strategies, we did not find evidence for SHP-2 having effects on rapid leptin-induced STAT3 tyrosine phosphorylation. This result is consistent with several reports showing that mutation of Tyr-985 of ObRb, which results in loss of SHP-2 binding, does not affect STAT3 tyrosine phosphorylation (32, 33, 40). Furthermore, we did not detect any effect by SHP-2 mutant proteins on STAT3-mediated gene transcription, by examining two different STAT-dependent promoters following 6 h of leptin treatment. We conclude that SHP-2 does not directly affect the STAT pathway by leptin receptors. On the other hand, we have recently demonstrated that SOCS-3 binds to Tyr-985 and that SOCS-3 requires this residue to mediate inhibition of ObRb signaling (33) (Fig. 8). We confirmed in these studies that mutation of Tyr-985 resulted in enhanced STAT transcription following 24 h of ligand treatment but not after that 6 h of treatment. Because the leptin receptor induces expression of SOCS-3 at the transcriptional level, these results provide a time-dependent mechanism that explains the increased STAT signaling after prolonged ligand treatment following mutation of Tyr-985 (53). We conclude from those studies that signaling effects that have previously been attributed to SHP-2, following mutagenesis of tyrosine docking sites, are in fact due to SOCS-3, as recently also shown for tyrosine 759 of gp130 (61, 62). The exact mechanism by which SOCS-3 inhibits leptin signaling after binding to Tyr-985 is unknown (Fig. 8). We also demonstrated functional competition between SHP-2 and SOCS-3 for Tyr-985 of ObRb (53). These data suggest that SHP-2 is an indirect positive regulator of STAT signaling, acting by preventing SOCS-3 from inhibiting leptin signaling via Tyr-985. In addition, it is conceivable that SOCS-3 can inhibit ERK signaling by two mechanisms; one involving prevention of SHP-2 binding to Tyr-985 and the other by inhibition of JAK activity after binding to Tyr-985. Combined, these data suggest that expression levels of SHP-2 and SOCS-3 are key factors in the regulation of intracellular leptin signaling.
We have earlier shown that ERK activation by leptin receptors is enhanced by JAK2 expression (26). However, specific ERK activation via the short leptin receptor, ObRa, is much weaker than that of ObRb, when the much higher expression of ObRa is taken into account (26). Similarly, leptin-stimulated JAK2 phosphorylation is more robust via ObRb than by ObRa (26). The fact that both JAK2 and ERK activation is lower via ObRa may be explained by the lack of the conserved Box-2 JAK-binding motif in ObRa, which is, however, present in ObRb and in other long cytokine receptors of the family (25, 63). The Box-2 motif is likely required for maximal JAK activation and for subsequent maximal stimulation of the ERK pathway. Thus, the more robust ERK signaling by ObRb, as compared with ObRa, is possibly caused by increased Box-2-mediated activation of JAK kinase activity, which likely leads to enhanced ERK activation via both the "alternative" JAK-mediated pathway and via increased phosphorylation of Tyr-985 followed by enhanced SHP-2 tyrosine phosphorylation. Interestingly, because ObRa does not contain Tyr-985, ERK signaling via this receptor is unlikely to be regulated by SHP-2 phosphorylation or by feedback inhibition by SOCS-3, as is the case of ObRb. The relevance of this observation is presently unknown.
We also show for the first time that leptin rapidly stimulates the mRNA expression of the zinc finger transcription factor, Egr-1, in the hypothalamus of mice. Our transfection results suggest that this regulation by leptin occurs by activation of the egr-1 promoter via activation of SHP-2 and of the ERK pathway. The role of the Egr-1 transcription factor in leptin action in the central nervous system, as well as the exact sites of activation within the hypothalamus, remains to be determined.
Our data demonstrate that SHP-2 is a positive regulator of ERK
activation and of ERK-dependent egr-1
transcription by leptin and suggest that this regulation occurs via two
mechanisms. One depends on Tyr-985 of ObRb and requires tyrosine
phosphorylation of SHP-2. The other pathway is independent of tyrosine
phosphorylation of both ObRb and of SHP-2. The phosphatase activity of
SHP-2 is required for both pathways. We conclude that both the adaptor function and the PTPase activity of SHP-2 are critical for ERK activation by leptin receptors. Finally, we speculate that SHP-2 may
also act as an indirect positive mediator of STAT signaling by
inhibition of SOCS-3 action via competition for tyrosine 985. SHP-2 and
SOCS-3 proteins are thus critical regulators of leptin receptor
signaling pathways, and combined our results imply a complex
interaction of multiple regulatory proteins at a single receptor site.
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ACKNOWLEDGEMENTS |
---|
Special thanks to Shlomo Melmed and Chris Auernhammer (UCLA) for providing the socs3-luciferase plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health (NIH) Grant DK-R37-28082 and by a grant from Eli Lilly (to J. S. F.).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.
¶ Supported by NIH Grant R01-DK-56731.
Supported by the Swiss National Science Foundation.
Recipient of National Research Service Award CA72144 from the
NIH and holder of an Anna D. Barker Fellowship in Basic Science from
the American Association for Cancer Research.
§§ Supported by NIH Grants RO1-CA49152 and DK-50693.
¶¶ Supported by a Harcourt General New Investigator Award.
To whom correspondence should be addressed: Division of
Endocrinology, Dept. of Medicine, Beth Israel Deaconess Medical Center, 99 Brookline Ave., Research North, Boston, MA 02215. Tel.:
617-667-2151; Fax: 617-667-2927; E-mail:
jflier@caregroup.harvard.edu.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M007439200
2 C. Bjørbæk, R. M. Buchholz, S. M. Davis, S. H. Bates, D. D. Pierroz, H. Gu, B. G. Neel, M. G. Myers, Jr., and J. S. Flier, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
ObRb, long leptin
receptor isoform;
ObRa, short leptin receptor isoform;
JAK, Janus tyrosine kinase;
SH2, src homology 2;
STAT, signal transducer and
activator of transcription;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase;
MEK, mitogen-activated
protein kinase/ERK kinase;
P-MAPK, phospho-MAPK;
EPO, erythropoietin;
ELR, EPOR-leptin receptor;
HA, hemagglutinin;
GAS, -interferon-activated sequence;
CHO, Chinese hamster ovary;
PAGE, polyacrylamide gel electrophoresis;
RT, reverse transcription;
PCR, polymerase chain reaction;
bp, base pair(s);
SHP-2 WT, wild-type SHP-2;
SHP-2 C-S, catalytically inactive SHP-2;
SHP-2 Y2F, SHP-2 variant with
two C-terminal GRB-2 tyrosine-binding sites mutated into phenylalanine;
CA, constitutively active;
DN, dominant negative;
pY, tyrosine
phosphorylation.
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REFERENCES |
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---|
1. | Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J. M. (1994) Nature 372, 425-432[CrossRef][Medline] [Order article via Infotrieve] |
2. | Pelleymounter, M. A., Cullen, M. J., Baker, M. B., Hecht, R., Winters, D., Boone, T., and Collins, F. (1995) Science 269, 540-543[Medline] [Order article via Infotrieve] |
3. | Halaas, J. L., Gajiwala, K. S., Maffei, M., Cohen, S. L., Chait, B. T., Rabinowitz, D., Lallone, R. L., Burley, S. K., and Friedman, J. M. (1995) Science 269, 543-546[Medline] [Order article via Infotrieve] |
4. | Campfield, L. A., Smith, F. J., Guisez, Y., Devos, R., and Burn, P. (1995) Science 269, 546-549[Medline] [Order article via Infotrieve] |
5. | Ahima, R. S., Prabakaran, D., Mantzoros, C., Qu, D., Lowell, B., Maratos-Flier, E., and Flier, J. S. (1996) Nature 382, 250-252[CrossRef][Medline] [Order article via Infotrieve] |
6. | Tartaglia, L. A., Dembski, M., Weng, X., Deng, N., Culpepper, J., Devos, R., Richards, G. J., Campfield, L. A., Clark, F. T., Deeds, J., Muir, C., Sanker, S., Moriarty, A., Moore, K. J., Smutko, J. S., Mays, G. G., Woolf, E. A., Monroe, C. A., and Tepper, R. I. (1995) Cell 83, 1263-1271[Medline] [Order article via Infotrieve] |
7. | Lee, G.-H., Proenca, R., Montez, J. M., Carroll, K. M., Darvishzadeh, J. G., Lee, J. I., and Friedman, J. M. (1996) Nature 379, 632-635[CrossRef][Medline] [Order article via Infotrieve] |
8. | Chen, H., Chatlat, O., Tartaglia, L. A., Woolf, E. A., Weng, X., Ellis, S. J., Lakey, N. D., Culpepper, J., Moore, K. J., Breitbart, R. E., Duyk, G. M., Tepper, R. I., and Morgenstern, J. P. (1996) Cell 84, 491-495[Medline] [Order article via Infotrieve] |
9. | Montague, C. T., Farooqi, I. S., Whitehead, J. P., Soos, M. A., Rau, H., Wareham, N. J., Sewter, C. P., Digby, J. E., Mohammed, S. N., Hurst, J. A., Cheetham, C. H., Earley, A. R., Barnett, A. H., Prins, J. B., and O'Rahilly, S. (1997) Nature 387, 903-908[CrossRef][Medline] [Order article via Infotrieve] |
10. | Clement, K., Vaisse, C., Lahlou, N., Cabrol, S., Pelloux, V., Cassuto, D., Gourmelen, M., Dina, C., Chambaz, J., Lacorte, J. M., Basdevant, A., Bougneres, P., Lebouc, Y., Froguel, P., and Guy-Grand, B. (1998) Nature 392, 398-401[CrossRef][Medline] [Order article via Infotrieve] |
11. | Maffei, M., Halaas, J., Ravussin, E., Pratley, R. E., Lee, G. H., Zhang, Y., Fei, H., Kim, S., Lallone, R., Ranganathan, S., et al.. (1995) Nat. Med. 1, 1155-1161[Medline] [Order article via Infotrieve] |
12. | Bjørbæk, C., Elmquist, J. K., Frantz, J. D., Shoelson, S. E., and Flier, J. S. (1998) Mol. Cell 1, 619-625[Medline] [Order article via Infotrieve] |
13. |
Elmquist, J. K.,
Ahima, R. S.,
Maratos-Flier, E.,
Flier, J. S.,
and Saper, C. B.
(1997)
Endocrinology
138,
839-842 |
14. | Elias, C. F., Aschkenasi, C., Lee, C., Kelly, J., Ahima, R. S., Bjørbæk, C., Flier, J. S., Saper, C. B., and Elmquist, J. K. (1999) Neuron 23, 775-786[CrossRef][Medline] [Order article via Infotrieve] |
15. | Stephens, T. W., Basinski, M., Bristow, P. K., Bue-Valleskey, J. M., Burgett, S. G., Craft, L., Hale, J., Hoffmann, J., Hsiung, H. M., Kriauciunas, A., MacKellar, W., Rosteck, P. R., Jr., Schoner, B., Smith, D., Tinsley, F. C., Zhang, X.-Y., and Heiman, M. (1995) Nature 377, 530-532[CrossRef][Medline] [Order article via Infotrieve] |
16. | Schwartz, M. W., Baskin, D. G., Bukowski, T. R., Kuijper, J. L., Foster, D., Lasser, G., Prunkard, D. E., Porte, D., Jr., Woods, S. C., Seeley, R. J., and Weigle, D. S. (1996) Diabetes 45, 531-535[Abstract] |
17. |
Ollmann, M. M.,
Wilson, B. D.,
Yang, Y. K.,
Kerns, J. A.,
Chen, Y.,
Gantz, I.,
and Barsh, G. S.
(1997)
Science
278,
135-138 |
18. | Kristensen, P., Judge, M. E., Thim, L., Ribel, U., Christjansen, K. N., Wulff, B. S., Clausen, J. T., Jensen, P. B., Madsen, O. D., Vrang, N., Larsen, P. J., and Hastrup, S. (1998) Nature 393, 72-76[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Thornton, J. E.,
Cheung, C. C.,
Clifton, D. K.,
and Steiner, R. A.
(1997)
Endocrinology
138,
5063-5066 |
20. |
Cheung, C. C.,
Clifton, D. K.,
and Steiner, R. A.
(1997)
Endocrinology
138,
4489-4492 |
21. |
Nillni, E. A.,
Vaslet, C.,
Harris, M.,
Hollenberg, A. N.,
Bjørbæk, C.,
and Flier, J. S.
(2000)
J. Biol. Chem.
275,
36124-36133 |
22. |
Ghilardi, N.,
Ziegler, S.,
Wiestner, A.,
Stoffel, R.,
Heim, M. H.,
and Skoda, R. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6231-6235 |
23. |
Bjørbæk, C.,
Elmquist, J. K.,
Michl, P.,
Ahima, R. S.,
van Bueren, A.,
McCall, A. L.,
and Flier, J. S.
(1998)
Endocrinology
139,
3485-3491 |
24. | Elmquist, J. K., Bjørbæk, C., Ahima, R. S., Flier, J. S., and Saper, C. B. (1998) J. Comp. Neurol. 395, 535-547[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Ghilardi, N.,
and Skoda, R. C.
(1997)
Mol. Endocrinol.
11,
393-399 |
26. |
Bjørbæk, C.,
Uotani, S.,
da Silva, B.,
and Flier, J. S.
(1997)
J. Biol. Chem.
272,
32686-32695 |
27. | Ihle, J. N. (1995) Nature 377, 591-594[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Darnell, J. E.
(1997)
Science
277,
1630-1635 |
29. | Stahl, N., Farruggella, T. J., Boulton, T. G., Zhong, Z., Darnell, J. E., Jr., and Yancopoulos, G. D. (1995) Science 267, 1349-1353[Medline] [Order article via Infotrieve] |
30. |
Baumann, H.,
Morella, K. K.,
White, D. W.,
Dembski, M.,
Bailon, P. S.,
Kim, H.,
Lai, C.-F.,
and Tartaglia, L. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8374-8378 |
31. |
White, D. W.,
Kuropatwinski, K. K.,
Devos, R.,
Baumann, H.,
and Tartaglia, L. A.
(1997)
J. Biol. Chem.
272,
4065-4071 |
32. |
Carpenter, L. R.,
Farruggella, T. J.,
Symes, A.,
Karow, M. L.,
Yancopoulos, G. D.,
and Stahl, N.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6061-6066 |
33. |
Banks, A. S.,
Davis, S. M.,
Bates, S. H.,
and Myers, M. G., Jr.
(2000)
J. Biol. Chem.
275,
14563-14572 |
34. | Vaisse, C., Halaas, J. L., Horvath, C. M., Darnell, J. E., Jr., Stoffel, M., and Friedman, J. M. (1996) Nat. Genet. 14, 95-97[Medline] [Order article via Infotrieve] |
35. |
Wang, Y.,
Kuropatwinski, K. K.,
White, D. W.,
Hawley, T. S.,
Hawley, R. G.,
Tartaglia, L. A.,
and Baumann, H.
(1997)
J. Biol. Chem.
272,
16216-16223 |
36. | Berti, L., Kellerer, M., Capp, E., and Haring, H. U. (1997) Diabetologia 40, 606-609[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Kim, Y. B.,
Uotani, S.,
Pierroz, D. D.,
Flier, J. S.,
and Kahn, B. B.
(2000)
Endocrinology
141,
2328-2339 |
38. |
Takahashi, Y.,
Okimura, Y.,
Mizuno, I.,
Iida, K.,
Takahashi, T.,
Kaji, H.,
Abe, H.,
and Chihara, K.
(1997)
J. Biol. Chem.
272,
12897-12900 |
39. | Neel, B. G., and Tonks, N. K. (1997) Curr. Opin. Cell Biol. 9, 193-204[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Li, C.,
and Friedman, J. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9677-9682 |
41. | Tang, T. L., Freeman, R. M., Jr., O'Reilly, A. M., Neel, B. G., and Sokol, S. Y. (1995) Cell 80, 473-483[Medline] [Order article via Infotrieve] |
42. | Bennett, A. M., Hausdorff, S. F., O'Reilly, A. M., Freeman, R. M., and Neel, B. G. (1996) Mol. Cell. Biol. 16, 1189-1202[Abstract] |
43. |
Shi, Z. Q.,
Lu, W.,
and Feng, G. S.
(1998)
J. Biol. Chem.
273,
4904-4908 |
44. | Gu, H., Pratt, J. C., Burakoff, S. J., and Neel, B. G. (1998) Mol. Cell. 2, 729-740[Medline] [Order article via Infotrieve] |
45. |
Kim, H.,
and Baumann, H.
(1999)
Mol. Cell. Biol.
19,
5326-5338 |
46. |
David, M.,
Zhou, G.,
Pine, R.,
Dixon, J. E.,
and Larner, A. C.
(1996)
J. Biol. Chem.
271,
15862-15865 |
47. | Ali, S., Chen, Z., Lebrun, J. J., Vogel, W., Kharitonenkov, A., Kelly, P. A., and Ullrich, A. (1996) EMBO J. 15, 135-142[Abstract] |
48. |
You, M., Yu, D. H.,
and Feng, G. S.
(1999)
Mol. Cell. Biol.
19,
2416-2424 |
49. |
Bjørbæk, C.,
Lavery, H. J.,
Bates, S. H.,
Olson, R. K.,
Davis, S. M.,
Flier, J. S.,
and Myers, M. G., Jr.
(2000)
J. Biol. Chem.
275,
40649-40657 |
50. |
Myers, M. G., Jr.,
Mendez, R.,
Shi, P.,
Pierce, J. H.,
Rhoads, R.,
and White, M. F.
(1998)
J. Biol. Chem.
273,
26908-26914 |
51. | Park, J. A., and Koh, J. Y. (1999) Neurochem. 73, 450-456[CrossRef] |
52. |
Hodge, C.,
Liao, J.,
Stofega, M.,
Guan, K.,
Carter-Su, C.,
and Schwartz, J.
(1998)
J. Biol. Chem.
273,
31327-31336 |
53. |
Bjørbæk, C.,
El-Haschimi, K.,
Frantz, J. D.,
and Flier, J. S.
(1999)
J. Biol. Chem.
274,
30059-30065 |
54. |
Auernhammer, C. J.,
Chesnokova, V.,
Bousquet, C.,
and Melmed, S.
(1998)
Mol. Endocrinol.
12,
954-961 |
55. | Bennett, A. M., Tang, T. L., Sugimoto, S., Walsh, C. T., and Neel, B. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7335-7339[Abstract] |
56. | Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J., Cooper, J. A., and Schlessinger, J. (1994) Mol. Cell. Biol. 14, 509-517[Abstract] |
57. |
Shi, Z.-Q., Yu, D.-H.,
Park, M.,
Marshall, M.,
and Feng, G.-S.
(2000)
Mol. Cell. Biol.
20,
1526-1536 |
58. | Fujioka, Y., Matozaki, T., Noguchi, T., Iwamatsu, A., Yamao, T., Takahashi, N., Tsuda, M., Takada, T., and Kasuga, M. (1996) Mol. Cell. Biol. 16, 6887-6899[Abstract] |
59. |
Tsuda, M.,
Matozaki, T.,
Fukunaga, K.,
Fujioka, Y.,
Imamoto, A.,
Noguchi, T.,
Takada, T.,
Yamao, T.,
Takeda, H.,
Ochi, F.,
Yamamoto, T.,
and Kasuga, M.
(1998)
J. Biol. Chem.
273,
13223-13229 |
60. |
Oh, E. S.,
Gu, H.,
Saxton, T. M.,
Timms, J. F.,
Hausdorff, S.,
Frevert, E. U.,
Kahn, B. B.,
Pawson, T.,
Neel, B. G.,
and Thomas, S. M.
(1999)
Mol. Cell. Biol.
19,
3205-3215 |
61. |
Nicholson, S. E.,
De Souza, D.,
Fabri, L. J.,
Corbin, J.,
Willson, T. A.,
Zhang, J. G.,
Silva, A.,
Asimakis, M.,
Farley, A.,
Nash, A. D.,
Metcalf, D.,
Hilton, D. J.,
Nicola, N. A.,
and Baca, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6493-6498 |
62. |
Schmitz, J.,
Weissenbach, M.,
Haan, S.,
Heinrich, P. C.,
and Schaper, F.
(2000)
J. Biol. Chem.
275,
12848-12856 |
63. | Murakami, M., Narazaki, M., Hibi, M., Yawata, H., Yasukawa, K., Hamaguchi, M., Taga, T., and Kishimoto, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11349-11353[Abstract] |