From the Neurosurgical Service, Molecular
Neuro-Oncology, the ¶ Program in Neuroscience, Molecular
Neurobiology, and the ** Department of Neurology, Massachusetts
General Hospital and Harvard Medical School, Boston, Massachusetts
02129, and the
Department of Pharmacology, Uniformed Services
University of the Health Sciences, Bethesda, Maryland 20814
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ABSTRACT |
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The src homology 2 (SH2) domain-containing protein-tyrosine phosphatase SHP-2 has been implicated as an important positive regulator of several mitogenic signaling pathways. SHP-2 has more recently been shown to be tyrosine phosphorylated and recruited to the gp130 component of the ciliary neurotrophic factor (CNTF) receptor complex upon stimulation with CNTF. CNTF does not, however, have a proliferative effect on responsive cells, but rather enhances the survival and differentiation of sympathetic, motor, and sensory neurons. In this study, expression of an interfering mutant of SHP-2 in the neuroblastoma cell line NBFL increased CNTF induction of a vasoactive intestinal peptide (VIP) reporter gene, and in cultures of sympathetic neurons, it resulted in an up-regulation of endogenous VIP and substance P (SP) gene expression. Members of the CNTF family of cytokines transmit their signal by activating signaling pathways involving both STAT and Fos-Jun transcription factors. In CNTF-stimulated NBFL cells that constitutively express the SHP-2 interfering mutant, there was increased and prolonged formation of STAT/DNA complexes, but decreased AP-1 binding activity, that mirrored a down-regulation of c-fos expression both at the mRNA and protein level. Taken together, these data indicate that SHP-2 has dual and opposing roles in a signaling cascade triggered by the same ligand, as illustrated by its ability to differentially regulate the levels of activity of both STAT and AP-1 transcription factors.
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INTRODUCTION |
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Ciliary neurotrophic factor (CNTF)1 promotes survival and differentiation in a variety of neuronal and glial cells in the vertebrate nervous system (1, 2). In cultured sympathetic neurons derived from superior cervical ganglia (SCG) of newborn rats, CNTF induces a switch from a noradrenergic to a cholinergic phenotype (3). In addition, CNTF increases expression of the genes encoding several peptide neurotransmitters, such as vasoactive intestinal polypeptide (VIP), somatostatin (SOM), and substance P (SP) (4, 5). In vivo, a similar noradrenergic to cholinergic switch and increased expression of VIP occur in a subset of sympathetic neurons when they contact the target sweat glands (3). The CNTF-induced neuronal differentiation observed in SCG is mimicked by some neuroblastoma-derived cell lines, such as NBFL, that share a similar embryonic origin with sympathetic neurons (6, 7).
The CNTF receptor (CNTFR) consists of a ligand-binding component,
CNTR
, and two signal transducing
receptor subunits, gp130 and
LIFR
, that lack any obvious catalytic motif in their cytoplasmic
domains but are constitutively associated with protein-tyrosine kinases
of the JAK (Janus kinase) family. Ligand-induced heterodimerization of
gp130 and LIFR
leads to tyrosine phosphorylation and activation of
the associated JAKs, which in turn phosphorylate the
receptor subunits and downstream signaling molecules (8, 9). The phosphotyrosine
residues on the
receptor subunits provide binding sites for SH2
domain-containing signaling molecules, and mutational analysis of the
cytoplasmic domain of gp130 has identified the SH2 domain docking sites
for Stat3 and SHP-2 (10).
Stat3 is a member of a family of transcription factors that, when
activated by tyrosine phosphorylation, homo- or heterodimerize and
translocate to the nucleus to direct transcriptional responses (see
Ref. 11 for review). Many of the genes that are coordinately regulated
by CNTF, including choline acetyltransferase (ChAT), VIP, and SP,
contain consensus STAT-binding sites (12). In particular, within the 5'
end of the VIP gene is a 180-base pair cytokine response element (CyRE)
that binds a complex composed of Stat1 and Stat3 and mediates
CNTF-stimulated VIP expression (12).
SHP-2 (SHPTP2, PTP1D, PTP2C, or Syp) is a protein-tyrosine phosphatase
that contains a C-terminal catalytic domain and a tandem array of two
SH2 domains at its N terminus (see Ref.13 for review). SHP-2 has been
implicated as an essential component in a variety of signaling cascades
(13, 14). In EGF, PDGF, insulin, and FGF pathways, SHP-2 acts as a
positive regulator in the activation of mitogen-activated protein
kinase (MAPK) and mitogenic signaling (15-21). Recently, a positive
role for SHP-2 has also been shown in JAK/STAT signal transduction
pathways triggered by prolactin or IFN- or -
(22, 23). However,
the finding that SHP-2 is a negative regulator of the RAS pathway in T
cell receptor signaling (24) suggests that SHP-2 may have either a
positive or negative role depending on the type of signaling pathway
involved.
The phosphotyrosine-dependent association SHP-2 displays with the gp130 component of the CNTF receptor complex suggests an important role for SHP-2 in this signaling pathway. We investigated this possibility by introducing an SHP-2 interfering mutant (N-P2) into NBFL cells and then examining CNTF-induced gene expression. We found that overexpression of the N-P2 mutant resulted in an up-regulation of CNTF-induced transcriptional activation of a VIP gene reporter plasmid, but a down-regulation of CNTF-induced c-fos expression. In NBFL cells that stably expressed the N-P2 mutant, gel shift analysis demonstrated that the N-P2 mutant prolongs STAT/DNA binding activity while decreasing AP-1/DNA binding activity in response to CNTF. In sympathetic neurons, overexpression of the N-P2 mutant resulted in an increase in the CNTF-induced expression of endogenous VIP and SP genes. Together, these data indicate that in NBFL cells SHP-2 regulates CNTF-induced gene expression by controlling the signaling pathways that activate both STAT and AP-1 transcription factors. Hence, it is likely that SHP-2 controls the neuronal gene expression induced by CNTF in sympathetic neurons through a similar molecular mechanism.
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EXPERIMENTAL PROCEDURES |
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Construction of Plasmids-- The various SHP-2 and SHP-1 interfering mutants were constructed using the polymerase chain reaction (PCR) and primers that resulted in PCR amplification of either the N-terminal SH2 domain or the colinear N- and C-terminal SH2 domains. For all interfering mutants, the 5' primer included sequences encoding the Flag epitope (DYKDDDDK). All mutants were cloned into the pRK5 mammalian expression vector (kindly provided by R. Derynck, University of California San Francisco, CA) under the control of the cytomeglovirus promoter, and pHSV-1 amplicon vector under the control of the HSV-1 IE 4/5 promoter (25) (kindly provided by R. Neve, McLean Hospital, Belmont, MA). All PCR-amplified DNAs were sequenced and found not to contain nonspecific Taq polymerase-induced mutations. The full-length SHP-2 CS mutant (kindly provided by B. Neel, Beth Israel Hospital, Boston, MA) was also cloned as an EcoRI fragment into pRK5.
Cell Culture and Transfection--
NBFL cells were maintained as
described previously (6). Cells were seeded at 1 × 106/10-cm, and 24 h later were cotransfected using the
calcium phosphate precipitation technique (12). For VIP promoter
activation studies, cells were cotransfected with 10 µg of VIP-luc
(luciferase) plasmid, 10 µg of pRK5 expression vector encoding one of
the SHP-2 or SHP-1 mutants, and 3 µg of pRSV-CAT to control for
transfection efficiency. For c-fos promoter activation
studies, cells were cotransfected with 10 µg of c-fos-CAT
plasmid, 10 µg of pRK5 expression vector encoding the N-P2 mutant,
and 1 µg of pRSV-luc to control for transfection efficiency.
Transfected cells were placed in serum-free medium for 12-24 h before
treatment with CNTF (25 ng/ml). Twelve hours later (VIP-luc) or 36 h later (c-fos-CAT), the cells were harvested, and
luciferase (26) and CAT (27) activities were assayed. For stable
transfectants, NBFL cells were cotransfected with 12 µg of a pRK5
expression vector encoding the N-P2 mutant and 2 µg of puromycin
resistance plasmid (pJ). Puromycin-resistant colonies were selected
in 1 µg of drug/ml for 3 weeks. Single colonies expressing high
levels of the N-P2 mutant were grown in medium supplemented with 1 µg
of puromycin.
Immunoprecipitation and Western Blotting-- NBFL cells were lysed for 20 min on ice in 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 2 mM Na3VO4, aprotinin (5 µg/ml), leupeptin (5 µg/ml), and phenylmethylsulfonyl fluoride (50 µg/ml). For immunoprecipitation, cell lysates were incubated with 4 µg of a C-terminal SHP-2 polyclonal antibody (Santa Cruz Biotechnology) for 1 h at 4 °C, and then with protein A-agarose for 1 h at 4 °C. Immunoprecipitation of the Flag epitope-tagged N-P2 mutant from lysates of NBFL transfected cells was performed with anti-Flag M1/M2 Affinity gel (Kodak) for 2 h at 4 °C. Immunoprecipitates were washed four times in lysis buffer and boiled for 5 min in 2× SDS sample loading buffer. Immunoprecipitates were then subjected to SDS-polyacrylamide gel electrophoresis (8% gel), transferred to nitrocellulose membrane, and probed with the indicated antisera. All the immunoblots were visualized using the enhanced chemiluminescence (ECL) Western blotting Detection System Kit (Amersham Pharmacia Biotech). For reprobing, the membranes were incubated for 30 min at 50 °C in stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol).
Electrophoretic Mobility Shift Assay (EMSA)--
Complementary
oligonucleotides with GGG or GATC overhangs were annealed and labeled
with [-32P]dCTP using MMLV reverse transcriptase (RT)
(Promega) or Klenow fragment (Promega). Complementary AP-1
oligonucleotides (Santa Cruz Biotechnology) had no overhang and were
labeled with [
-32P]ATP using T4 polynucleotide kinase.
Nuclear extracts were prepared, and binding reactions were carried out
as described previously (12). For supershift experiments, nuclear
extracts were preincubated with 2 µg of poly(dI-dC) and anti-Stat1
(Transduction Laboratories), or anti-Stat3 (C-20; Santa Cruz
Biotechnology), or control antibodies for 15 min on ice. After the
addition of the probe (~20,000 cpm), the binding reactions were
continued for 20 min at room temperature. Samples were then resolved on
a 5% non-denaturing polyacrylamide gel in 0.5 × Tris borate/EDTA
buffer at 200 V. The sequences of the complementary nucleotides used
(SIE (SIEm67), G3, and noncanonical AP-1 (m3CyB)) have been reported
(12, 31).
Immunocytochemistry-- Sympathetic neurons were fixed with 4% paraformaldehyde for 30 min. The Flag epitope-tagged mutants were detected by M5 antibody (25 µg/ml; Kodak) followed by the APAAP immunoalkaline phosphatase procedure (APAAP Kit; DAKO).
Packaging of HSV-1 Amplicon Vectors and Infection of Neuronal Cultures-- pHSV-N-P2, pHSV-N-P1, and pHSV-LacZ amplicon DNAs were packaged into HSV-1 particles as described previously (25) with the following modifications. Plasmid DNAs were transfected into permissive Vero 2-2 cells using LipofectAMINE (Life Technologies, Inc.). One day later, cells were infected with dl5.2 helper virus (32). Virus were passaged and amplified three times to optimize helper:amplicon ratios. Amplified virus were purified and concentrated on sucrose gradients. pHSV-N-P2 and pHSV-N-P1 amplicon titers were determined by immunocytochemistry using M5 anti-Flag antibody (Kodak) on BHK cells. Helper virus titers were determined by plaque formation on Vero 2-2 cells. Titers of the viral stocks were 2 × 104 infectious amplicon particles/µl and 1 × 105 dl5.2 helper particles/µl, with an amplicon to helper ratio of 2:10. The pHSV-LacZ virus was used at the same helper virus titer as the helper virus titer of the other two stocks to control for viral cytotoxicity. Sympathetic neurons were infected on the 3rd day of culture with 0.5 µl of each viral stock for 3 h. Twenty-four h after infection, the neurons were stimulated with CNTF (10 ng/ml) for 12 h.
Quantitative Reverse Transcriptase-PCR-- To obtain a quantitative estimate of SP and VIP gene transcripts, we performed a quantitative RT-PCR assay (33), using neurofilament M (NF-M) as an internal standard. Total RNA was isolated from sympathetic neurons cultured in each well using the TRIzol reagent (Life Technologies, Inc.). Contaminating genomic DNA was removed by incubation with 10 units of DNase for 30 min at 37 °C in the presence of 40 units of RNasin (Promega). RNA was phenol/chloroform extracted and ethanol-precipitated. An average of 1 µg of RNA was obtained from each cell sample; half was used as control for genomic DNA contamination during PCR reactions and half was reverse transcribed as follows. Total RNA was mixed with 0.5 µg of oligo(dT) (Life Technologies, Inc.), 0.5 mM dNTP, and 1× RT buffer (Promega) in a final volume of 19 µl and incubated at 65 °C for 5 min. After 10 min at 37 °C, 200 units of MMLV reverse transcriptase were added. The solution was incubated for 50 min at 37 °C, and the RT was then inactivated by heating to 95 °C for 5 min. The consistency of reverse transcription was validated by comparing the results of PCR analyses of 0.2, 0.5, and 1 µg of total RNA; reverse transcription was found to be quantitative because the PCR amplification products of SP, VIP, and NF-M were directly proportional to the amounts of input RNA.
The cDNAs obtained from the reverse transcription reaction were used as template for PCR amplification. The sequence of each pair of primers has been reported (5, 34). Each cDNA was amplified in the presence of [ ![]() |
RESULTS |
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SHP-2 Is a Negative Signal Regulator in CNTF-induced VIP Gene Transcription in NBFL Cells-- To determine whether SHP-2 may play a role in CNTF-induced gene expression, we first used the human neuroblastoma cell line, NBFL, as a model system. NBFL cells respond to CNTF by coordinately inducing multiple neuropeptide genes, in a similar way to sympathetic neurons (6). To remove endogenous SHP-2 activity from the CNTF signaling cascade in NBFL cells, we used SHP-2 interfering mutants that lack the catalytic domain (Fig. 1A). SHP-2 mutants should compete with endogenous SHP-2 for binding to the SHP-2 phosphotyrosine containing binding site on gp130. NBFL cells were cotransfected with SHP-2 mutants (N-P2, NC-P2, or CS-P2) and a CNTF-responsive luciferase reporter (VIP-luc) (6), which contains 1929 base pairs upstream of the transcription start site of the VIP gene. As a control for specificity of the SHP-2 mutants, we used the corresponding mutant forms of the related tyrosine phosphatase SHP-1 (N-P1 and NC-P1; Fig. 1A). While SHP-2 is broadly expressed, SHP-1 is mainly expressed in hematopoietic cells (see Ref. 13 for review). CNTF-stimulated VIP-luc reporter activity was ~11-fold in NBFL cells transfected with the parental expression vector alone (Fig. 1B, CT). Transfection of either the colinear N- and C-terminal SH2 domains (NC-P2) or the N-terminal SH2 domain (N-P2) of SHP-2 enhanced CNTF-stimulated VIP-luc activity ~2-fold over the CT value, without affecting the basal level. The effect of the SHP-2 mutants was specific, because overexpression of the corresponding mutant forms (NC-P1 and N-P1) of SHP-1 did not significantly alter basal or CNTF-induced VIP-luc transcription over the CT value. Transfection of the full-length but catalytically inactive dominant negative SHP-2 mutant (CS-P2), in which the essential catalytic cysteine residue has been replaced with serine (21), enhanced CNTF-stimulated VIP-luc activity ~3-fold over the CT value. The stronger effect of CS-P2 compared with the other two SHP-2 mutants could reflect the ability of the CS mutant to tightly bind and "trap" substrates in addition to competing for binding to gp130 (21, 35). These transfection studies using catalytically inactive SHP-2 mutants indicate that endogenous SHP-2 plays a negative role in the transmission of the CNTF signal in NBFL cells.
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The N-P2 Interfering Mutant Specifically Associates with the CNTF
Receptor Complex--
Further experiments were performed using the
N-P2 interfering mutant because its effect on CNTF-induced VIP
expression in NBFL cells is substantial and comparable to the effect of
the other mutants. To confirm that the N-P2 mutant interacts
specifically with the CNTF receptor complex, we compared the pattern of
phosphotyrosine-containing proteins that coimmunoprecipitate with
endogenous SHP-2 with those that coimmunoprecipitate with the N-P2
interfering mutant following CNTF stimulation. We found that
tyrosine-phosphorylated bands of ~190 and ~130 kDa, which
correspond to LIFR and gp130 (36), coimmunoprecipitated with
endogenous SHP-2 in CNTF-treated NBFL cells (Fig. 1C). Two
additional tyrosine-phosphorylated proteins of ~90 and ~68 kDa also
coimmunoprecipitated with the CNTF receptor complex. The ~68-kDa
phosphoprotein comigrated with SHP-2 (Fig. 1C); however, the
~90 kDa phosphoprotein is of unknown identity (the protein was not
recognized by antibodies to Stat1
or Stat3; data not shown).
Interestingly, a 90-kDa tyrosine-phosphorylated protein named SIRP
1
has recently been characterized, which associates with SHP-2 upon EGF,
PDGF, or insulin stimulation (37). Whether the 90-kDa protein that
coimmunoprecipitates with SHP-2 upon CNTF stimulation might be SIRP
1
still remains to be determined. Similar to that observed with
endogenous SHP-2, tyrosine-phosphorylated gp130 and LIFR
coimmunoprecipitated with the Flag-tagged N-P2 interfering mutant after
CNTF stimulation (Fig. 1C;
Flag lanes). The
90-kDa phosphoprotein, however, was not detected in the
immunoprecipitates, indicating that the 90-kDa phosphoprotein does not
associate with SHP-2 through the N-terminal SH2 domain.
N-P2 Mutant Prolongs DNA Binding of STAT Complexes in Response to
CNTF--
The VIP gene contains a 180 base pairs CyRE within its
5'-flanking sequence that binds activated STAT after CNTF stimulation and which mediates induction of the VIP gene (12, 31). To investigate
how SHP-2 may regulate CNTF-dependent VIP transcriptional induction, we examined whether SHP-2 affects the ability of activated STAT to bind to elements within the CyRE using NBFL clonal cell lines
that stably express the N-P2 mutant. NBFL cells were transfected with
an expression vector containing the N-P2 interfering mutant and
puromycin-resistant clones were established and characterized for the
level of expression of the mutant protein. Several clones were found
that expressed the N-P2 mutant at levels comparable with that of the
endogenous SHP-2 (Fig. 2A). In
the following experiments, similar results were obtained with different
clones; therefore, only data from one representative clone, NBFL-N2,
are reported. Parental NBFL and NBFL-N2 cells were serum-starved for 12 h and then stimulated with CNTF for increasing times. Nuclear extracts were prepared before and after CNTF treatment, and the degree
of STAT/DNA binding activity was assessed by EMSA using either the
high-affinity variant of the c-fos sis-inducible element (SIE) sequence (38) or the acute phase response element (APRE)-like site of the CyRE (G3) (12) as a probe. In parental NBFL cells, CNTF
treatment resulted in the stimulation of SIE binding activity of the
STATs, which at 30 min was primarily represented by complex A (Fig.
2B). Supershift analysis confirmed this complex as being composed of Stat3 homodimers (Fig. 2B), as described
previously (38, 39). The two faster migrating complexes B and C were much less abundant. Complex C reacted almost quantitatively with Stat1 antibody, while complex B reacted with both anti-Stat1
and
anti-Stat3 antibodies (Fig. 2B). None of the complexes were altered in the presence of irrelevant antibody. A small amount of
complex A was still present 4 h after CNTF treatment, while complexes B and C were undetectable after 1 h. EMSA performed with
NBFL-N2 nuclear extracts revealed an increase in the amount and
duration of CNTF-induced binding of STAT proteins to the SIE probe
(Fig. 2B); most notably in the Stat3-containing complex A,
as shown by supershift analysis (Fig. 2B). When the G3 site within the CyRE was used as a probe in EMSA with nuclear extracts from
NBFL or NBFL-N2 cells, complexes A, B, and C were visible after 30 min
of CNTF stimulation (Fig. 2C). Supershift assays confirmed
the identity of the most retarded complex A as a Stat3 homodimer, while
the two faster migrating complexes B and C were Stat1
/Stat3
heterodimers and Stat1
homodimers, respectively (Fig.
2C). While there was no detectable difference in the amount of CNTF-induced G3/STAT complexes in nuclear extracts from parental NBFL and NBFL-N2 cells at 30 min, there was a prolonged duration of
binding of the Stat3-containing complex A in NBFL-N2 cells, which at
4 h was still visible as a band of similar intensity of that
observed at 1 h.
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N-P2 Mutant Decreases AP-1 Binding Activity and c-fos Expression in Response to CNTF-- Previous studies have shown that in addition to activating the STATs, CNTF and related cytokines activate members of the MAPK family as well as the Fos-Jun transcription factors (40-45). Furthermore, an AP-1 complex (referred to as Complex IV) containing c-Fos, JunB, and JunD has been described previously as binding a noncanonical AP-1 site within the VIP CyRE in a CNTF-dependent manner (31). This AP-1 partially overlaps with a C/EBP-like motif, responsible for binding to a complex (Complex III) present in nuclear extracts from untreated NBFL cells (31). Because the AP-1-like binding site appears to be important for full CNTF-dependent transcriptional activation mediated by the CyRE, we asked whether AP-1 binding activity was altered in NBFL-N2 cells. Nuclear extracts from parental and NBFL-N2 cells were incubated with either a consensus AP-1 binding site or the noncanonical AP-1 site of the CyRE as a probe. As expected, an increase in binding of nuclear proteins to both probes was observed after CNTF stimulation in parental NBFL cells, reaching a maximal level of binding after ~1 h (Fig. 3, A and B). The addition of c-Fos antibody to the EMSA performed with the two AP-1 binding site probes inhibited complex binding, confirming that c-Fos is a major component of the AP-1 complex and complex IV (Fig. 3, A and B). CNTF-induced binding of AP-1 complexes to both probes was substantially reduced when nuclear extract from NBFL-N2 was used (Fig. 3, A and B). No detectable differences in the formation of the uninducible complex III were observed between NBFL and NBFL-N2 cells.
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SHP-2 Coordinately Down-regulates CNTF Induction of Endogenous VIP and SP Genes in Sympathetic Neurons-- The VIP gene is one of several genes that are coregulated by CNTF in NBFL cells. The neurotransmitter gene expression induced by CNTF in NBFL cells mimics that which occurs in sympathetic neurons, suggesting that a common molecular mechanism mediates the response to CNTF in both NBFL cells and sympathetic neurons. Therefore, we examined whether SHP-2 may also play a role in CNTF-induced expression of the endogenous VIP gene in cultured sympathetic neurons. In addition, to address whether SHP-2 may control in a coordinate manner the transcriptional activation that occurs in CNTF-induced neuronal differentiation of sympathetic neurons, we also examined the role of SHP-2 in the expression of the SP gene.
Expression of SHP-2 in sympathetic neurons was confirmed by means of immunofluorescent staining and RT-PCR (data not shown). In time course (up to 72 h) and dose-response experiments (10-25 ng/ml CNTF), we found that 10 ng/ml CNTF induced a substantial and quantifiable increase of VIP and SP mRNA levels in as little as 12 h. VIP and SP gene expression was determined by a quantitative RT-PCR assay, using NF-M as an internal standard (Fig. 4, A and B). Similar results were obtained when VIP and SP mRNA levels were normalized to constitutively expressed
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DISCUSSION |
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Our studies show that SHP-2 negatively regulates CNTF-induction of VIP and SP gene expression while at the same time positively regulating c-fos expression. These data indicate that SHP-2 has dual and opposing roles in a signaling cascade triggered by the same ligand and clearly demonstrate that SHP-2 is a multifunctional protein in the regulation of the pathways activated by cytokines or growth factors.
The enhancement of CNTF-induced STAT binding activity observed in NBFL
cells that overexpress the SHP-2 interfering mutant suggests that the
negative function of SHP-2 could be at the level of the STATs or the
STAT activating JAK kinases. Possible targets for SHP-2 phosphatase
activity may be Jak1 or Jak2 since these kinases are activated by
tyrosine phosphorylation upon CNTF stimulation in NBFL cells (data not
shown) and a direct association of SHP-2 with the JAKs has recently
been shown (50-52). Inhibition of JAK-STAT signaling by SHP-2 through
dephosphorylation of JAKs has been proposed in the interleukin-6
(IL-6)-triggered signaling pathway (51). In addition, a similar
mechanism has been suggested for the related tyrosine phosphatase
SHP-1, which plays a major role in terminating the erythropoietin- and
/
interferon-stimulated JAK-STAT pathway (23, 53). Alternatively,
because SHP-2 associates directly with gp130, it may dephosphorylate
phosphotyrosine docking sites on gp130. One obvious possibility is the
Stat3 binding site on gp130 (10). Lastly, because maximal activation of
Stat1
and Stat3 appears to be dependent on serine phosphorylation
(54-56), it is possible that SHP-2 modulates STAT activation by
controlling kinases of the MAPK family. Cytokines of the CNTF family
induce both tyrosine phosphorylation and H7 (a serine/threonine kinase inhibitor)-sensitive phosphorylation of Stat3 (57). Indeed, in NBFL
cells, induction of VIP gene expression by CNTF is abolished by H7 (12,
42). A link between SHP-2 and MAPK activation has been shown in
mitogenic signaling pathways, with SHP-2 being generally a positive,
but sometimes a negative regulator of MAPK activity (58). MAPKs can be
activated by cytokines in a cell-dependent manner (59, 60),
and several lines of evidence suggest that JAKs may be required for
activation of both STATs and ERK/MAPKs by cytokines, providing
mechanisms for the optimization of gene induction (61-64).
Both the AP-1 and STAT binding elements present in the CyRE of the VIP gene cooperate to achieve maximal rates of transcription upon CNTF stimulation (12, 31). What is puzzling, however, is that while SHP-2 negatively regulates STAT binding activity, it in parallel positively regulates AP-1 binding activity by controlling levels of c-Fos. This observation raises the possibility that SHP-2 controls other regions, as yet unidentified, within the VIP CyRE, resulting in its down-regulation.
In the context of CNTF signaling, the down-regulation that SHP-2 exerts on VIP expression is in striking contrast with the up-regulation SHP-2 has on c-fos expression in NBFL cells. Multiple regulatory elements are present in the c-fos promoter, including the SIE and SRE. In our experiments, although overexpression of the N-P2 interfering mutant significantly increased STAT binding to the SIE, the actual effect on c-fos was to decrease expression. Therefore, it appears that the positive effect SHP-2 has on CNTF induction of c-fos expression is not through STATs, but more likely through regulation of transcription factors that bind other regulatory elements in the c-fos promoter, such as the SRE. Indeed, a number of studies have indicated only a minor contribution of the SIE to induction of c-fos expression (see Ref. 63, and references within) but a major role for the SRE in a variety of physiological settings (38, 63, 65).
A requirement of SHP-2 in the activation of c-fos
transcription has been reported previously in insulin (15), PDGF (66), and -thrombin (20) triggered pathways. Expression of
c-fos is rapidly increased in many cell types in response to
several mitogens (67), and therefore the up-regulation of
c-fos that is dependent on SHP-2 might be related to the
mitogenic role that SHP-2 has on these cell types. However, because
CNTF is not a mitogenic factor for either NBFL cells or progenitor
sympathetic neurons (68, 69), but rather a factor that can induce
differentiation, the induction of c-fos is likely to be
needed in the context of neuronal differentiation.
The negative regulation that SHP-2 exerts on CNTF-induced expression of the SP and VIP genes is likely to be mediated through a common mechanism since STAT consensus binding sites are present in the promoter region of both genes (70, 71). It is conceivable then that SHP-2 may have a similar effect on the expression of other genes that contain STAT binding sites and that are coordinately regulated by CNTF. Indeed, STAT binding sites have also been identified within the promoter regions of the SOM, cholecystokinin, enkephalin, and ChAT genes. Together with VIP and SP, these neuropeptide and neurotransmitter synthetic enzyme genes define the cholinergic phenotype in sympathetic neurons (72). Therefore it is possible that SHP-2 is present in these neurons to monitor neurocytokine-induced cholinergic differentiation.
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ACKNOWLEDGEMENTS |
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We thank B. Neel for providing the SHP-1 and SHP-2 plasmids, Rachel Neve for the HSV amplicon and helper virus, and Peter Pechan and for helpful suggestions with the HSV amplicon system.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO1 NS27514-08.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 a fellowship from the Associazione Italiana per la Ricerca sul Cancro, Rome, Italy and the Amgen Medical Discovery Fellowship.
Supported in part by Massachusetts General Hospital
Neurosurgical Service and to whom correspondence should be addressed: Molecular Neuro-Oncology, Neurosurgical Service, 149 13th St., Massachusetts General Hospital and Harvard Medical School, Boston, MA
02129. Tel.: 617-726-5510; Fax: 617-726-5079; E-mail:
reeves{at}helix.mgh.harvard.edu.
1 The abbreviations used are: CNTF, ciliary neurotrophic factor; SH2, src homology 2; VIP, vasoactive intestinal peptide; SCG, superior cervical ganglia; SOM, somatostatin; SP, substance P; CNTFR, CNTF receptor; EMSA, electrophoretic mobility shift assay; JAK, Janus kinase; ChAT, choline acetyltransferase; CyRE, cytokine response element; MAPK, mitogen-activated protein kinase; RT-PCR, reverse transcriptase-polymerase chain reaction; MMLV, Moloney murine leukemia virus; SIE, sis-inducible element; APRE, acute phase response element; SRE, serum response element; NF-M, neurofilament M; CAT, chloramphenicol acetyltransferase; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor.
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REFERENCES |
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