From the Departments of Microbiology and Immunology
and
Physiology, University of Michigan Medical School, Ann
Arbor, Michigan 48109 and the ¶ Department of Molecular Biology,
Max-Planck-Institute für Biochemie,
82152 Martinsried, Germany
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ABSTRACT |
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SIRPs (signal-regulatory proteins) are a family
of transmembrane glycoproteins that were identified by their
association with the Src homology 2 domain-containing protein-tyrosine
phosphatase SHP-2 in response to insulin. Here we examine whether
SIRP and SHP-2 are signaling molecules for the receptors for growth
hormone (GH), leukemia inhibitory factor (LIF), or interferon-
(IFN
), cytokine receptor superfamily members that bind to and
activate Janus kinase 2 (JAK2). In 3T3-F442A fibroblasts, GH rapidly
stimulates tyrosyl phosphorylation of both SIRP
and SHP-2 and
enhances association of SHP-2 with SIRP
. Consistent with JAK2
binding and phosphorylating SIRP
in response to GH, co-expression of
SIRP
and JAK2 in COS cells results in tyrosyl phosphorylation of
SIRP
and JAK2 association with SIRP
. LIF does not stimulate
tyrosyl phosphorylation of SIRP
but stimulates greater tyrosyl
phosphorylation of SHP-2 than GH. Additionally, LIF enhances
association of SHP-2 with the gp130 subunit of the LIF receptor
signaling complex. IFN
, which stimulates JAK2 to a greater extent
than LIF, is ineffective at stimulating tyrosyl phosphorylation of
SIRP
or SHP-2. These results suggest that SIRP
is a signaling
molecule for GH but not for LIF or IFN
. Differential phosphorylation
of SIRP
and SHP-2 may contribute to the distinct physiological
effects of these ligands.
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INTRODUCTION |
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The protein tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) is a member of a protein-tyrosine phosphatase family characterized by two tandem SH21 domains in the N
terminus and a catalytic domain in the C terminus. This family also
includes the hematopoietic cell phosphatase SHP-1 and the
Drosophila phosphatase Csw (1-4). Unlike SHP-1, SHP-2 is
ubiquitously expressed in vertebrate cells and tissues (5, 6). SHP-2
plays a critical role in development because disruption of the
shp2 gene in mice results in embryonic lethality of mice homozygous for the mutant shp2 (7, 8). At the cellular
level, SHP-2 has been implicated in signaling by receptor tyrosine
kinases and cytokine receptors. In response to ligand, the SH2 domains of SHP-2 associate with IRS-1, insulin receptor, EGF receptor, PDGF
receptor, or erythropoietin receptor (5, 9-12). Binding of the SH2
domains of SHP-2 to tyrosyl phosphorylated signaling molecules is
thought to regulate SHP-2 phosphatase activity because phosphopeptides
corresponding to SHP-2 binding sites in PDGF receptor and IRS-1
stimulate SHP-2 phosphatase activity (13, 14). In addition to binding
tyrosyl phosphorylated signaling molecules in response to ligand, SHP-2
is tyrosyl phosphorylated in response to epidermal growth factor and
PDGF as well as ligands for cytokine receptors that activate the Janus
tyrosine kinases JAK1 and JAK2, such as erythropoietin, IL-11, IL-3,
granulocyte-macrophage colony stimulating factor, IFN/
,
prolactin, and ciliary neurotrophic factor (5, 11, 13, 15-20).
The tyrosines in SHP-2 that are phosphorylated by PDGF receptor, JAK1,
or JAK2 are within consensus binding sites for the adapter protein Grb2
and have been shown to mediate Grb2-SHP-2 association in response to
PDGF, IL-3, or granulocyte-macrophage colony-stimulating factor (17,
21-23). Thus, SHP-2 has been hypothesized to play a positive role in
signal transduction by serving as an adapter protein between the
receptor and Grb2, linking SHP-2 phosphorylation to the
Grb2-SOS-Ras-MAPK pathway (22, 24). However, the role of SHP-2 in
growth factor and cytokine receptor signaling pathways may be more
complex than that of an adapter molecule. The catalytic activity of
SHP-2 is critical for its ability to modulate certain cellular
responses to ligands for some receptor tyrosine kinases and cytokine
receptors. For example, SHP-2 phosphatase activity is required for
fibroblast growth factor-induced Xenopus development, EGF-induced cell cycle progression, and PDGF-induced mitogenesis (25-27). Additionally, overexpression of catalytically inactive SHP-2
but not of wild-type SHP-2 inhibits ligand-stimulated reporter gene
activity or MAPK activity in response to insulin, EGF, prolactin, or
IFN/
(19, 20, 26, 28-30).
How phosphatase-inactive SHP-2 negatively regulates signaling is poorly
understood. One possible mechanism is that overexpression of inactive
SHP-2 prevents dephosphorylation by endogenous SHP-2 of a negative
regulator of signaling. In support of this model, overexpression of
catalytically inactive but not of active SHP-2 results in hyper tyrosyl
phosphorylation of an SHP-2-associated 115-kDa protein in response to
insulin in NIH3T3 and CHO cells (28, 31, 32). The rat and human
SHP-2-associated 115-kDa proteins have been cloned and designated
SHPS-1 (33) and SIRP (34), respectively, and are both substrates for
SHP-2. SIRPs are a family of transmembrane glycoproteins that are
divided into two subgroups, based on the presence (SIRP) or the
absence (SIRP
) of a cytoplasmic domain. SIRP
proteins contain
four potential tyrosine phosphorylation sites and a proline-rich region
in their cytoplasmic region. The tyrosine phosphorylated cytoplasmic
domain is required for growth factor-induced association of SHP-2 with SIRP (34).
Four SIRPs, SIRP1, SIRP
2, SIRP
3, and SIRP
1, have been
cloned. The best characterized is SIRP
1. SIRP
1 is believed to be
a negative regulator of growth factor signaling because overexpression of wild-type but not mutant SIRP
1 lacking the SHP-2 binding region inhibits growth factor-induced MAPK activation and cell proliferation (34). Whether SIRP
family members are regulated by cytokine receptor
signaling is unknown. We therefore examined the role of SIRP
in
signaling initiated by GH, LIF, or IFN
, ligands that activate the
Janus tyrosine kinases (35-37).
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EXPERIMENTAL PROCEDURES |
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Materials--
Recombinant 22,000 human GH was a gift from Eli
Lilly. Murine recombinant LIF was from R & D Systems. Murine
recombinant IFN was from Sigma. The mammalian expression vector prk5
encoding either murine JAK2 or kinase-inactive K882E JAK2 was kindly
provided by J. Ihle (St. Jude Children's Research Hospital, Memphis,
TN). Endoglycosidase-F/N-Glycosidase F, Triton X-100,
leupeptin, and aprotinin were from Boehringer Mannheim. Recombinant
protein A-agarose was from Repligen. The ECL detection system and
anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase
were from Amersham. Prestained protein molecular weight standards were
from Life Technologies, Inc.
Antisera--
Anti-SHP-2 antibody (SHP-2) raised against a
peptide corresponding to amino acids 576-593 of human SHP-2 (Santa
Cruz) was used for immunoprecipitations at a dilution of 1:250.
Monoclonal
SHP-2 antibody raised against a peptide corresponding to
amino acids 1-177 of human SHP-2 (Transduction Laboratories) was used for immunoblotting at a dilution of 1:2500. Anti-phosphotyrosine antibody 4G10 (
PY) (Upstate Biotechnology, Inc.) was used at 1:7500
for Western blotting. Antibody to gp130 raised against amino acids
895-914 of murine gp130 (Santa Cruz) was used at 1:2500 for Western
blotting. Antibody to GHR (
GHR) raised against recombinant rat
GH-binding protein, was kindly provided by W. Baumbach (American Cyanamid, Princeton, NJ) and was used at a dilution of 1:1000 for
immunoprecipitation. Antibody to JAK2 raised against a peptide corresponding to amino acids 758-776 of murine JAK2 was used at a
dilution of 1:1000 for immunoprecipitation and 1:15,000 for Western
blotting (38). Antibody to SIRP (
SIRP) raised against a glutathione
S-transferase fusion protein containing amino acids 336-503
of human SIRP
1 was used at dilutions of 1:1000 and 1:4000 for
immunoprecipitation and Western blotting, respectively (34).
Transfection and Cell Culture--
The stock of 3T3-F442A cells
was provided by H. Green (Harvard University). 3T3-F442A cells were
cultured as described previously (39). COS-7 cells were transfected
with prk5 expression vectors encoding the indicated cDNAs by
calcium phosphate precipitation (40). cDNAs for SIRP1 (10 µg),
murine JAK2 (2.5 µg), or kinase-inactive K882E JAK2 (2.5 µg) were
used for transient transfection. Empty prk5 expression vector was added
to ensure equivalent amounts of DNA in each transfection. After 24 h, cells were washed twice with Dulbecco's modified Eagle's medium
and incubated for an additional 24 h with Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 1 mM
L-glutamine and antibiotics. Cell lysates were prepared
48 h post-transfection.
Immunoprecipitation, Western Blotting, and Protein
Deglycosylation--
Confluent 3T3-F442A fibroblasts were incubated in
serum-free medium (41). GH, LIF, or IFN were then added at 37 °C
for the indicated times at the indicated concentrations. COS or
3T3-F442A cells were washed twice with ice-cold 10 mM
sodium phosphate, pH 7.4, 150 mM NaCl, 1 mM
Na3VO4 and solubilized in lysis buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 150 mM
NaCl, 2 mM EGTA, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Cell lysates
were centrifuged at 12,000 × g for 10 min, and the
supernatants were incubated on ice for 2 h with the indicated
antibody. Immune complexes were collected with protein A-agarose for
1 h at 8 °C, washed three times with 50 mM Tris, pH
7.5, 0.1% Triton X-100, 137 mM NaCl, 2 mM
EGTA, and boiled for 5 min in a mixture of lysis buffer and 5×
SDS-polyacrylamide gel electrophoresis sample buffer (250 mM Tris, pH 6.8, 10% SDS, 10%
-mercaptoethanol, and
40% glycerol). Samples were resolved by SDS-polyacrylamide gel
electrophoresis followed by Western blot analysis with the indicated
antibodies using the ECL detection system (41). Blots were either
directly reprobed with antibody as indicated or stripped in stripping
buffer (100 mM
-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl) at 55 °C for 30 min and then reprobed with
the indicated antibody. For deglycosylation experiments, immunoprecipitated proteins were deglycosylated as described previously (34). Samples were analyzed by Western blotting as described above.
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RESULTS |
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GH Stimulates Tyrosyl Phosphorylation of SHP-2 and an
SHP-2-associated 120-kDa Protein--
We first examined whether GH
treatment induces tyrosyl phosphorylation of SHP-2. 3T3-F442A cells
were incubated with GH for various times, and SHP-2 was
immunoprecipitated using SHP-2. GH induces a rapid and transient
tyrosyl phosphorylation of SHP-2, with tyrosyl phosphorylation apparent
at 2.5 min after GH treatment (the earliest time tested) and decreasing
to near basal levels 60 min after GH stimulation (Fig.
1, lanes 1-6). Reprobing the blot with
SHP-2 indicated that the amount of SHP-2 did not change (Fig. 1, lower panel). A prominent tyrosyl phosphorylated
protein with an Mr of ~120,000 (p120)
co-precipitated with SHP-2. p120 displayed a rapid and transient time
course of tyrosyl phosphorylation in response to GH, similar to that of
SHP-2. To determine if GH enhances the association of p120 with SHP-2
or stimulates tyrosyl phosphorylation of p120 constitutively associated
with SHP-2, we first had to determine the identity of p120.
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The SHP-2-associated p120 Is a Glycoprotein with the Migration
Properties of SIRP--
The SHP-2-associated p120 migrates with a
size appropriate for either GHR or a SIRP
protein. To distinguish
between these proteins,
SHP-2,
SIRP, or
GHR immunoprecipitates
from GH-treated 3T3-F442A cells were deglycosylated, and proteins were
visualized by immunoblotting with
PY. In the absence of
endoglycosidase, the SHP-2-associated p120 more closely co-migrates
with SIRP
than with GHR (Fig. 2,
compare lanes 1, 3, and 5).
Deglycosylation of
SIRP immunoprecipitates reduces the
Mr of SIRP
to ~60,000 (Fig. 2, lane
2), whereas deglycosylation of
GHR immunoprecipitates reduces
the Mr of GHR to ~90,000 (Fig. 2, lane
6), in agreement with previous results (34, 42). Deglycosylation
of
GHR immunoprecipitates revealed an additional 130-kDa
phosphoprotein that is believed to be GHR-associated JAK2 (Fig. 2,
lane 6) (35). Importantly, deglycosylation of
SHP-2
immunoprecipitates results in reduction of the majority of
SHP-2-associated p120 to 60 kDa, a size appropriate for a SIRP
protein (Fig. 2, lane 4). A small amount of deglycosylated protein from
SHP-2 immunoprecipitates also co-migrates with GHR (Fig. 2, lane 4). These data suggest that SHP-2 associates
with both GHR and SIRP
, with SIRP
being the major
SHP-2-associated tyrosyl phosphorylated protein in GH-treated 3T3-F442A
cells.
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GH Stimulates Tyrosyl Phosphorylation of SIRP and
Association of SIRP
with SHP-2--
To confirm that the
SHP-2-associated p120 protein is SIRP
and to determine if GH
stimulates SHP-2 association with SIRP
,
SHP-2 immunoprecipitates
from untreated or GH-treated cells were immunoblotted with
SIRP.
Because the specificity of this antibody for SIRP
1, SIRP
2, and
SIRP
3 has not been determined, we shall refer to the SIRP that this
antibody Western blots or immunoprecipitates as SIRP
. As shown in
Fig. 3A, lanes 1 and 2, GH stimulates the association of SIRP
with SHP-2.
No GHR was detectable by immunoblotting
SHP-2 immunoprecipitates
with
GHR (data not shown), as predicted from the small amount of
tyrosyl phosphorylated GHR that seems to co-precipitate with
SHP-2
(Fig. 2). Stripping and reprobing the blot in Fig. 3A
(lanes 1 and 2) with
PY (Fig. 3A,
lanes 3 and 4) shows that SIRP
co-migrates
with the tyrosyl phosphorylated p120 in
SHP-2 immunoprecipitates,
providing additional evidence that the tyrosyl phosphorylated 120-kDa
protein that associates with SHP-2 is SIRP
. An additional tyrosyl
phosphorylated protein of 130 kDa, a mass appropriate for JAK2, also
co-precipitated with
SHP-2 (Fig. 3A, lane 4).
To determine whether GH enhances tyrosyl phosphorylation of SIRP
,
SIRP
was immunoprecipitated from cells that were treated with or
without GH and immunoblotted with
PY. As shown in Fig.
3B, GH enhances tyrosyl phosphorylation of SIRP
. The
results of Fig. 3 therefore indicate that GH promotes tyrosyl
phosphorylation of SIRP
and its association with SHP-2.
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JAK2 Tyrosyl Phosphorylates and Associates with SIRP1--
The
GH-activated tyrosine kinase JAK2 was thought to be the most likely
candidate for the SIRP
tyrosine kinase. To determine if JAK2 tyrosyl
phosphorylates SIRP
proteins, we transiently expressed in COS-7
cells JAK2 or kinase-inactive JAK2 in which the critical lysine 882 in
the ATP binding region is replaced with glutamate (JAK2 K/E) or
SIRP
1. As shown in Fig. 4,
co-expression of SIRP
1 with JAK2 (lane 3), but not
kinase-inactive JAK2 (lane 5), results in tyrosyl
phosphorylation of SIRP
1. JAK2 associates with SIRP
1, as shown by
co-precipitation by
SIRP of a tyrosyl phosphorylated protein with a
size appropriate for JAK2 in cells overexpressing wild-type JAK2 (Fig.
4, lane 3). The identity of this phosphoprotein as JAK2 was
confirmed by immunoblotting with antibody to JAK2 (data not shown).
Upon reprobing the nitrocellulose membrane with
SIRP, multiple bands
corresponding to SIRP
1 were apparent at 60-80 kDa in cells
transfected with SIRP
1 (Fig. 4, lanes 2, 3,
and 5, lower panels). Because deglycosylation
of
SIRP immunoprecipitates reduces the multiple bands to a single
60-kDa band (data not shown), the multiple bands most likely represent glycosylation intermediates of overexpressed SIRP
1 in COS cells.
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LIF Stimulates Tyrosyl Phosphorylation of SHP-2 and
SHP-2-associated LIFR and gp130 but IFN
Stimulates Tyrosyl
Phosphorylation of Neither SHP-2 nor of SHP-2-associated
Proteins--
Because JAK2 is activated in response to LIF and IFN
as well as GH (35-37), we compared the abilities of GH, LIF, and
IFN
to induce tyrosyl phosphorylation of SHP-2 and SHP-2-associated proteins such as SIRP
. 3T3-F442A cells were incubated with 25 ng/ml
LIF or 10 ng/ml IFN
, concentrations shown previously to induce
maximal JAK2 tyrosyl
phosphorylation.2 Although
LIF stimulates tyrosyl phosphorylation of JAK2 poorly in comparison
with GH (Fig. 5, lanes 1 and
4), LIF induces a more robust tyrosyl phosphorylation of
SHP-2 than GH (Fig. 5, lanes 2, 3, 5 and 6). Our results support previous findings that IL-6, oncostatin M, or LIF, ligands that utilize gp130 in their receptor signaling complexes, stimulate tyrosyl phosphorylation of a protein that migrates with a size appropriate for SHP-2 (18). LIF did not
increase the amount of tyrosyl phosphorylated SIRP
associated with
SHP-2 (Fig. 5, lanes 5 and 6). Instead, LIF
stimulates tyrosyl phosphorylation of two other SHP-2-associated
proteins that migrate at sizes appropriate for gp130 and LIFR
. In
contrast to GH and LIF, IFN
does not stimulate tyrosyl
phosphorylation of SHP-2 or SHP-2-associated proteins such as SIRP
,
even though IFN
promotes a more robust tyrosyl phosphorylation of
JAK2 than LIF does (Fig. 5, lanes 7-9).
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LIF Stimulates SHP-2 Association with gp130--
To determine
whether LIF stimulates association of SHP-2 with gp130 or tyrosyl
phosphorylation of gp130 constitutively associated with SHP-2, SHP-2
immunoprecipitates from untreated or LIF-treated cells were
immunoblotted with antibody to gp130. As shown in Fig. 6 (lanes 1 and 2),
LIF stimulates association of SHP-2 with gp130. Reprobing this blot
with
PY demonstrates that gp130 and the SHP-2-associated 130-kDa
phosphoprotein co-migrate (Fig. 6, lanes 3 and
4). In this experiment, LIF induces tyrosyl phosphorylation
of SHP-2-associated SIRP
to a small extent. However, LIF-induced
tyrosyl phosphorylation of SHP-2-associated SIRP
was not
reproducible, occurring in only three out of ten experiments.
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GH, but Not LIF nor IFN, Potently Stimulates Tyrosyl
Phosphorylation of SIRP
--
To determine whether SIRP
is
tyrosyl phosphorylated in response to LIF or IFN
, 3T3-F442A cells
were treated with GH, LIF, or IFN
, and
SIRP immunoprecipitates
were immunoblotted with
PY. Consistent with the previous data (Fig.
5, lanes 2 and 3), GH potently stimulates tyrosyl
phosphorylation of SIRP
(Fig. 7,
lanes 1 and 2). However, neither LIF nor IFN
stimulate tyrosyl phosphorylation of SIRP
(Fig. 7, lanes
3 and 4). These results suggest that GH, but not LIF or
IFN
, regulates SIRP
.
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DISCUSSION |
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SIRP Is a Signaling Molecule for GH--
Our findings that GH
induces tyrosyl phosphorylation of SIRP
and association of SHP-2
with SIRP
demonstrate that SIRP
is involved in GHR signaling. To
our knowledge, this is the first evidence that SIRPs are involved in
cytokine receptor signaling. JAK2 is the most likely candidate for the
tyrosine kinase that phosphorylates SIRP
in response to GH. JAK2 is
potently activated by GH (35), and co-expression of wild-type but not
kinase-inactive JAK2 with SIRP
1 in COS cells results in tyrosyl
phosphorylation of SIRP
1 (Fig. 4). The association of SIRP
1 with
JAK2 in COS cells may not be dependent on phosphorylated tyrosines in
either JAK2 or SIRP
1, because neither JAK2 nor SIRP
1 contains a
known phosphotyrosine binding domain (e.g., SH2 or PTB).
However, SIRP
1 contains a proline-rich region (PKQPAPKP) that
conforms to the consensus sequence
(hydrophobic-XXX-aliphatic-PXP) of the
proline-rich box1 region of cytokine receptors that mediates
JAK2-cytokine receptor association (43, 44) Therefore, it is tempting
to speculate that the association of JAK2 with SIRP
involves binding of JAK2 to the proline-rich region of SIRP
. Whether or not
GH-induced tyrosyl phosphorylation of SIRP
in 3T3-F442A cells is a
consequence of a direct interaction of JAK2 with SIRP
, as suggested
by the COS cell transfection experiments, or involves an additional
protein(s) remains to be determined. Interestingly, not all ligands
that activate JAK2 induce detectable levels of tyrosyl phosphorylated SIRP
in 3T3-F442A cells. The ability of GH, but not LIF or IFN
, to potently stimulate tyrosyl phosphorylation of SIRP
may be a
consequence of the much greater ability of GH to stimulate JAK2 tyrosyl
phosphorylation (Fig. 5 and Refs. 45 and 46). Alternatively, tyrosyl
phosphorylation of SIRP
may require a factor(s) in addition to JAK2
that is recruited by GHR but not the receptors for LIF or IFN
.
GH and LIF Promote Association of SHP-2 Primarily with SIRP and
gp130, Respectively--
In this paper we demonstrate that GH
stimulates SHP-2 association with SIRP
, coincident with GH-induced
SIRP
tyrosyl phosphorylation. SIRP
contains four potential
binding sites of the SH2 domains of SHP-2, based upon the
phosphotyrosine-containing motif
tyrosine-hydrophobic-X-hydrophobic defined for the
N-terminal SH2 domain of SHP-2 (47). Because mutation of these
cytoplasmic tyrosines in SIRP
abolishes SIRP
-SHP-2 association in
response to insulin or EGF (34), it is likely that SIRP
-SHP-2
association in response to GH is mediated by phosphotyrosines in
SIRP
and SH2 domains in SHP-2. GHR also contains three potential
SHP-2 binding motifs, and JAK2 contains three potential SHP-2 binding
motifs. The presence of a small amount of tyrosyl phosphorylated GHR
(Fig. 2) and JAK2 (Fig. 3A) in
SHP-2 immunoprecipitates
suggests that some SHP-2 also binds to GHR-JAK2 complexes in response
to GH. Although JAK2 contains a consensus binding site for SHP-2 SH2
domains, the interaction of the SH2-domains of SHP-2 with GHR-JAK2 is
thought to be mediated primarily via GHR because JAK2 binds to a
glutathione S-transferase fusion protein containing the SH2
domains of SHP-2 only when JAK2 is co-expressed with
GHR.3 Similarly, the
GH-dependent increase in the amount of SHP-2
co-precipitated with
IRS-2 suggests that a small amount of SHP-2
also binds to IRS proteins (48). The amount of SHP-2 associated with
IRS-2 is probably a small subset of total SHP-2 because we did not
observe IRS-2 in
SHP-2 immunoprecipitates.
SHP-2 Is Tyrosyl Phosphorylated in Response to GH and LIF, but Not
IFN--
GH and LIF, but not IFN
, stimulate the tyrosyl
phosphorylation of SHP-2, with LIF being more effective than GH. Thus,
the levels of tyrosyl phosphorylation of SHP-2 induced by GH, LIF, or
IFN
do not correspond to the relative levels of JAK2 tyrosyl phosphorylation induced by these ligands. One explanation of this apparent discrepancy is that the levels of SHP-2 tyrosyl
phosphorylation may reflect the differential abilities of the ligands
to recruit SHP-2 to cytokine receptor-JAK signaling complexes, where
SHP-2 can be tyrosyl phosphorylated by JAKs. For example, in response to LIF, SHP-2 appears to be primarily recruited into the
gp130-LIFR
-JAK signaling complex where it is likely to be in
sufficiently close proximity to LIFR
-gp130-associated JAKs to be
highly phosphorylated. Consistent with this model, mutation of the
SHP-2 binding site in gp130 abolishes neurotrophin-3-induced tyrosyl
phosphorylation of SHP-2 by JAKs in cells overexpressing TrkC-gp130
chimeric proteins (49). In the case of GH, the majority of SHP-2
appears to be recruited to tyrosyl phosphorylated SIRP
. This subset
of SHP-2 may not be in close enough proximity to JAK2 to be
phosphorylated. The SHP-2 that is phosphorylated may reflect the
smaller amount of SHP-2 that is recruited to GHR-JAK2 complexes. The
greater binding of SHP-2 to SIRP
compared with GHR-JAK2 may reflect
a higher affinity of SHP-2 for SIRP
than for GHR or a greater degree of phosphorylation of SHP-2 binding motifs within SIRP
compared with
GHR. Consistent with the lack of association of SHP-2 with tyrosyl
phosphorylated IFN
receptor-JAK signaling complexes, IFN
does not
induce SHP-2 tyrosyl phosphorylation in 3T3-F442A cells.
Role of SIRP and SHP-2 in GH, LIF, and IFN
Signaling--
Our findings that GH stimulates SIRP
tyrosyl
phosphorylation and the association of SHP-2 with SIRP
suggests that
SIRP
plays a role in signaling by GH and potentially other members of the cytokine receptor superfamily. Overexpression of SIRP
1, but
not mutant SIRP
1, which cannot bind SHP-2, inhibits insulin and
EGF-induced DNA synthesis and MAPK activation (34), suggesting that
SIRP
may be a negative regulator of these cellular functions. These
effects are mimicked by overexpression of catalytically inactive SHP-2.
The observation that overexpression of inactive SHP-2 results in a
dramatic accumulation of SHP-2 at the cell membrane (32), coincident
with enhanced SIRP
tyrosyl phosphorylation, raises the possibility
that a major function of SIRP
is to recruit SHP-2 to the cell
membrane, where it dephosphorylates SIRP
and other
membrane-associated proteins. Binding of the SH2 domains of SHP-2 to
phosphorylated tyrosine-containing motifs has been shown to stimulate
SHP-2 phosphatase activity (13, 14). In support of SHP-2 being
activated by binding to SIRP
, SHP-2 associated with SIRP
via
SHP-2 SH2 domains dephosphorylates SIRP
(32, 34).
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ACKNOWLEDGEMENTS |
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We thank L. Rui and Drs. L. S. Argetsinger, J. Herrington, and J. A. VanderKuur for helpful suggestions. We thank B. Hawkins for assistance with the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK 48283 and DK 34171 (to C. C.-S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Loeb Predoctoral Fellowship from the University of Michigan Comprehensive Cancer Center.
** To whom correspondence should be addressed. Fax: 734-647-9523; E-mail: cartersu{at}umich.edu.
1
The abbreviations used are: SH2, Src homology 2;
GH, growth hormone; hGH, human growth hormone; GHR, growth hormone
receptor; LIF, leukemia inhibitory factor; LIFR, leukemia inhibitory
factor receptor
; gp130, glycoprotein 130; IFN
, interferon-
;
IL, interleukin; EGF, epidermal growth factor; PDGF, platelet-derived
growth factor; JAK, Janus kinase; IRS, insulin receptor substrate;
MAPK, mitogen-activated protein kinase;
SHP-2, anti-SHP-2 antibody;
PY, anti-phosphotyrosine antibody 4G10;
GHR, antibody to GHR;
SIRP, antibody to SIRP.
2 L. S. Argetsinger and C. Carter-Su, unpublished data.
3 M. R. Stofega and C. Carter-Su, unpublished data.
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REFERENCES |
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