Involvement of the Src Homology 2-containing Tyrosine Phosphatase
SHP-2 in Growth Hormone Signaling*
Sung-Oh
Kim
§,
Jing
Jiang
¶,
Woelsung
Yi
§,
Gen-Sheng
Feng
, and
Stuart J.
Frank
§¶**
From the
Department of Medicine, Division of
Endocrinology and Metabolism, and the § Department of Cell
Biology, University of Alabama at Birmingham and the ¶ Veterans
Administration Medical Center, Birmingham, Alabama 35294 and the
Department of Biochemistry and Molecular Biology, Walther
Oncology Center, Indiana University School of Medicine and Walther
Cancer Institute, Indianapolis, Indiana 46202-5121
 |
ABSTRACT |
Growth hormone (GH) signaling
requires activation of the GH receptor (GHR)-associated tyrosine
kinase, JAK2. JAK2 activation by GH is believed to facilitate
initiation of various pathways including the Ras, mitogen-activated
protein kinase, STAT, insulin receptor substrate (IRS), and
phosphatidylinositol 3-kinase systems. In the present study, we explore
the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling.
GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that
homologously express GHRs, resulted in tyrosine phosphorylation of
SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and
by affinity precipitation with a glutathione S-transferase
fusion protein incorporating the SH2 domains of SHP-2, GH induced
formation of a complex of tyrosine phosphoproteins including SHP-2,
GHR, JAK2, and a glycoprotein with properties consistent with being a
SIRP-
-like molecule. A reciprocal binding assay using IM-9 cells as
a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but
not SHP-1) with a glutathione S-transferase fusion
incorporating GHR cytoplasmic domain residues 485-620, but only if the
fusion was first rendered tyrosine-phosphorylated.
GH-dependent tyrosine phosphorylation of SHP-2 was also observed
in murine 32D cells (which lack IRS-1 and -2) stably transfected with
the GHR. Further, GH-dependent anti-SHP-2
coimmunoprecipitation of the Grb2 adapter protein was detected in both
3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement
of SHP-2 in GH signaling may not require IRS-1 or -2. Finally,
GH-induced transactivation of a c-Fos enhancer-driven luciferase
reporter in GHR- and JAK2-transfected COS-7 cells was significantly
reduced when a catalytically inactive SHP-2 mutant (but not wild-type
SHP-2) was coexpressed; in contrast, expression of a catalytically
inactive SHP-1 mutant allowed modestly enhanced GH-induced
transactivation of the reporter in comparison with that found with
expression of wild-type SHP-1. Collectively, these biochemical and
functional data imply a positive role for SHP-2 in GH signaling.
 |
INTRODUCTION |
Growth hormone (GH)1 is
a powerful growth-promoting and metabolic regulatory polypeptide
hormone (1). Recent developments have allowed significant understanding
of the initiating steps in GH-induced signal transduction in various
target cells and tissues. The GH receptor (GHR) is a single
membrane-spanning glycoprotein in the hematopoietin receptor
superfamily that has a ligand-binding external domain and a
350-residue cytoplasmic domain, which is required for GH
signaling (2, 3).
Rapid dimerization of GHRs by GH promotes acute activation of the
nonreceptor tyrosine kinase, JAK2, an obligate step in GH signaling (4,
5). JAK2's physical and functional association with the GHR requires
membrane-proximal GHR regions that include the receptor's proline-rich
Box1 element (6-8) and parts of the amino-terminal one-half of JAK2
(9, 10) (a noncatalytic region of the kinase molecule (11)). Various
signaling pathways (including the activation of STAT molecules
(12-19); the activation of Ras, phosphatidylinositol 3-kinase, and MAP
kinase (20-23); and the recently described involvement of the IRS-1
and -2 molecules (24-27)) are accessed as a result of these proximal
events.
Intense interest has of late been focused on molecules that regulate
the early activation steps of the GHR signal pathway and of similar
pathways utilized by other cytokine, growth factor, and immune
receptors. In particular, the role(s) played by the SH2-containing
protein-tyrosine phosphatases, SHP-1 and SHP-2, in modulating signaling
have attracted attention. SHP-1 has been implicated as a negative
regulator (signal attenuator) in various signaling systems, including
the erythropoietin receptor (28), interleukin-3 receptor (29),
colony-stimulating factor-1 receptor (30), T-cell receptor (31), Fc
receptors (32), and, very recently, the GHR (33). While similar in
structure and catalytic activity to SHP-1, SHP-2 has been proposed to
have a positive role in signaling via the platelet-derived growth
factor receptor (34), insulin receptor (35-37), and prolactin receptor
(38), among others. The propensity of SHP-2 to interact with activated growth factor and cytokine receptors either directly (via its SH2
domains with phosphotyrosine residue(s) of the receptors themselves (39-44)) or indirectly (via its SH2 domains with phosphotyrosine residues of IRS molecules (45, 46)) or via nonphosphotyrosine-mediated interactions with JAKs (47) is thought to be important in its ability
to regulate receptor signaling.
Herein we explore the potential roles of SHP-2 in GHR signaling. We
observe in cell lines biochemical evidence of SHP-2 involvement in GH
signaling, in the settings of both homologous and heterologous expression of the GHR. Further, by comparing wild-type SHP-2 and a
catalytically inactive SHP-2 mutant, we uncover a positive effect of
SHP-2 on GH-induced gene activation. Both the biochemical and functional aspects of the involvement of SHP-2 in GH signaling that we
observe appear to be independent of IRS proteins.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Recombinant hGH was kindly provided by Eli Lilly
Co. Routine reagents were purchased from Sigma unless otherwise noted.
Restriction endonucleases were obtained from New England Biolabs
(Beverly, MA).
Cells, Cell Culture, and Generation of Stable
Transfectants--
COS-7 and IM-9 cells were maintained as described
previously (6). NIH 3T3-F442A cells (48), kindly provided by Dr. H. Green (Harvard University) and Dr. C. Carter-Su (University of Michigan), were cultured in Dulbecco's modified Eagle's medium (4.5 g/liter glucose) (Cellgro, Inc.) supplemented with 10% calf serum
(Biofluids, Rockville, MD) and 50 µg/ml gentamicin sulfate, 100 units/ml penicillin, and 100 µg/ml streptomycin (all Biofluids). Factor-dependent murine promonocytic 32D cells, kindly
provided by Dr. A. Kraft (University of Colorado), were maintained in
RPMI 1640 (Biofluids) supplemented with 10% fetal bovine serum
(Biofluids) and 10% (v/v) of the conditioned medium of confluent
WEHI-3B cells (also a gift of Dr. Kraft) (a source of interleukin-3)
and antibiotics, as above.
Pools of stably transfected 32D-rGHR cells were prepared by
electroporation of 32D cells (2 × 107/ml in
complete medium; 250 V, 960 microfarads in a GenePulser (Bio-Rad)
electroporator) with the rabbit GHR (rGHR) cDNA either in the
pRc/CMV expression plasmid, as described previously (6), or in the pSX
plasmid, as described below. Selection was either in the presence of
G418 (0.8 mg/ml, Life Technologies, Inc.) for pRc/CMV or in the
presence of RPMI 1640 supplemented with 10% fetal bovine serum and
antibiotics. 32D cells expressing the rGHR (but not untransfected 32D
cells), whether selected in G418 or not, were able to be maintained in
bovine serum without added interleukin-3. (Further, serum-starved
32D-rGHR cells exhibited hGH-dependent growth in
proliferation assays).2 In
addition to their achievement of GH factor dependence, the presence of
stable transfectants was verified by anti-GHRcyt
immunoblotting of multiple independently transfected pools, as
described below.
Antibodies--
Anti-JAK2 peptide antiserum, directed at
residues 758-776 of murine JAK2 (11) (used for immunoblotting)
(Upstate Biotechnology, Inc., Lake Placid, NY); 4G10 monoclonal
antiphosphotyrosine (APT) antibody (Upstate Biotechnology); monoclonal
anti-GST antibody (Santa Cruz Biotechnology, Santa Cruz, CA); mAb 5 monoclonal anti-GHR external domain antibody (Agen, Queensland,
Australia); and SHP-1, SHP-2, and Grb2 monoclonal antibodies (all from
Transduction Laboratories) were all purchased commercially. The
anti-JAK2 serum used for immunoprecipitation was raised in rabbits
against a GST fusion protein incorporating residues 746-1129 of the
murine JAK2, as has been described (49). Anti-GHRcyt serum
(50), directed at the residue 317-620 region of the human GHR
cytoplasmic domain, anti-SHP-2 serum (39), and anti-SHP-1 serum (Ref.
31; a kind gift of Dr. M. Thomas, Washington University) have been
described.
The anti-GHRext mouse monoclonal antibody (IgG1-
) was
raised against a GST fusion protein incorporating residues 1-245 of the rGHR (2) (GST/rGHR1-245) (see below for plasmid
construction) and screened by enzyme-linked immunosorbent assay at the
University of Alabama Multipurpose Arthritis Center Hybridoma Facility
(Dr. M. Accaviti). The antibody was purified from ascites using the Affi-Gel Protein A MAPS II kit (Bio-Rad), according to the
manufacturer's suggestions.
Plasmid Construction--
The pSX plasmid (a kind gift of
Dr. J. Bonifacino (National Institutes of Health) and Dr. K. Arai, DNAX Institute of Molecular and Cellular Biology) drives
eukaryotic protein expression from the SR
promoter, which is
composed of the SV40 early promoter and the R-U5 segment of the human
T-cell lymphotrophic virus-1 long terminal repeat (51). pSX was derived
by replacement of a transcriptionally silent region of the pcDL-SR
plasmid with a multiple cloning site encoding the following restriction
enzyme recognition sites: SacI, EcoRV,
BglII, PvuII, XbaI, NotI,
KpnI, EcoRI, PstI, SmaI,
and XhoI. The rGHR cDNA (2) (a kind gift of Dr. W. Wood,
Genentech, Inc.) was ligated into pSX using the XbaI and
KpnI sites. The ligation of rGHR into the pRC/CMV expression plasmid (InVitrogen) has been described (9). The preparation of the
wild-type and CS463 mutant murine SHP-2 cDNAs (47) and of the
wild-type and CS mutant murine SHP-1 cDNAs (Ref. 31; a kind gift of
Dr. M. Thomas, Washington University) have been described; each
was ligated into pSX at the EcoRI site. The p2FTL c-fos-luciferase reporter plasmid has been described
(52).
The generation of GST-SHP-1-SH2 and GST-SHP-2-SH2 fusion proteins has
been described (39, 53). To generate the cDNA encoding the
GST/rGHR1-245 fusion protein, the region of the rGHR cDNA encoding residues 1-245 was amplified by polymerase chain reaction with the 5
-primer incorporating an EcoRI
restriction site and the 3
-primer incorporating a termination codon
and an XhoI restriction site 3
to the residue 245 codon.
The resulting polymerase chain reaction fragment was ligated into pGEX
4T (Pharmacia Biotech Inc.) using the EcoRI and
XhoI restriction sites. Correct assembly of the cDNA
fragment was verified by dideoxy sequencing and by specific
immunoblotting of the bacterially expressed fusion protein with the mAb
5 anti-GHR external domain monoclonal antibody (not shown).
The preparation of the cDNA encoding the
GST/hGHR485-620 fusion protein was accomplished using our
previously described modified pGEX2T (Pharmacia) vector, called
pGEX2TRS, which contains multiple cloning sites convenient for
incorporation of fragments of the cytoplasmic domain of human GHR
(hGHR) (50). Our isolation of the hGHR cDNA from IM-9 cells has
also been described, as has the generation of a plasmid encoding GST
fused to hGHR residues 317-620 (all but the proximal 47 residues of
the cytoplasmic domain) (50). (The numbering system used for the rGHR
and hGHR is that of Ref. 2, in which the full-length GHR includes
residues 1-620.) From GST/hGHR317-620, we used an
endogenous SmaI site to ligate the fragment encoding hGHR
residues 485-620 into pGEX2TRS.
To generate the GST/hGHR485-620/E tyrosine-phosphorylated
version of the GST/hGHR485-620 fusion protein in bacteria, we used the methods of Pawson and colleagues (54), previously described. In brief, Escherichia coli harboring the plasmids
encoding GST/hGHR485-620 or GST only (to generate GST/E)
were infected with a
phage incorporating the cDNA for the
kinase domain for the Elk tyrosine kinase (a generous gift from Dr. T. Pawson, Lunenfeld Cancer Institute, Toronto, Canada), which is
inducible by isopropyl-1-thio-
-D-galactopyranoside. Infectants harboring the lysogenic form of the phage were selected by
virtue of the phage's temperature sensitivity (lytic at 42 °C, lysogenic at 32 °C).
Isopropyl-1-thio-
-D-galactopyranoside induction of these
infectants allowed for high level production of both the GST fusions
and
/Elk and thus resulted in tyrosine phosphorylation of the target
fusions at any sites recognized by Elk.
Cell Stimulation, Protein Extraction, and GST Fusion Binding
Assays--
Serum starvation of 3T3-F442A and IM-9 cells was
accomplished by substitution of 0.5% (w/v) BSA (fraction V; Boehringer
Mannheim) for serum in their respective culture media for 16-20 h
prior to experiments. 32D-rGHR cells were serum-starved similarly, but for only 4 h prior to stimulation. Unless otherwise noted, hGH was
used at a final concentration of 500 ng/ml, and stimulations were
performed at 37 °C. Details of the hGH treatment protocol have been
described (6). Briefly, for IM-9 and 32D-rGHR cells, cells were
stimulated in suspension at 1 × 107/ml in binding
buffer (BB, consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM
MgCl2, 0.1% (w/v) BSA, and 1 mM dextrose).
Stimulations were terminated, and cells were collected by
centrifugation (800 × g for 1 min at 4 °C) and
aspiration of the BB. 3T3-F442A cells were stimulated in confluent
150 × 20-mm dishes (Falcon) in BB. Stimulations were terminated
by washing the cells once with ice-cold phosphate-buffered saline
containing 0.4 mM sodium orthovanadate (PBS-vanadate) and
then harvesting by scraping in PBS-vanadate; pelleted cells were
collected by brief centrifugation. For each cell type, pelleted cells
were solubilized for 15 min at 4 °C in fusion lysis buffer (FLB)
(1% (v/v) Triton X-100, 150 mM NaCl, 10% (v/v) glycerol,
50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM PMSF, 1 mM sodium
orthovanadate, 10 mM benzamidine, 10 µg/ml aprotinin), as
indicated. After centrifugation at 15,000 × g for 15 min at 4 °C, the detergent extracts were subjected to either
immunoprecipitation or affinity precipitation or were directly
electrophoresed, as indicated below.
For affinity precipitation of extracts with GST fusion proteins, fusion
protein induction and affinity purification on glutathione-agarose beads (Pharmacia) were performed as described previously (6). Bacterial
extract containing roughly 5 µg of each of the indicated fusions
bound to glutathione-agarose beads was incubated with FLB extract from
3T3-F442A cells, 32D rGHR cells, or IM-9 cells. After incubation for
2 h at 4 °C, the beads were washed extensively with FLB and
eluted in reduced Laemmli SDS sample buffer. 90% of the eluate was
resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 7%
gels and immunoblotted sequentially with antibodies as indicated. For
normalization of loading of the fusion proteins on the beads, 5% of
the eluate was resolved on a 10% gel and immunoblotted with anti-GST
antibody. Where indicated, fractions of the cell extracts that were
subjected to glutathione-agarose binding were also electrophoresed and
sequentially probed.
Immunoprecipitation, Enzymatic Deglycosylation, Electrophoresis,
and Immunoblotting--
For immunoprecipitation, the rabbit antisera
described above were used at the following volumes per precipitation:
anti-JAK2 (directed at residues 746-1129), 8 µl;
anti-GHRcyt, 3 µl; anti-SHP-1 and anti-SHP-2-1 µl for
both. For the immunoprecipitation of the GHR with the monoclonal
anti-GHRext antibody, 0.6 µg of purified antibody was
used per precipitation and 1 mM dithiothreitol was added to
the cell extract prior to precipitation. Protein A-Sepharose (Pharmacia) or, for anti-GHRext, protein G-Sepharose
(Pharmacia) was used to adsorb immune complexes, and after extensive
washing with lysis buffer, Laemmli sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated.
For enzymatic deglycosylation experiments, immunoprecipitation or
affinity precipitation was first performed, as above. Precipitated proteins were eluted by boiling the protein A-Sepharose or
glutathione-agarose beads in 0.5% SDS, 3% 2-mercaptoethanol for 5 min. Deglycosylation in 0.125 ml was accomplished by adjusting the
buffer to include the following: 50 mM NaOAc, pH 5.5, 50 mM EDTA, 0.4% (v/v) Nonidet P-40, 6.4 mM
phenylmethylsulfonyl fluoride, 0.1% SDS, 0.6% 2 mercaptoethanol, and
0.3 units of endoglycosidase F/N-Glycosidase F (Boehringer Mannheim) at 37 °C for 16 h. Nondeglycosylated controls were
subject to the same treatment but without the addition of the
glycosidase mixture. After the addition of SDS sample buffer, the
proteins were subjected to SDS-PAGE.
Resolution of proteins under reduced conditions by SDS-PAGE, Western
transfer of proteins, and blocking of Hybond-ECL (Amersham, Inc.) with
2% BSA were performed as described previously (6, 19). Immunoblotting
with antibodies 4G10 (1:2500), mAb 5 (1:1000), monoclonal anti-SHP-2
(1:2000), monoclonal anti-SHP-1 (1:500), anti-GST (1:1000), anti-Grb2
(1:1000), anti-GHRcyt (1:2000), or anti-JAK2 peptide
antiserum with horseradish peroxidase-conjugated anti-mouse or
anti-rabbit secondary antibodies (1:2000) and ECL detection reagents
(all from Amersham Corp.) and stripping and reprobing of blots were
accomplished according to the manufacturer's suggestions.
c-fos-Luciferase Transactivation Assay--
COS-7 cells (6 × 106/dish) were transfected in 10 ml of Dulbecco's
modified Eagle's medium in 100 × 20-mm dishes (Falcon) by the
calcium phosphate precipitation method as described previously (9).
Each dish was transfected with both 10 µg of pSX rGHR and 3 µg of
pSX mJak2 along with either 10 µg of wild-type mSHP-2 or mSHP-2 CS463
cDNA in pSX or 20 µg of wild-type mSHP-1 or mSHP-1 CS453 cDNA
in pSX. For samples in which no exogenous protein-tyrosine phosphatase
was expressed, 10 or 20 µg of pSX (for SHP-2 or SHP-1 experiments,
respectively) was added to equalize the amount of total DNA added in
each transfection. p2FTL was added at 5 µg/dish. The
-galactosidase-expressing plasmid, pCH110 (Pharmacia), was added at
5 µg/dish for internal transfection control purposes. At 18-20 h
after transfection, the cells were split into 12-well plates. Serum
starvation (substitution of 0.5% BSA for fetal bovine serum in the
medium) was begun at 24 h after transfection and continued for
18-20 h prior to the addition of hGH (500 ng/ml) or vehicle for 5 h. Stimulations (performed in triplicate) were terminated by aspiration
of the medium and the addition of luciferase lysis buffer; luciferase
activity was assayed as has been described previously (9), and a
standard
-galactosidase spectrophotometric assay was performed on
the same extracts. (No systematic variation in basal or GH-induced
-galactosidase activity was observed with any combination of
expressed proteins.)
Densitometric Analysis--
Densitometry of ECL immunoblots was
performed using a solid state video camera (Sony-77, Sony Corp.) and a
28-mm MicroNikkor lens over a lightbox of variable intensity (Northern
Light Precision 890, Imaging Research Inc., Toronto, Canada).
Quantification was performed using a Macintosh II-based image analysis
program (Image 1.49, developed by W. S. Rasband, Research Services
Branch, NIMH, National Institutes of Health, Bethesda, MD). The ratio
of tyrosine-phosphorylated SHP-2 (Ptyr SHP-2) to total SHP-2 in
anti-SHP-2 precipitates of GH-stimulated 32D-rGHR cells was determined
by dividing the relative densitometric signal in APT blots of
anti-SHP-2 precipitates by the relative signal in the same stripped and
anti-SHP-2 mAb-reprobed precipitates. In each case, this ratio is
normalized to that seen at a 5-min stimulated point within the same
experiment, which was always the maximal tyrosine phosphorylation seen
of specifically precipitated SHP-2.
 |
RESULTS |
GH Promotes Association of Tyrosine-phosphorylated GHR and JAK2 and
SHP-2 in NIH 3T3-F442A Cells--
We first examined biochemically the
involvement of SHP-2 in GH signaling in murine NIH3T3-F442A
fibroblasts. This cell line has been shown to be GH-responsive with
regard to activation and tyrosine phosphorylation of JAK2 and tyrosine
phosphorylation of the GHR, SHC, MAP kinase, IRS-1, IRS-2, and STAT1,
-3, and -5 (see Ref. 55 and references therein); further, it can be converted from the fibroblast to the adipocyte phenotype in part in
response to GH (48). As has been observed by others (5), APT
immunoblotting of detergent extracts of GH-treated cells resolved by
SDS-PAGE revealed the presence of multiple GH-induced tyrosine phosphoproteins, including among others a band that, based on specific
immunoblotting, exactly comigrated with SHP-2 (data not shown).
We investigated the potential GH-induced tyrosine phosphorylation of
SHP-2 by immunoprecipitation experiments. Portions of detergent
extracts of 3T3-F442A fibroblasts stimulated with GH for various
durations were specifically immunoprecipitated with anti-SHP2 serum
(Fig. 1A, lanes
1-5, with nonimmune serum precipitation controls in lanes
6 and 7). APT immunoblotting of the eluates of these
immunoprecipitates revealed GH-enhanced tyrosine phosphorylation of
SHP-2 itself, as confirmed by stripping and reprobing of the same
precipitates with a monoclonal anti-SHP-2 antibody (Fig. 1C,
lanes 1-7). This enhanced SHP-2 tyrosine phosphorylation
was detected in anti-SHP-2 precipitates with as little as 1-5 min of
GH treatment.

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Fig. 1.
GH promotes tyrosine phosphorylation of SHP-2
and its association with other tyrosine phosphoproteins in NIH
3T3-F442A fibroblasts. A, tyrosine phosphoproteins present
in specific anti-SHP-2, anti-GHRcyt, and anti-JAK2
precipitates of GH-treated cells. FLB extracts from 3T3-F442A cells
(one 150 × 20-mm dish (roughly 2 × 107 cells)
per condition) treated with hGH for the indicated durations or exposed
to vehicle only (untreated (U)) were immunoprecipitated with
anti-SHP-2 (lanes 1-5), nonimmune (NI)
(lanes 6 and 7), anti-GHRcyt (lanes 8 and 9), and anti-JAK2 (lanes
10 and 11) sera. Eluates were resolved by SDS-PAGE and
APT-immunoblotted. The positions of tyrosine-phosphorylated SHP-2 and
of a nonspecific (NS) tyrosine phosphoprotein present in
each precipitate are indicated. The position of tyrosine-phosphorylated
JAK2 is indicated by a bar. The positions of migration of
the GHR (bracket) and three tyrosine-phosphorylated forms of
the GHR (arrowhead and two arrows above it within
the bracket) are also indicated (see text and Fig.
1B, lanes 14 and 15). Positions of
prestained molecular mass markers, in kDa, are indicated on the
left. B, specific detection of the GHR in
untreated and GH-treated cells. The relevant region of the blot shown
in Fig. 1A, lanes 6-9 (nonimmune and
anti-GHRcyt precipitates) was stripped and reprobed with
anti-GHRcyt. The position of specifically detected GHR is
indicated by the bracket. The arrowhead and
two arrows above it indicate slower migrating forms of the
GHR that appear in response to GH treatment (and are consistent with
being phosphorylated GHR forms). C, verification of the
presence of specifically precipitated SHP-2 in anti-SHP-2 precipitates.
The relevant region of the blot shown in Fig. 1A,
lanes 1-7 (anti-SHP-2 and nonimmune precipitates), was
stripped and reprobed with anti-SHP-2 monoclonal antibody. The position
of specifically detected SHP-2 is indicated. The experiment shown in
panels A-C is representative of three such
experiments.
|
|
Interestingly, in addition to tyrosine-phosphorylated SHP-2, components
of a roughly 100-125-kDa cluster of GH-induced tyrosine phosphoproteins observed in unprecipitated cell extracts were also
specifically detected in the anti-SHP-2 precipitates (Fig. 1A, lanes 2-5). Increased tyrosine
phosphorylation of these anti-SHP-2-coprecipitated proteins was
detected in response to as little as 1 min of GH exposure. Since their
migration in SDS gels was similar to those of the GHR and JAK2 (as in
Ref. 5), the identity (or identities) of the anti-SHP-2-coprecipitated
tyrosine phosphoprotein(s) was initially investigated by comparison to
the tyrosine phosphoproteins that specifically precipitated with
anti-GHRcyt and anti-JAK2 sera.
As expected, immunoprecipitations with both anti-GHRcyt and
anti-JAK2 sera (but not with nonimmune serum) contained GH-induced tyrosine phosphoproteins in the 100-125-kDa range (Fig. 1A,
lanes 6-11). In both immunoprecipitates, this cluster could
be resolved into two dominant components: a sharp band at 120 kDa
(labeled JAK2), most prominent in the anti-JAK2 precipitate,
and a diffuse band (bracket), more prominent in the
anti-GHRcyt precipitate. Stripping and reprobing of
this blot with anti-JAK2 serum confirmed that the sharp 120-kDa band
was JAK2 (not shown), as has been shown by others (5). Stripping and
reprobing with anti-GHRcyt (Fig. 1B, lanes
12-15) identified the GHR in the anti-GHRcyt
precipitates of both the unstimulated and stimulated cells
(bracketed region). Notably, the GHR in
anti-GHRcyt precipitates from GH-treated cells migrated as
a set of bands with retarded electrophoretic mobility compared with
GHRs from unstimulated cells (compare within the bracket the
GH-induced appearance of the bands marked with an arrowhead
and the two arrows above it in lane 15 with the
faster migrating collection of bands in lane 14). The most
dominant focused band in the anti-GHRcyt immunoblot of the
anti-GHRcyt precipitate (arrowhead, lane
15) exactly comigrated with the most intense band observed in the
bracketed region of the APT blot of the same precipitate
(arrowhead, Fig. 1A, lane 9) and in
the anti-JAK2 precipitate (arrowhead, Fig. 1A,
lane 11) of GH-treated cells. This GH-induced retardation in
GHR migration and the enhanced APT reactivity of the immunologically
detected GHRs are reflective of the significant GH-induced tyrosine
phosphorylation of the receptor in these cells. (The presence of both
the tyrosine-phosphorylated GHR and JAK2 in the unstimulated samples
indicated a small degree of basal activation of GHRs in these cells,
despite overnight serum starvation. Such basal activation was a
variable finding.)
Despite the very similar migration of the anti-SHP-2-coprecipitated
tyrosine phosphoproteins with the tyrosine-phosphorylated GHR, we were
not able to directly blot the GHR in the anti-SHP2 precipitates with
anti-GHRcyt, our only anti-receptor antibody capable of
immunoblotting (not shown). Since we considered that this was likely to
be due at least in part to the GHR's low abundance in the anti-SHP-2
immunoprecipitate, we sought to further characterize possible
GH-induced SHP-2-associated tyrosine phosphoproteins by virtue of their
affinity for the SHP-2 SH2 domains. As diagrammed in Fig.
2A, SHP-2 contains two tandem
SH2 domains (SH2-N and SH2-C) amino-terminal to its catalytic domain.
We explored whether the SHP-2 region containing these domains might be
capable of interacting with the 100-125-kDa cluster of GH-induced
tyrosine phosphoproteins seen in the anti-SHP-2 immunoprecipitate by
using GST alone or each of the two fusions indicated in Fig.
2A in an affinity precipitation experiment. GST/SHP-2-SH2
incorporates both (N and C) SH2 domains of SHP-2 fused to GST. As a
control, we used a fusion (designated GST/SHP-1-SH2) incorporating the
analogous SH2 domains of the related protein-tyrosine phosphatase,
SHP-1.

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Fig. 2.
Interaction of SHP-1 and SHP-2 SH2-containing
GST fusion proteins with the 3T3-F442A cell-derived GHR and JAK2.
A, diagram of GST fusions used in the experiments in Fig. 2,
B-D compared with SHP-1 and SHP-2. The relative positions
of the SH2-N and SH2-C domains of the related protein-tyrosine
phosphatases SHP-1 and SHP-2 are indicated, in each case amino-terminal
to the catalytic domains of each molecule. GST, either by itself or as
part of the GST/SHP-1-SH2 or GST/SHP-2-SH2 fusions, is indicated by the
cross-hatched ovoid moiety. GST/SHP-1-SH2 incorporates residues 1-296
of SHP-1 (53); GST/SHP-2-SH2 incorporates residues 2-216 of SHP-2
(39). B, FLB extracts of 3T3-F442A cells treated with hGH
(+) or vehicle ( ) for 10 min prior to extraction were affinity-precipitated with the indicated GST fusions (lanes
1-6) or immunoprecipitated with anti-GHRcyt
(lanes 9 and 10) or anti-JAK2 (lanes
11 and 12). Eluates of the precipitates (one 150 × 20-mm dish equivalent/lane) or unprecipitated extracts (lanes
7 and 8; 0.1 dish equivalent/lane) were resolved by
SDS-PAGE and APT-immunoblotted. The positions of
tyrosine-phosphorylated JAK2 and GHR are indicated, as in Fig. 1,
A and B. C and D, the
relevant regions of Fig. 2B, lanes 1-8, were
sequentially stripped and reprobed with anti-GHRcyt (C) and anti-JAK2 (D). The positions of the GHR
and its tyrosine-phosphorylated forms (arrowhead and
two arrows above it) and of JAK2 are indicated. Note the
GH-dependent association of tyrosine-phosphorylated GHR forms and tyrosine-phosphorylated JAK2 with GST/SHP-2-SH2 and the
non-GH- and non-phosphotyrosine-dependent association of
JAK2 with GST/SHP-1-SH2. The experiment shown in panels B-D
is representative of three such experiments.
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Detergent extracts from either untreated or GH-stimulated 3T3-F442A
cells were affinity-precipitated with each GST fusion or GST alone or
were immunoprecipitated, as above, with anti-GHRcyt or
anti-JAK2. In Fig. 2B, eluates from each precipitate and
unfractionated extracts were resolved by SDS-PAGE and
APT-immunoblotted. Although the exposure is lighter, the pattern of
tyrosine phosphoproteins present in the immunoprecipitates (lanes
9-12) and in the extracts (lanes 7-8) is the same as
that seen in the experiment in Fig. 1. Notably, GST/SHP-2-SH2
precipitated a complex of GH-induced tyrosine phosphoproteins in the
100-125-kDa range similar to those immunoprecipitated with
anti-GHRcyt (Fig. 2B, compare lanes 5 and 6 with lanes 9 and 10). Despite
the presence of equivalent amounts of GST and each fusion in each
precipitate (data not shown), these same GH-induced tyrosine
phosphoproteins were only minimally precipitated with GST/SHP-1-SH2
(lanes 3 and 4) and not detected at all in GST
precipitates (lanes 1-2).
Sequential stripping and reprobing of the relevant region of the blot
with anti-GHRcyt (Fig. 2C) and anti-JAK2 (Fig.
2D) confirmed the dramatic GH-dependent
enrichment of these molecules in the GST/SHP-2-SH2 precipitate in
comparison with the GST and GST/SHP-1-SH2 precipitates. Notably, only
GHRs exhibiting the most retarded migration (and thus likely being the
most phosphorylated) in the extract of GH-stimulated cells
(arrowhead and the two arrows above it in Fig.
2C, lane 8) were detected in the GST/SHP-2-SH2
precipitate (the same region of lane 6). Although the
interactions of both the tyrosine-phosphorylated GHR and JAK2 with the
GST/SHP-2-SH2 fusion were clearly GH-dependent, we cannot
determine from this experiment if either interaction was
direct.3
Because we could not directly immunoblot the GHR in anti-SHP-2
immunoprecipitates, we further investigated the composition of the
100-125-kDa GH-induced tyrosine phosphoprotein complex precipitated by
both anti-SHP-2 and GST/SHP-2-SH2 by performing deglycosylation
experiments. The GHR is extensively N-glycosylated and, upon
complete enzymatic deglycosylation, displays a characteristic enhanced electrophoretic mobility (2, 57). We compared the electrophoretic pattern of the tyrosine phosphoproteins from
GH-stimulated 3T3-F442A cells that were present in anti-SHP-2,
anti-GHRcyt, and GST/SHP-2-SH2 (or control nonimmune)
precipitates treated with or without the combination of
endoglycosidase-F and N-glycosidase-F (Fig.
3), as under "Experimental
Procedures." Anti-GHRcyt immunoblotting (Fig.
3A) revealed the expected enhanced migration to the 95-kDa region of the GHRs (denoted by the brackets and
arrows) precipitated by both anti-GHRcyt and
GST/SHP-2-SH2 as a result of apparently complete enzymatic
deglycosylation. Notably, the inclusion in the deglycosylated
anti-GHRcyt precipitates of a slightly faster migrating
complement of GHRs (denoted by the arrow) relative to those
detected in the GST/SHP-2-SH2 precipitate reflects the inclusion in the
immunoprecipitate of some non-tyrosine-phosphorylated receptors (more
apparent, as expected, in the deglycosylated immunoprecipitate from
unstimulated versus GH-stimulated cells (lane 2 versus
lane 4)), whereas only the more slowly migrating
tyrosine-phosphorylated GHRs (largely included in the
brackets, as below) were present in the deglycosylated
affinity precipitate (lane 6).

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Fig. 3.
Characterization of GH-induced
SHP-2-associated tyrosine phosphoproteins by enzymatic
deglycosylation. A, enzymatic deglycosylation of
immunoprecipitated and affinity-precipitated GHRs. 3T3-F442A cells were
treated without (lanes 1 and 2) or with
(lanes 3-6) GH for 10 min, as indicated. Detergent extracts (one 150 × 20-mm dish/condition) were
anti-GHRcyt-immunoprecipitated (lanes 1-4) or
GST/SHP-2-SH2 affinity-precipitated (lanes 5 and 6), as in Figs. 1 and 2. Precipitates were treated with or
without endoglycosidase F/N-glycosidase F (+ Glycosidase or Glycosidase), as under
"Experimental Procedures," prior to resolution by SDS-PAGE and
immunoblotting with anti-GHRcyt. Positions of glycosylated and deglycosylated GHRs are indicated. Brackets correspond
to the GHR cohort that includes slower migrating
tyrosine-phosphorylated receptors (see panel B);
arrows indicate faster migrating (and non-tyrosine-phosphorylated)
GHRs. NS indicates nonspecific bands seen in immunoblots of
affinity precipitates, as determined by their presence in blots of the
same filter with nonspecific serum (not shown). B, enzymatic
deglycosylation of immunoprecipitated and affinity-precipitated
GH-induced tyrosine phosphoproteins. In lanes 7-12, the
blot in A (lanes 1-6) was stripped and reprobed with APT.
Anti-SHP-2 precipitates (lanes 13-16) and nonimmune precipitates (lanes 17 and 18) of FLB extracts
from GH-treated cells were also deglycosylated, as indicated, prior to
SDS-PAGE and APT immunoblotting. Lanes 15 and 16 (longer exposure of lanes 13 and 14) and
lanes 17 and 18 are overexposed relative to the other lanes. The positions of tyrosine-phosphorylated glycosylated and
deglycosylated GHRs, SHP-2, and a protein that changes migration from
roughly 100-125 kDa to 65 kDa with deglycosylation (65 kDa degly) are indicated. The experiment in panels A and
B is representative of four such experiments.
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Stripping and reprobing of this blot with APT (Fig. 3B)
revealed that the 100-125-kDa and 95-kDa tyrosine phosphoproteins present in the nondeglycosylated (lane 9) and deglycosylated
(lane 10) anti-GHRcyt precipitates,
respectively, of GH-treated cells were identified as the
tyrosine-phosphorylated GHR by virtue of their exact comigration
with the immunoblotted GHR from the same samples (Fig. 3A,
lanes 3 and 4). As expected, the sharp 120-kDa GH-induced tyrosine phosphoprotein present in the
anti-GHRcyt precipitates (lanes 9 and
10), as well as in the GST/SHP-2-SH2 precipitates
(lanes 11 and 12), which was confirmed to be JAK2 by specific anti-JAK2 immunoblotting (not shown), did not display a
change in its migration in the deglycosylated samples, consistent with
JAK2 being a non-N-glycosylated cytosolic protein. However, the migration of the majority of the GH-induced 100-125-kDa tyrosine phosphoproteins in the complex specifically present in the
GST/SHP-2-SH2 and anti-SHP-2 precipitates (lanes 11 and
12 and lanes 13 and 14) did
change upon enzymatic deglycosylation.
The deglycosylated 100-125-kDa tyrosine phosphoprotein complex from
the GST/SHP-2-SH2 precipitate (lane 12) largely resolved into two specific clusters of bands. An intense 95-kDa deglycosylated band exactly comigrated with the deglycosylated GHR present in the
anti-GHRcyt blot of the same precipitate (lane
6), identifying it as the tyrosine-phosphorylated GHR.
Notably, however, a substantial portion of the deglycosylated
GH-induced tyrosine phosphoproteins present in the GST/SHP-2-SH2 precipitate (lane 12) resolved to a 65-kDa band (denoted in
Fig. 3 as 65 kDa degly) not present in the
anti-GHRcyt precipitate (lane 10). Anti-SHP-2
immunoblotting (not shown) confirmed that this band is not a form of
SHP-2 (which, as indicated, migrates slightly slower than the 65-kDa
protein) generated during the deglycosylation process; SHP-2 is not
N-glycosylated. This same deglycosylated 65-kDa GH-induced
tyrosine phosphoprotein was also detected in a deglycosylated
anti-SHP-2 precipitate (lane 14); indeed, the 65-kDa
deglycosylated tyrosine phosphoprotein appeared to account for the
majority of the anti-SHP-2-precipitated GH-induced 100-125-kDa
tyrosine phosphoprotein complex present prior to deglycosylation (lane 13). Longer exposure (lanes 15 and
16) of the same samples seen in lanes 13 and
14 and of a nonimmune precipitation control (lanes
17 and 18) did reveal in the deglycosylated anti-SHP-2 precipitate (lane 16) a relatively small, but specifically
precipitated, degree of 95-kDa GH-induced tyrosine phosphoprotein that
exactly comigrates with the deglycosylated tyrosine-phosphorylated GHR. This relatively small yield of tyrosine-phosphorylated GHR so identified in the GH-induced anti-SHP-2-precipitated 100-125-kDa tyrosine phosphoprotein complex is consistent with our inability to
detect it by anti-GHRcyt blotting. While we do not yet know the identity of the major component of the GH-induced 100-125-kDa tyrosine phosphoprotein complex present in anti-SHP-2 precipitates, we
note that its migration upon deglycosylation (roughly 65 kDa) is very
similar to that exhibited by deglycosylated members of the SIRP-
family, a recently discovered SHP-2-associated
tyrosine-phosphorylated glycoprotein family (58-60).
Tyrosine-phosphorylated GHR Can Specifically Interact with
Non-tyrosine-phosphorylated SHP-2--
The data in Figs. 1-3 indicate
that in 3T3-F442A cells, GH promotes the formation of a
phosphotyrosine-dependent complex of proteins that includes
SHP-2 and the tyrosine-phosphorylated GHR and JAK2 (in addition to at
least one other tyrosine phosphoprotein, which, when deglycosylated,
becomes the 65-kDa deglycosylated protein). In other experiments (not
shown), anti-SHP2 coimmunoprecipitation of a complex of GH-induced
tyrosine phosphoproteins of 115-140 kDa was also detected in the human
IM-9 B-lymphoblastoid cell line, which, like 3T3-F442A cells, also
homologously expresses GHRs (61) and displays various responses to GH
including activation of JAK2 (6, 57) and tyrosine phosphorylation of
JAK2, the GHR, and STAT5 (6, 57, 62). Notably, however, in IM-9 cells, this GH-induced association of SHP-2 with tyrosine phosphoproteins that
probably include the GHR and JAK2 was not accompanied by detectable
GH-induced tyrosine phosphorylation of SHP-2 (not shown).
We tested whether tyrosine phosphorylation of the GHR cytoplasmic
domain could confer specific association with SHP-2 and whether SHP-2
must be tyrosine-phosphorylated to allow such an interaction. To
examine this, we prepared a bacterially expressed GST fusion
incorporating residues 485-620 of the hGHR (the C-terminal 40% of the
receptor cytoplasmic domain), designated GST/hGHR485-620. In addition, we prepared a tyrosine-phosphorylated derivative of this
fusion, designated GST/hGHR485-620/E, by inducing its
expression in bacteria that were infected with a bacteriophage
,
which directs an inducible high level expression of the Elk protein-tyrosine kinase (54). As negative controls, we induced the
production of GST alone and of GST/E, in which GST was induced in the
presence of the coinduced Elk kinase. Detergent extracts from
unstimulated IM-9 cells were affinity-precipitated with each GST fusion
or the control GST molecules and eluates from each precipitate, and
unfractionated extracts were resolved by SDS-PAGE and immunoblotted
with anti-SHP-2 monoclonal antibody (Fig.
4A). No SHP-2 was detected in
the GST, GST/E, or GST/hGHR485-620 precipitates; specific
interaction was, however, detected between GST/hGHR485-620/E and SHP-2 (Fig. 4A,
lane 4). As a further control, this blot was reprobed with
anti-SHP-1 monoclonal antibody (Fig. 4B). Despite amply
detected SHP-1 in the cell extract (lane 5), SHP-1 was not
observed in any of the affinity precipitates. The same experiment
performed with detergent extracts from both unstimulated and
GH-stimulated 3T3-F442A cells yielded an identical result and indicated
no change in the amount of SHP-2 precipitated by the
GST/hGHR485-620/E fusion relative to prior GH stimulation of the cells (not shown). Further, JAK2 was also not detected in the
GST/hGHR485-620/E precipitates for either IM-9 or 3T3-F442A cells (not shown).4
We conclude that tyrosine phosphorylation of residue(s) in the distal
GHR cytoplasmic domain can allow specific interaction with SHP-2
independent of SHP-2 tyrosine phosphorylation. At present, it is
impossible to conclude definitively that this interaction is
direct.

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Fig. 4.
A tyrosine-phosphorylated GST/hGHR fusion
protein specifically interacts with SHP-2. A and
B, FLB extract from unstimulated serum-starved IM-9 cells
was affinity-precipitated with each of the indicated GST fusions.
Eluates of the precipitates (2 × 107 cells/lane,
lanes 1-4) and unprecipitated extract (2 × 106 cells, lane 5) were resolved by SDS-PAGE and
immunoblotted sequentially with monoclonal antibodies to SHP-2
(A) and SHP-1 (B). Note the interaction of SHP-2
(and not SHP-1) with only the GST/hGHR485-620/E tyrosine-phosphorylated version of the GST/hGHR485-620
fusion. As in the text, fusions induced in the presence of the Elk
tyrosine kinase activity are indicated by the E following their
designation.
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GH-induced Tyrosine Phosphorylation of SHP-2 Can Occur in the
Absence of IRS-1 and IRS-2--
The association of SHP-2 with
tyrosine-phosphorylated IRS proteins in response to insulin stimulation
of cells is a well documented phenomenon (45, 46). Since GH treatment
of various cells, including 3T3-F442A cells, leads to tyrosine
phosphorylation of IRS-1 and IRS-2 (24-27), we sought to determine
whether involvement of SHP-2 in GH signaling required the presence of
either of these two proteins. To address this issue, we used the murine
factor-dependent promonocytic cell line, 32D. 32D cells
have been shown to lack IRS-1 and IRS-2 at the protein and mRNA
levels (63). After confirming by 125I-hGH binding and
cross-linking studies (not shown) that these cells also lacked
endogenous GHRs, we prepared several independently transfected isolates
of 32D cells stably expressing rGHR. As described under "Experimental
Procedures," each 32D-rGHR isolate exhibited GH-dependent
proliferation,5 similar to
that observed by others for other factor-dependent lines
transfected with the GHR (3).
After a 4-h period of GH and serum deprivation, 32D-rGHR cells
responded to acute stimulation with GH with the appearance of a pattern
of tyrosine phosphoproteins in cell extracts similar to that seen for
3T3-F442A and IM-9 cells (Fig.
5A). Again, specific immunoprecipitation and/or immunoblotting identified the 120-kDa, 90-kDa, and diffuse 115-130-kDa tyrosine phosphoproteins as JAK2, STAT5, and the rGHR (not shown), respectively, as indicated. Another tyrosine phosphoprotein (Fig. 5A, lane 6,
asterisk) was also easily detectable in extracts of
GH-stimulated 32D-rGHR cells. Anti-SHP-2 (but not anti-SHP-1) serum
precipitated this GH-induced tyrosine phosphoprotein, identifying it as
SHP-2 (compare Fig. 5A, lanes 1 and 2 and lanes 3 and 4). This finding was confirmed by
stripping and reprobing sequentially with anti-SHP-2 and anti-SHP-1
monoclonal antibodies (not shown).

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Fig. 5.
GH promotes tyrosine phosphorylation of SHP-2
in 32D-rGHR cells, which lack IRS-1 and -2. A,
GH-induced tyrosine phosphorylation in 32D-rGHR cells. FLB extracts of
32D-rGHR cells treated with hGH or vehicle (U) for 5 min
were immunoprecipitated with either anti-SHP-1 (lanes 1 and
2) or anti-SHP-2 (lanes 3 and 4).
Eluates from the immunoprecipitates (1 × 107
cells/lane) and unprecipitated extracts (lanes 5 and
6; 1 × 106 cells/lane) were resolved by
SDS-PAGE and APT-immunoblotted. Positions of the GH-induced
tyrosine-phosphorylated GHR, JAK2, STAT5, and a protein marked by an
asterisk are indicated (lane 6). The position of
specifically precipitated tyrosine-phosphorylated SHP-2 (lane
4), which comigrates with the asterisked protein, is
also indicated. The experiment shown is representative of four such
experiments. B-F, time course of GH-induced tyrosine phosphorylation of SHP-2 in 32D-rGHR cells. FLB extracts
from cells stimulated with hGH for the indicated durations were
immunoprecipitated with anti-SHP-2 (B and C) or
anti-GHRext monoclonal antibody (D and E). Eluates from each precipitate (1 × 107
cells/lane for each) were resolved by SDS-PAGE. Anti-SHP-2 precipitates were sequentially immunoblotted with APT (B) and anti-SHP-2
monoclonal antibody (C). Anti-GHRext
precipitates were sequentially immunoblotted with APT (D)
and anti-GHRcyt (E). The positions of 115 kDa
(arrow) and 90 kDa (arrowhead) tyrosine
phosphoproteins coprecipitated with anti-SHP-2 are indicated in
B. The positions of the fully glycosylated rGHR
(brackets in D and E) and
underglycosylated rGHR (arrowhead in E) forms
(see Ref. 19) are also indicated. (Note the slight loss of overall
precipitated rGHR in lane 5 of D and
E.) F, densitometric analysis, as under
"Experimental Procedures," of the time course of GH-induced
tyrosine phsophorylation of specifically precipitated SHP-2 (0 min,
n = 3; 0.5 min, n = 2; 2 min,
n = 2; 5 min, n = 3; 15 min,
n = 1; 20 min, n = 1). Where
appropriate, values are mean ± range.
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The time course of GH-induced SHP-2 tyrosine phosphorylation was
examined in a separate experiment (Fig. 5, B-F). APT
blotting of anti-SHP-2 precipitates of extracts from 32D-rGHR cells
indicated that GH treatment for as little as 30 s led to
detectably increased SHP-2 tyrosine phosphorylation with maximal levels
being achieved by 5 min (Fig. 5B). Stripping and reprobing
of this blot with anti-SHP-2 monoclonal antibody verified equivalent
amounts of SHP-2 in each sample (Fig. 5C). Quantitative
densitometric analysis of data from representative experiments is shown
in Fig. 5F and further substantiates this time course.
Interestingly, a basally tyrosine-phosphorylated diffuse protein of
roughly 115 kDa was coprecipitated with anti-SHP-2 serum (arrow, Fig. 5B). In some experiments, this
protein exhibited a degree of dephosphorylation in response to GH. The
migration of this protein in SDS gels was similar to that of the GH
receptor; however, specific precipitation of the GH receptor with our
monoclonal antibody to the receptor external domain followed by
immunoblotting with APT and anti-GHRcyt (Fig. 5,
D and E) indicated that the receptor was not
significantly tyrosine-phosphorylated basally. Thus, the 115-kDa
tyrosine phosphoprotein coprecipitated with anti-SHP-2 could not be
identified as the GH receptor; we do not know its exact identity. Nor
do we yet know the identity of another diffuse tyrosine phosphoprotein
of roughly 90 kDa coprecipitated with anti-SHP-2 (arrowhead,
Fig. 5B) that was also observed to variably undergo
GH-induced tyrosine dephosphorylation. Based on their coprecipitability
with anti-SHP-2 and their migration in SDS gels, these proteins
also may be related to similarly migrating tyrosine
phosphoproteins recently described by Gu et al. (64) in
32D and other murine factor-dependent cells (see
"Discussion").
GH-induced Coimmunoprecipitation of Grb2 with SHP-2--
Various
studies link SHP-2 to growth factor-induced activation of the MAP
kinase pathway and, in particular, to inclusion of the adapter protein
Grb2 in complex with SHP-2 (65-68). Given the GH-induced changes in
the tyrosine phosphorylation and/or association state of SHP-2 noted
above in both 3T3-F442A and 32D-rGHR cells, we examined whether GH also
induced association of Grb2 with SHP-2 (Fig.
6). 3T3-F442A (Fig. 6A) or
32D-rGHR (Fig. 6B) cells were treated with or without GH for
10 min. Detergent extracts of each were subjected to
immunoprecipitation with anti-SHP-2 or nonimmune serum or were not
immunoprecipitated prior to SDS-PAGE. Anti-Grb2 immunoblotting (Fig. 6,
bottom of A and B) revealed the
specific GH-induced inclusion of Grb-2 in the SHP-2 immunoprecipitates in both cell types, while there was no GH-induced change in total Grb2
present in the detergent extracts. Stripping and reprobing with
anti-SHP-2 (Fig. 6, top of A and B)
verified the presence of equivalent amounts of SHP-2 in the detergent
extracts and in the specific anti-SHP-2 precipitates, independent of GH
stimulation. Thus, GH promotes inclusion of Grb2 in anti-SHP-2
precipitates independent of whether IRS-1 and/or IRS-2 is expressed in
the cell.

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Fig. 6.
GH-induced association of Grb2 with
SHP-2. 3T3-F442A (A) or 32D-rGHR (B) cells
were treated without or with GH for 5 min. FLB extracts were either
immunoprecipitated with anti-SHP-2 or nonimmune serum or not
precipitated (extract) prior to resolution by SDS-PAGE and
immunoblotting with anti-Grb2 (bottom) or anti-SHP-2 (top) monoclonal antibodies. The positions of specifically
detected SHP-2 and Grb-2 are indicated. This experiment is
representative of two such experiments.
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SHP-2 Can Positively Affect GH-induced Transcriptional
Activation--
These findings led us to ask whether a functional GH
signal might be influenced by a change in the integrity of SHP-2
enzymatic activity. To address this issue, we examined in transient
transfectants the effects of overexpression of either wild-type or
catalytically inactive SHP-2 molecules on GH-induced transactivation of
a luciferase reporter gene driven by two tandem copies of a
c-fos enhancer fragment (52). We have previously shown that
transient expression of both the GHR and JAK2 is required for efficient
GH-induced transactivation of this reporter in COS-7 cells and that a
kinase-deficient JAK2 cannot mediate this response (9).
As shown in the representative experiment in Fig.
7A, expression of the
transfected rGHR and murine JAK2 in COS-7 cells without any transfected
SHP-2 mediated a substantial GH-induced increase in luciferase activity
measured in the cell extracts. When wild-type murine SHP-2 was
coexpressed with the rGHR and JAK2, GH-induced c-fos-mediated transactivation was only modestly increased.
(The degree of change in GH-induced transactivation with expression of
exogenous wild-type SHP-2 was variable among multiple experiments performed.) Notably, however, expression of a murine SHP-2 molecule that was rendered catalytically inactive (the so-called CS SHP-2) resulted in decreased GH-induced transactivation. When directly compared in three separate experiments, expression of the CS SHP-2 consistently led to significantly suppressed GH-induced
c-fos-mediated transactivation (average decline of 53%, as
indicated in Fig. 7B) when compared with matched expression
of WT SHP-2. As a specificity control, we performed similar experiments
using either WT SHP-1 or an analogously catalytically inactive SHP-1
mutant (the so-called CS SHP-1) (Fig. 7C). When again
directly compared with expression of WT SHP-1 in three separate
experiments, expression of the CS SHP-1 (unlike the findings with WT
and CS SHP-2) led to moderately increased GH-induced
c-fos-mediated transactivation (average increase of 21%).
The SHP-2 results cannot, therefore, be attributed simply to the
overexpression of any SH2-containing protein-tyrosine phosphatases in
this system. These results suggest that SHP-2, perhaps by virtue of its
presence in a signaling complex including the GHR, JAK2, and Grb2,
exerts a positive effect on a functional aspect of GH signaling, the
transactivation of a c-fos-driven reporter.

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Fig. 7.
SHP-2 positively influences GH-induced
c-fos-luciferase transactivation. A, as detailed
under "Experimental Procedures," COS-7 cells were transfected with
cDNAs encoding a c-fos enhancer-driven luciferase
reporter, the rGHR, and JAK2 and either pSX vector only (No
SHP-2), the wild-type (WT) SHP-2, or a catalytically inactive (CS) SHP-2 mutant. -Fold increases in luciferase
activity in cell extracts in response to hGH treatment (5 h) are
plotted. The data shown are from a representative experiment, in which the mean ± S.E. of triplicates is indicated for each
condition. B, data from three independent experiments (each
performed in triplicate with S.E. less than 10% of the mean for each
triplicate) such as those in A are plotted for the WT SHP-2
compared with CS SHP-2. GH-induced -fold increases in normalized
luciferase activity for each of the WT SHP-2-expressing samples are
assigned values of 100%. Mean luciferase activity for the CS SHP-2
mutant in three independent experiments was 47% (error bars
indicate range, 40-54%) that of WT SHP-2 (p < 0.001 for the difference between WT and CS SHP-2). C, experiments
were performed such as those in B, except that WT SHP-1 or
CS SHP-1 were expressed, as under "Experimental Procedures." Data
from three independent experiments (each performed in triplicate with
S.E. less than 10% of the mean for each triplicate) are plotted for
the WT SHP-1 compared with CS SHP-1. GH-induced -fold increases in
normalized luciferase activity for each of the WT SHP-1-expressing
samples are assigned values of 100%. Mean luciferase activity for the
CS SHP-1 mutant in three independent experiments was 121% (error
bars indicate range) that of WT SHP-1 (p < 0.05 for the difference between WT and CS SHP-1).
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DISCUSSION |
GH, like various other peptide hormones and cytokines, initiates
transmembrane signaling by causing the activation of a complex of
molecules, the nidus of which is the dimeric GHR itself. We now know
some details of the physical and functional architecture of this array
of GHR-associated molecules. The nonreceptor tyrosine kinase, JAK2, is
a critical component of the GHR signaling complex; intact JAK2
enzymatic activity is required for GH signaling (9, 55). Although the
stability of the complex may be affected by GH treatment (5),
association of the GHR and JAK2 does not appear to require either JAK2
activation or GHR dimerization (6). Consistent with this, the major
interaction between the GHR and JAK2 via the receptor's Box 1 region
and region(s) in the amino-terminal one-half of JAK2 is
non-phosphotyrosine-dependent (6, 9).
Other signaling molecules activated by GH appear to at least
transiently form associations with the GHR-JAK2 complex. For example,
we have observed a low basal degree of association of STAT3 and STAT5b
with the GHR. In this case, the degree of association between STAT5b,
in particular, and the receptor is markedly enhanced by JAK2-catalyzed
GHR tyrosine phosphorylation (19), which apparently allows this STAT to
itself become a better target for JAK2-induced tyrosine phosphorylation
and activation as a transcriptional activator. While less completely
understood, the physical association of IRS molecules with the GHR-JAK2
complex has been speculated to be largely via JAK2 (26, 27); the
dependences for GH stimulation or for tyrosine phosphorylation of
either JAK2 or of IRS-1 or -2 for their physical associations to occur
are currently unknown.
Our present data implicate another important signaling molecule, SHP-2,
as a functional component of the GHR-JAK2-associated signaling complex.
We observe both in 3T3-F442A cells homologously expressing the receptor
and in 32D cells heterologously expressing the GHR an acute and
pronounced GH-induced tyrosine phosphorylation of SHP-2, the kinetics
of which are consistent with being catalyzed by JAK2. One question that
arises pertains to the nature of SHP-2's interaction with the GHR-JAK2
complex. Is SHP-2 directly physically interacting with either the GHR
or JAK2, and if so, is the interaction(s) phosphotyrosine-dependent? While we do not yet have
complete answers to these questions, several results favor the
interactions being somewhat complicated and mixed. Previous detailed
mapping studies in COS cells overexpressing JAK2 and SHP-2 indicated a
specific non-phosphotyrosine-dependent interaction of the
two molecules mediated by a region of SHP-2 C-terminal to the SH2
domains and the amino terminus of JAK2 (47). Such data would favor the
likelihood that SHP-2 might associate basally to some degree with the
inactive GHR-JAK2 complex via this interaction with
JAK2.6
Our data indicate, however, that additional GH- and
phosphotyrosine-dependent interaction between SHP-2 and the
GHR-JAK2 complex can occur via the SH2-containing region of SHP-2. We
do not yet have definitive evidence as to whether this interaction is
directly between SHP-2 and the tyrosine-phosphorylated GHR or whether
it is less direct and mediated by tyrosine-phosphorylated SIRP-
-like molecules or other molecules. We think it unlikely that significant GH-dependent direct interaction between SHP-2 and
tyrosine-phosphorylated JAK2 occurs, given that the JAK2-SHP-2 mapping
studies referred to above (47), in which no GHR or other cytokine
receptor was expressed, gave no evidence of a
phosphotyrosine-dependent interaction between the two
molecules. Our finding that a GST fusion incorporating the
tyrosine-phosphorylated C-terminal 40% of the GHR cytoplasmic domain
(residues 485-620), but not an unphosphorylated version of the same
fusion, specifically interacted with SHP-2 (and not SHP-1) from
extracts of IM-9 cells argues that at least part of the SHP-2
interaction with the GHR-JAK2 complex may be mediated via the
receptor's distal tail; further studies in which mutagenized receptors
are reconstituted will be required to confirm and extend this
finding.
Although a relatively significant amount of tyrosine-phosphorylated GHR
was recovered in a GST/SHP-2-SH2 precipitate of GH-stimulated 3T3-F442A
cells, we also note that much less was recovered in the anti-SHP-2
precipitate of the same extract and that this relative decrease was at
the expense of the potential SIRP-
-like tyrosine-phosphorylated glycoprotein enriched in the immunoprecipitate. Similar findings were
observed in 32D-rGHR cells with regard to the basally
tyrosine-phosphorylated diffuse protein at roughly 115 kDa enriched in
anti-SHP-2 precipitates. This 115-kDa protein may be similar to a
glycoprotein (referred to as p135) recently described by Gu et
al. (64) in anti-SHP-2 precipitates of murine
factor-dependent cells. In our work, this tyrosine
phosphoprotein, by virtue of its significant basal tyrosine phosphorylation, can be differentiated from the GHR, which only became
significantly tyrosine-phosphorylated in the 32D-rGHR cells in response
to GH. It has been noted by others that SHP-2 in some systems may
exhibit a higher affinity for SIRP
-like proteins than for receptors
(such as tyrosine-phosphorylated epidermal growth factor or insulin
receptors) whose function is influenced by SHP-2 (58, 69). Our finding
that the tyrosine-phosphorylated GHR is more readily detected in
GST-SHP-2-SH2 precipitates than in anti-SHP-2 immunoprecipitates may
reflect the relatively less stable SHP-2-GHR association in these cells
in the presence of a more pronounced SHP-2-SIRP
-like protein
association. Further studies with direct reacting antibodies that
detect SIRP-
-like proteins and p135 may shed light on whether the
complex of tyrosine-phosphorylated proteins coprecipitated with
anti-SHP-2 from 3T3-F442A and 32D-rGHR cells might indeed include such
molecules.
While GH has been clearly shown to elicit IRS-1 and -2 tyrosine
phosphorylation and consequent activation of phosphatidylinositol 3-kinase (26, 27), our results in 32D-rGHR cells indicate that IRS-1
and -2 are not necessary for GH-induced tyrosine phosphorylation of
SHP-2 or its association with Grb2. Since at least transient inclusion
of SHP-2 in a complex containing the GHR and JAK2 probably underlies
SHP-2's ability to undergo this tyrosine phosphorylation, we also
conclude that such a complex can form in the absence of IRS-1 and -2 proteins. We are currently investigating whether expression of these
proteins, however, can affect the degree or stability of GHR-JAK2-SHP-2
complex(es).
At a functional level, the results of our studies comparing expression
of wild-type versus catalytically inactive SHP-2 in the
context of cotransfection of the GHR and JAK2 indicate that intact
SHP-2 catalytic activity contributes positively to GH-induced transcriptional activation of a c-fos enhancer-driven
luciferase reporter gene. In these experiments, enhancement of this
GH-induced signal by transfection of wild-type SHP-2 was variable;
COS-7 cells have ample endogenous
SHP-2,7 which probably
couples to the transfected GHR and JAK2. However, substitution of the
CS SHP-2 for the wild-type SHP-2 in the transfection mix substantially
reduced the GH-induced transactivation. A similar degree of inhibition
of ligand-induced transactivation of a c-fos SRE-driven
luciferase reporter was recently reported to occur with substitution of
a catalytically inactive SHP-2 for wild-type SHP-2 in the
proteinase-activated receptor-2 (a G protein-coupled receptor whose
activation results in SHP-2 tyrosine phosphorylation) signaling system
(70). Recent studies of prolactin receptor signaling have also
implicated a positive role for SHP-2 in that catalytically inactive or
SH2 domain mutant SHP-2 molecules also exhibited a roughly 50-65%
decrease in prolactin-induced transactivation of a reporter driven, in
that case, by the
-casein gene promoter (presumably at least
partially dependent on STAT5 signaling) (38).
We do not yet know the biochemical basis for the positive role for
SHP-2 in GH signaling that we observe. Given that COS-7 cells do not
have immunologically detectable IRS-1 or -2,7 this effect
of SHP-2 does not appear to depend on either of those proteins. We do
note that the enhancer driving our luciferase reporter contains both
the c-fos SRE and SIE regions and might thus be affected by
various GH-induced signaling intermediates, including members of the
MAP kinase and STAT1/3 activation pathways. Indeed, expression of
catalytically inactive SHP-2 has been shown to result in diminished
insulin-induced MAP kinase activation (35). We think it unlikely, based
on published studies (47) and our unpublished preliminary results, that
JAK2 itself is a significant target for SHP-2 catalytic activity and
that changes in overall JAK2 activation status underlie the defective
signaling conferred by the catalytically inactive SHP-2. It is
appealing to speculate, however, that the inclusion of SHP-2 in a
physical complex with the GHR and JAK2 that we have observed herein may have a role in allowing its enzymatic activity to discretely influence signaling through the complex. Further studies that identify relevant targets of activated GHR-JAK2 complex-associated SHP-2 will undoubtedly help decipher the mechanism of its positive influence.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Drs. J. Ihle, W. Wood, M. Thomas, W. Chen, J. Kudlow, C. Carter-Su, H. Green, A. Kraft,
J. Bonifacino, K. Arai, and T. Pawson for contribution of cells,
plasmids, and reagents and Eli Lilly Co. for providing the hGH. We
appreciate helpful conversations with Drs. J. Kudlow, A. Paterson, E. Chin, L. Liang, J. Goldsmith, and L. Justement. J. Goldsmith's
contribution to development of the anti-GHRext monoclonal
antibody is also acknowledged.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant DK46395 (to S. J. F.) and a Veterans Administration Merit Review award (to S. J. 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.
**
To whom correspondence should be addressed: University of
Alabama at Birmingham, Room 756, DREB, UAB Station, Birmingham, AL
35294. Tel.: 205-934-9856; Fax: 205-934-4389; E-mail:
frank{at}endo.dom.uab.edu.
1
The abbreviations used are: GH, growth hormone;
GHR, GH receptor; SH2, Src homology 2; hGH, human GH; hGHR, human GHR;
rGHR, rabbit GHR; APT, antiphosphotyrosine; mAb, monoclonal antibody; GST, glutathione S-transferase; BSA, bovine serum albumin; FLB, fusion
lysis buffer; PAGE, polyacrylamide gel electrophoresis; MAP,
mitogen-activated protein; IRS, insulin receptor substrate; WT, wild
type.
2
S. O. Kim, W. Yi, and S. J. Frank,
unpublished data.
3
Interestingly, we note that a low level of JAK2
was specifically present in the GST/SHP-1-SH2 precipitate independent
of GH stimulation (Fig. 2D, lanes 3 and
4); this probably indicates that the small amount of
tyrosine-phosphorylated GHR and JAK2 detected in this precipitate (Fig.
2B, lane 4) may have resulted from a non-GH- and
non-phosphotyrosine-dependent associability of JAK2 with
that region of SHP-1, as has also been seen by others (56).
4
Because of the intense tyrosine phosphorylation
of the fusion itself and of fusion multimers (not shown), we were
unable to evaluate the presence in GST/hGHR485-620/E
precipitates of a protein from GH-treated cells corresponding to the
deglycosylated 65-kDa tyrosine phosphoprotein seen in the 3T3-F442A
cells.
5
S. O. Kim, J. Jiang, W. Yi, and
S. J. Frank, unpublished observations.
6
A similar situation has been observed for SHP-1
and JAK2 in that a non-phosphotyrosine-dependent
interaction of the N-terminal SH2 domain-containing region of that
phosphatase with JAK2 has been mapped (56); indeed, our GST fusion
binding data (Fig. 2D, lanes 3 and 4)
reflected this interaction in that a low level of non-GH- and
non-phosphotyrosine-dependent association between GST/SHP-1-SH2 (but not GST/SHP-2-SH2) and JAK2 was observed.
7
S. O. Kim, J. Jiang, W. Yi, G. S. Feng, and S. J. Frank, unpublished observations.
 |
REFERENCES |
-
Isaksson, O. G. P.,
Eden, S.,
and Jansson, J.-O.
(1985)
Annu. Rev. Physiol.
47,
483-499[CrossRef][Medline]
[Order article via Infotrieve]
-
Leung, D. W.,
Spencer, S. A.,
Cachianes, G.,
Hammonds, R. G.,
Collins, C.,
Henzel, W. J.,
Barnard, W. J.,
Waters, M. J.,
Wood, W. I.
(1987)
Nature
330,
537-543[CrossRef][Medline]
[Order article via Infotrieve]
-
Colosi, P.,
Wong, K.,
Leong, S. R.,
Wood, W. I.
(1993)
J. Biol. Chem.
268,
12617-12623[Abstract/Free Full Text]
-
Cunningham, B. C.,
Ultsch, M.,
de Vos, A. M.,
Mulkerrin, M. G.,
Clauser, K. R.,
Wells, J. A.
(1991)
Science
254,
821-825[Medline]
[Order article via Infotrieve]
-
Argetsinger, L. S.,
Campbell, G. S.,
Yang, X.,
Witthuhn, B. A.,
Silvennoinen, O.,
Ihle, J. N.,
Carter-Su, C.
(1993)
Cell
74,
237-244[Medline]
[Order article via Infotrieve]
-
Frank, S. J.,
Gilliland, G.,
Kraft, A. S.,
Arnold, C. S.
(1994)
Endocrinology
135,
2228-2239[Abstract]
-
VanderKuur, J. A.,
Wang, X.,
Zhang, L,
Campbell, G. S.,
Allevato, G.,
Billestrup, N.,
Norstedt, G.,
Carter-Su, C.
(1994)
J. Biol. Chem.
269,
21709-21717[Abstract/Free Full Text]
-
Sotiropoulos, A.,
Perrot-Applanat, M.,
Dinerstein, H.,
Pallier, A.,
Postel-Vinay, M.-C.,
Finidori, J.,
and Kelly, P. A.
(1994)
Endocrinology
135,
1292-1298[Abstract]
-
Frank, S. J.,
Yi, W.,
Zhao, Y.,
Goldsmith, J. R.,
Gilliland, G.,
Jiang, J.,
Sakai, I.,
Kraft, A. S.
(1995)
J. Biol. Chem.
270,
14776-14785[Abstract/Free Full Text]
-
Tanner, J. W.,
Chen, W.,
Young, R. L.,
Longmore, G. D.,
Shaw, A. S.
(1995)
J. Biol. Chem.
270,
6523-6530[Abstract/Free Full Text]
-
Silvennoinen, O.,
Witthuhn, B. A.,
Quelle, F. W.,
Cleveland, J. L.,
Yi, T.,
Ihle, J. N.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8429-8433[Abstract/Free Full Text]
-
Meyer, D. J.,
Campbell, G. S.,
Cochran, B. H.,
Argetsinger, L. S.,
Larner, A. C.,
Finbloom, D. S.,
Carter-Su, C.,
Schwartz, J.
(1994)
J. Biol. Chem.
269,
4701-4704[Abstract/Free Full Text]
-
Campbell, G. S.,
Meyer, D. J.,
Raz, R.,
Levy, D. E.,
Schwartz, J.,
Carter-Su, C.
(1995)
J. Biol. Chem.
270,
3974-3979[Abstract/Free Full Text]
-
Gronowski, A. M.,
and Rotwein, P.
(1994)
J. Biol. Chem.
269,
7874-7878[Abstract/Free Full Text]
-
Gronowski, A. M.,
Zhong, Z.,
Wen, Z.,
Thomas, M. J.,
Darnell, J. E.,
Rotwein, P.
(1995)
Mol. Endocrinol.
9,
171-177[Abstract]
-
Gouilleux, F.,
Pallard, C.,
Dusanter-Fourt, I.,
Wakao, H.,
Haldosen, L.-A.,
Norstedt, G.,
Levy, D.,
and Groner, B.
(1995)
EMBO J.
14,
2005-2013[Abstract]
-
Wood, T. J. J.,
Sliva, D.,
Lobie, P. E.,
Pircher, T. J.,
Gouilleux, F.,
Wakao, H.,
Gustafsson, J.-A.,
Groner, B.,
Norstedt, G.,
Haldosen, L.-A.
(1995)
J. Biol. Chem.
270,
9448-9453[Abstract/Free Full Text]
-
Ram, P. A.,
Park, S.-H.,
Choi, H. K.,
Waxman, D. J.
(1996)
J. Biol. Chem.
271,
5929-5940[Abstract/Free Full Text]
-
Yi, W.,
Kim, S.-O.,
Jiang, J.,
Park, S.-H.,
Kraft, A. S.,
Waxman, D. J.,
Frank, S. J.
(1996)
Mol. Endocrinol.
10,
1425-1443[Abstract]
-
VanderKuur, J.,
Allevato, G.,
Billestrup, N.,
Norstedt, G.,
and Carter-Su.
(1995)
J. Biol. Chem.
270,
7587-7593[Abstract/Free Full Text]
-
Moller, C.,
Hansson, A.,
Enberg, B.,
Lobie, P. E.,
Norstedt, G.
(1992)
J. Biol. Chem.
267,
23403-23408[Abstract/Free Full Text]
-
Campbell, G. S.,
Pang, L.,
Miyasaka, T.,
Saltiel, A. R.,
Carter-Su, C.
(1992)
J. Biol. Chem.
267,
6074-6080[Abstract/Free Full Text]
-
Winston, L. A.,
and Bertics, P. J.
(1992)
J. Biol. Chem.
267,
4747-4751[Abstract/Free Full Text]
-
Souza, S. C.,
Frick, G. P.,
Yip, R.,
Lobo, R. B.,
Tai, L.-R.,
Goodman, H. M.
(1994)
J. Biol. Chem.
269,
30085-30088[Abstract/Free Full Text]
-
Ridderstrale, M.,
Degerman, E.,
and Tornqvist, H.
(1995)
J. Biol. Chem.
270,
3471-3474[Abstract/Free Full Text]
-
Argetsinger, L. S.,
Hsu, G. W.,
Myers, M. G., Jr.,
Billestrup, N.,
White, M. F.,
Carter-Su, C.
(1995)
J. Biol. Chem.
270,
14685-14692[Abstract/Free Full Text]
-
Argetsinger, L. S.,
Norstedt, G.,
Billestrup, N.,
White, M. F.,
Carter-Su, C.
(1996)
J. Biol. Chem.
271,
29415-29421[Abstract/Free Full Text]
-
Klingmuller, U.,
Lorenz, U.,
Cantley, L. C.,
Neel, B. G.,
Lodish, H. F.
(1995)
Cell
80,
729-738[Medline]
[Order article via Infotrieve]
-
Yi, T. L.,
Mui, A. L. F.,
Krystal, G.,
and Ihle, J. N.
(1993)
Mol. Cell. Biol.
13,
7577-7586[Abstract]
-
Chen, H. E.,
Chang, S.,
Trub, T.,
and Neel, B. G.
(1996)
Mol. Cell. Biol.
16,
3685-3597[Abstract]
-
Plas, D. R.,
Johnson, R.,
Pingel, J. T.,
Mathews, R. J.,
Dalton, M.,
Roy, G.,
Chan, A. C.,
Thomas, M. L.
(1996)
Science
272,
1173-1176[Abstract]
-
D'Ambrosio, D.,
Hippen, K. L.,
Minskoff, S. A.,
Mellman, I.,
Pani, G.,
Siminovitch, K. A.,
Cambier, J. C.
(1995)
Science
268,
293-297[Medline]
[Order article via Infotrieve]
-
Hackett, R. H.,
Wang, Y.-D.,
Sweitzer, S.,
Feldman, G.,
Wood, W. I.,
Larner, A. C.
(1997)
J. Biol. Chem.
272,
11128-11132[Abstract/Free Full Text]
-
Bennett, A. M.,
Tang, T. L.,
Sugimoto, S.,
Walsh, C. T.,
Neel, B. G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7335-7339[Abstract]
-
Milarski, K. L.,
and Saltiel, A. R.
(1994)
J. Biol. Chem.
269,
21239-21243[Abstract/Free Full Text]
-
Xiao, S.,
Rose, D. W.,
Sasoka, T.,
Maegawa, H.,
Burke, T. R., Jr.,
Roller, P. P.,
Shoelson, S. E.,
Olefsky, J. M.
(1994)
J. Biol. Chem.
269,
21244-21248[Abstract/Free Full Text]
-
Yamauchi, K.,
Milarski, K. L.,
Saltiel, A. R.,
Pessin, J. E.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
664-668[Abstract]
-
Ali, S.,
Chen, Z.,
Lebrun, J.-J.,
Vogel, W.,
Dharitonenkov, A.,
Kelly, P. A.,
Ullrich, A.
(1996)
EMBO J.
15,
135-142[Abstract]
-
Feng, G.-S.,
Hui, C.-C.,
and Pawson, T.
(1993)
Science
259,
1607-1611[Medline]
[Order article via Infotrieve]
-
Vogel, W.,
Lammers, R.,
Huang, J.,
and Ullrich, A.
(1993)
Science
259,
1611-1614[Medline]
[Order article via Infotrieve]
-
Lechleider, R. J.,
Sugimoto, S.,
Bennett, A. M.,
Kashishian, A. S.,
Cooper, J. A.,
Shoelson, S. E.,
Walsh, C. T.,
Neel, B. G.
(1993)
J. Biol. Chem.
268,
21478-21481[Abstract/Free Full Text]
-
Kazlauskas, A.,
Feng, G.-S.,
Pawson, T.,
and Valius, M.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
6939-6942[Abstract]
-
Fuhrer, D. K.,
Feng, G.-S.,
and Yang, Y.-C.
(1995)
J. Biol. Chem.
270,
24826-24830[Abstract/Free Full Text]
-
Tauchi, T.,
Damen, J. E.,
Toyama, K.,
Feng, G.-S.,
Broxmeyer, H. E.,
Krystal, G.
(1996)
Blood
87,
4495-4501[Abstract/Free Full Text]
-
Kuhne, M. R.,
Pawson, T.,
Lienhard, G. E.,
Feng, G.-S.
(1993)
J. Biol. Chem.
268,
11479-11481[Abstract/Free Full Text]
-
Sun, X. J.,
Crimmons, D. L.,
Myers, M. G.,
Malpeix, M.,
White, M. F.
(1993)
Mol. Cell. Biol.
13,
7418-7428[Abstract]
-
Yin, T.,
Shen, R.,
Feng, G.-S.,
and Yang, Y.-C.
(1997)
J. Biol. Chem.
272,
1032-1037[Abstract/Free Full Text]
-
Kehinde, O.,
and Green, H.
(1976)
Cell
7,
105-113[Medline]
[Order article via Infotrieve]
-
Zhao, Y.,
Wagner, F.,
Frank, S. J.,
Kraft, A. S.
(1995)
J. Biol. Chem.
270,
13814-13818[Abstract/Free Full Text]
-
Frank, S. J.,
Gilliland, G.,
and Van Epps, C.
(1994)
Endocrinology
135,
148-156[Abstract]
-
Takebe, Y.,
Seiki, M.,
Fujisawa, J.-I.,
Hoy, P.,
Yokota, K.,
Arai, K.-I.,
Yoshida, M.,
and Arai, N.
(1988)
Mol. Cell. Biol.
8,
466-453[Medline]
[Order article via Infotrieve]
-
Chen, W. S.,
Lazar, C. S.,
Poenie, M.,
Tsien, R. Y.,
Gill, G. N.,
Rosenfeld, M. G.
(1987)
Nature
328,
820-823[CrossRef][Medline]
[Order article via Infotrieve]
-
Kozlowski, M.,
Mlinaric-Rascan, I.,
Feng, S.-S.,
Shen, R.,
Pawson, T.,
and Siminovitch, K. A.
(1993)
J. Exp. Med.
178,
2157-2163[Abstract]
-
Reedijk, M.,
Liu, X.,
van der Geer, P.,
Letwin, K.,
Waterfield, M. D.,
Hunter, T.,
Pawson, T.
(1992)
EMBO J.
11,
1365-1372[Abstract]
-
Carter-Su, C.,
Schwartz, J.,
and Smit, L. S.
(1996)
Annu. Rev. Physiol.
58,
187-207[CrossRef][Medline]
[Order article via Infotrieve]
-
Jiao, H.,
Berrada, K.,
Yang, W.,
Tabrizi, M.,
Platanias, L. C.,
Yi, T.
(1996)
Mol. Cell. Biol.
16,
6985-6992[Abstract]
-
Silva, C. M.,
Weber, M. J.,
and Thorner, M. O.
(1993)
Endocrinology
132,
101-108[Abstract]
-
Yamauchi, K.,
Ribon, V.,
Saltiel, A. R.,
Pessin, J. E.
(1995)
J. Biol. Chem.
270,
17716-17722[Abstract/Free Full Text]
-
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]
-
Kharitonenkov, A.,
Chen, Z.,
Sures, I.,
Wang, H.,
Schilling, J.,
and Ullrich, A.
(1997)
Nature
386,
181-186[CrossRef][Medline]
[Order article via Infotrieve]
-
Lesniak, M. A.,
Gorden, P.,
Roth, J.,
and Gavin, J. R., III
(1974)
J. Biol. Chem.
249,
1661-1667[Abstract/Free Full Text]
-
Silva, C. M.,
Lu, H.,
and Day, R. N.
(1996)
Mol. Endocrinol.
10,
508-518[Abstract]
-
Sun, X. J.,
Pons, S.,
Wang, L.-M.,
Zhang, Y.,
Yenush, L.,
Burks, D.,
Myers, M. G., Jr.,
Glasheen, E.,
Copeland, N. G.,
Jenkins, J. A.,
Pierce, J. H.,
White, M. F.
(1997)
Mol. Endocrinol.
11,
251-262[Abstract/Free Full Text]
-
Gu, H.,
Griffin, J. D.,
and Neel, B. G.
(1997)
J. Biol. Chem.
272,
16421-16430[Abstract/Free Full Text]
-
Li, W.,
Nishimura, R.,
Kashishian, A.,
Batzer, A. G.,
Kim, W. J. H.,
Cooper, J. A.,
Schlessinger, J.
(1994)
Mol. Cell. Biol.
14,
509-517[Abstract]
-
Bennett, A. M.,
Tang, T. L.,
Sugimoto, S.,
Walsh, C. T.,
Neel, B. G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7335-7339[Abstract]
-
Tauchi, T.,
Feng, G.-S.,
Shen, R.,
Hoatlin, M.,
Bagby, G. C., Jr.,
Kabat, D.,
Lu, L.,
Broxmeyer, H. E.
(1995)
J. Biol. Chem.
270,
5631-5635[Abstract/Free Full Text]
-
Kharitonenkov, A.,
Schnekenburger, J.,
Chen, Z.,
Knyazev, P.,
Ali, S.,
Zwick, E.,
White, M.,
and Ullrich, A.
(1995)
J. Biol. Chem.
270,
29189-29193[Abstract/Free Full Text]
-
Yamauchi, K.,
and Pessin, J. E.
(1995)
J. Biol. Chem.
270,
14871-14874[Abstract/Free Full Text]
-
Yu, Z.,
Ahmad, S.,
Schwartz, J.-L.,
Banville, D.,
and Shen, S.-H.
(1997)
J. Biol. Chem.
272,
7519-7524[Abstract/Free Full Text]
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