Involvement of the Src Homology 2-containing Tyrosine Phosphatase SHP-2 in Growth Hormone Signaling*

Sung-Oh KimDagger §, Jing JiangDagger , Woelsung YiDagger §, Gen-Sheng Fengpar , and Stuart J. FrankDagger §**

From the Dagger  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 par  Department of Biochemistry and Molecular Biology, Walther Oncology Center, Indiana University School of Medicine and Walther Cancer Institute, Indianapolis, Indiana 46202-5121

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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-alpha -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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
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Introduction
Procedures
Results
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References

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-kappa ) 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 SRalpha 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-SRalpha 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 lambda  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-beta -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-beta -D-galactopyranoside induction of these infectants allowed for high level production of both the GST fusions and lambda /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 beta -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 beta -galactosidase spectrophotometric assay was performed on the same extracts. (No systematic variation in basal or GH-induced beta -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
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Procedures
Results
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References

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.

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.

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-alpha 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 lambda , 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.

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.

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.

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).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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-alpha -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-alpha -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 SIRPalpha -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-SIRPalpha -like protein association. Further studies with direct reacting antibodies that detect SIRP-alpha -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 beta -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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