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Endocrinology Vol. 143, No. 12 4856-4867
Copyright © 2002 by The Endocrine Society

ARTICLE

A Role for Grb2-Associated Binder-1 in Growth Hormone Signaling

Sung-Oh Kim, Kimberly Loesch, Xiangdong Wang, Jing Jiang, Lin Mei, Jess M. Cunnick, Jie Wu and Stuart J. Frank

Department of Medicine, Division of Endocrinology and Metabolism (S.-O.K., X.W., J.J., S.J.F.), Department of Cell Biology (K.L., S.J.F.), and Departments of Neurobiology, Pathology, and Physical Medicine and Rehabilitation, Civitan International Research Center (L.M.), University of Alabama, Birmingham, Alabama 35294; Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute and Department of Interdisciplinary Oncology, University of South Florida (J.M.C., J.W.), Tampa, Florida 33612; and Veterans Affairs Medical Center (S.J.F.), Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Dr. Stuart J. Frank, University of Alabama, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012. E-mail: frank{at}endo.dom.uab.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH signaling begins with activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, Janus kinase-2. GH-induced Janus kinase-2 activation leads to engagement of several signaling pathways, including the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase, phosphoinositol 3-kinase, and signal transducer and activator of transcription-5 (STAT5) pathways. Previous work suggests that ERK activation in response to GH may be modulated by several proteins acting as docking molecules, including the epidermal growth factor receptor (EGFR) and insulin receptor substrate-1. In this study we investigate potential roles for the pleckstrin homology (PH) domain-containing insulin receptor substrate-like protein, Grb-2-associated binder-1 (Gab1), in GH signaling. We find in 3T3-F442A preadipocytes that GH promotes tyrosine phosphorylation of Gab1 and its association with SHP2, an Src homology 2-containing cytoplasmic tyrosine phosphatase. The Grb2 adapter protein, in contrast, is specifically coimmunoprecipitated with Gab1, even in the absence of GH exposure. Using a COS-7 cell transient reconstitution system, we observed that GH-induced Gab1 tyrosine phosphorylation is dependent on the Gab1 PH domain, whereas GH-induced coimmunoprecipitation of SHP2 requires tyrosine 627 of Gab1, as previously reported for EGF-induced Gab1-SHP2 association. Deletion of the Gab1 PH domain significantly attenuates GH-induced ERK activation and trans-activation of a c-fos enhancer-driven reporter construct compared with wild-type Gab1 in this system. In contrast, GH-induced STAT5 tyrosine phosphorylation and STAT5-dependent trans-activation are similar in cells expressing wild-type or PH domain-deleted Gab1. Notably, neither the ERK nor the STAT5 GH-dependent signaling outcome is affected by expression of the Gab1 mutant with tyrosine 627 changed to phenylalanine. Finally, we observed GH-dependent translocation of a wild-type, but not a PH domain-deleted, Gab1-green fluorescent protein chimera from the cytoplasm to the plasma membrane. Our results suggest selective involvement of Gab1 in GH-induced ERK activation and implicate the Gab1 PH domain as critical in this involvement.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS A 22-kDa polypeptide that exerts growth-promoting, metabolic, and differentiative effects on its target cells and tissues (1). The GH receptor (GHR) is a transmembrane glycoprotein belonging to the cytokine receptor superfamily (2). Binding of GH leads to activation and autophosphorylation of the GHR-associated cytoplasmic tyrosine kinase Janus kinase-2 (JAK2) (3, 4). JAK2 activation promotes tyrosine phosphorylation of the GHR and other cellular proteins, fostering the activation of signal transducers and activators of transcription (STATs) and other pathways, including the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), and phosphoinositol 3-kinase (PI3K) pathways (5, 6). STAT5 (particularly STAT5b) is implicated in several aspects of GH action, including growth, sexually dimorphic hepatic gene expression, IGF-I gene expression, and adipocyte differentiation (7, 8, 9). GH-induced STAT5 activation requires its tyrosine phosphorylation and phosphorylation of a tyrosine residue(s) within the GHR cytoplasmic domain (10, 11, 12, 13, 14, 15).

ERK1 and -2 are variably activated in response to GH in several cell systems, including fibroblasts, preadipocytes, rat hepatocytes, GHR-transfected Chinese hamster ovary (CHO) cells, COS cells, and factor-dependent promonocytes (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). ERK activation may be relevant in several aspects of GH signaling. For example, augmentation of GH-induced ERK activity in factor-dependent promonocytes by expression of insulin receptor substrate-1 (IRS-1) is associated with enhanced GH-induced proliferation (23, 25). In contrast, GH-induced ERK activation in 3T3-F442A preadipocytes leads to serine/threonine phosphorylation of ErbB-2, a transmembrane tyrosine kinase member of the epidermal growth factor receptor (EGFR) family that heterodimerizes with the EGFR in response to EGF (27). In these cells, ERK pathway activation-dependent phosphorylation renders ErbB-2 less susceptible to activation and may contribute to GH’s inhibition of EGF-induced proliferation (27, 28). Further studies suggested that acute GH stimulation of c-fos gene expression is related to GH-induced activation of the ERK pathway (29, 30).

Several potential upstream activators of the MEK1-ERK pathway are engaged by GH-induced signaling. Activation of JAK2 by GH is required for GH-induced ERK activation (18, 20, 21, 22, 31), and the kinetics of GH-induced Shc-Grb2-Sos-Ras-Raf pathway engagement suggest that it is a mechanism of ERK activation (32, 33). Yamauchi et al. (34) showed that GH-induced JAK2-mediated tyrosine phosphorylation of the EGFR at a Grb2 association site can enhance GH-stimulated ERK activation and c-fos expression, suggesting that EGFR in that instance acts as a docking protein for activation of the ERK pathway. IRS proteins are cytoplasmic docking molecules that are tyrosine phosphorylated in response to a number of growth factors and cytokines, including GH (35, 36, 37, 38, 39). In previous studies, IRS-1 expression in IRS-deficient cells augmented GH-induced ERK activation, but did not influence GH-induced STAT5 tyrosine phosphorylation (25).

The IRS proteins (IRS-1 to -4) share structural and functional similarities with other large cytoplasmic docking proteins, including Daughter of Sevenless, fibroblast growth factor receptor substrate-2, Grb2-associated binder 1 (Gab1), and Gab2. These proteins have been implicated as important in various signaling systems (reviewed in Refs. 40 and 41). Structurally, each contains an N-terminal pleckstrin homology (PH) domain, lacks enzymatic activity, and possesses multiple tyrosine residues that become phosphorylated after activation of certain intrinsic tyrosine kinase and cytokine receptors. These phosphorylated sites can inducibly associate with Src homology 2 (SH2) domain-containing proteins to affect downstream signaling. To date, IRS-1, -2, and -3 are the only known members of this family to undergo GH-induced tyrosine phosphorylation.

Gab1 is a widely distributed protein that was first identified by virtue of its constitutive association with the Grb2 adapter protein, which is mediated by interaction of a Grb2 SH3 domain with a proline-rich sequence in Gab1 (42, 43). It is critically involved in allowing hepatocyte growth factor (HGF)-induced epithelial branching morphogenesis, and its targeted deletion in vivo results in cardiac, placental, and skin developmental defects and death in utero (44, 45). Gab1 is involved in signaling from various receptors other than the HGF receptor, including IL-3, IL-6, interferon-{alpha} and -{gamma}, insulin, EGF, and B cell antigen receptors (42, 46, 47, 48). In some instances, Gab1’s PH domain may be critical by interacting with PI3K-generated lipid metabolites to localize Gab1 to the plasma membrane, where it may engage with signaling complexes. Gab1’s potentiation of the ERK pathway activation is prominent for certain stimuli, including HGF, platelet-derived growth factor, and EGF, and for stimuli using the gp130 cytokine receptor (45). Notably, EGF-induced ERK activation, in particular, appears to rely on an EGF-induced association of SHP2, an SH2-containing cytoplasmic tyrosine phosphatase, with Gab1 phosphotyrosines 627 and 659, resulting in enzymatic activation of SHP2 (49, 50, 51).

In this study we investigate the involvement of Gab1 in GH signaling. We found in 3T3-F442A preadipocytes that GH promotes tyrosine phosphorylation of Gab1 and its association with SHP2. Using a transient reconstitution system, we observed that GH-induced Gab1 tyrosine phosphorylation is dependent on the Gab1 PH domain, whereas GH-induced coimmunoprecipitation of SHP2 requires tyrosine 627 of Gab1, as previously reported for EGF-induced Gab1-SHP2 association. Deletion of the Gab1 PH domain significantly attenuates GH-induced ERK activation and trans-activation of a c-fos enhancer-driven reporter construct compared with wild-type (WT) Gab1 in this system, but notably neither signaling outcome is affected by expression of the Gab1 mutant with tyrosine 627 changed to phenylalanine. Further, we observed GH-dependent translocation of a WT, but not a PH domain-deleted, Gab1-enhanced green fluorescent protein (Gab1-EGFP) chimera from the cytoplasm to the plasma membrane. Our results suggest potential involvement of Gab1 in GH-induced ERK activation and implicate the PH domain as important in this involvement.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant hGH was provided by Eli Lilly \|[amp ]\| Co. (Indianapolis, IN). Recombinant human EGF was purchased commercially (Collaborative Biomedical Products, Bedford, MA). Routine reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted. Restriction endonucleases were obtained from New England Biolabs, Inc. (Beverly, MA).

Antibodies
4G10 monoclonal antiphosphotyrosine (anti-pY) antibody (Upstate Biotechnology, Inc., Lake Placid, NY), anti-Gab1 polyclonal antibody (Upstate Biotechnology, Inc.), anti-SHP-2 monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Grb2 monoclonal antibody (Transduction Laboratories, Inc., Lexington, KY), anti-STAT5 monoclonal antibody (Transduction Laboratories, Inc.), anti-phospho-STAT5 affinity-purified rabbit antibody [recognizing the tyrosine phosphorylated form (Y694) of STAT5; Zymed Laboratories, Inc., South San Francisco, CA], anti-HA monoclonal antibody (Roche, Indianapolis, IN), anti-MAPK affinity-purified rabbit antibody (directed at residues 333–367 of rat ERK1; recognizes both ERK1 and ERK2; Upstate Biotechnology, Inc.), and anti-active MAPK affinity-purified rabbit antibody (recognizing the dually phosphorylated Thr183 and Tyr185 residues that correspond to the active forms of ERK1 and ERK2; Promega Corp., Madison, WI) were all purchased commercially. Anti-FLAG M2 affinity gel (Sigma; monoclonal anti-FLAG antibody covalently coupled to agarose) was used for immunoprecipitation of FLAG-tagged Gab1 proteins.

Plasmid construction
C-Terminally FLAG-tagged human Gab1 WT and the mutant Y627F (Tyr627 replaced with Phe) in the pcDNA3.1 expression vector have been previously described (49), as have pSX rabbit (rb) GHR and pSX murine (m) JAK2 (52). The Gab1 delPH mutant was generated by PCR using WT Gab1 (Gab1 WT) cDNA as a template and the following primers: sense, 5'-GGGGTACCATGAATCCAACAGAAGAAGATCCTGTG-3'; and antisense, 5'-CGGGATCCTCCTGAATGCCAAGAGTTTCCAG-3'. The PCR product was ligated into pcDNA3.1/Gab1 WT via the KpnI and BamHI sites to replace the WT sequence with the PH domain deletion. PCR fidelity and the presence of the mutations were confirmed by dideoxy-DNA sequencing. Retroviral vectors (below) encoding the WT and delPH mutant Gab1 molecules fused to EGFP (48) were provided by Dr. Michael Gold (University of British Columbia, Vancouver, British Columbia, Canada). The plasmid encoding human EGFR, pXGen/EGFR, was provided by Dr. Paul Bertics (University of Wisconsin, Madison, WI). The plasmids encoding the c-fos and STAT5-dependent luciferase reporter plasmids, p2FTL (22, 53), pRc/CMV ERK2tag (22), and serine protease inhibitor (Spi)-GAS-like element (GLE)-luciferase (luc) (54) (provided by Dr. William Lowe, Northwestern University, Chicago, IL), have been previously described.

Cell culture, transfection, and retroviral infection
3T3-F442A cells (55), provided by Drs. H. Green (Harvard University, Boston, MA) and C. Carter-Su (University of Michigan, Ann Arbor, MI), were cultured in DMEM (4.5 g/liter glucose; Cellgro, Inc., Herndon, VA) supplemented with 10% calf serum (Biofluids, Rockville, MD) and 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Biofluids). COS-7 cells were grown in DMEM (1.0 g/liter glucose) supplemented with 10% fetal bovine serum (Biofluids). Cells were transfected in 150 x 20-mm dishes by the calcium phosphate precipitation method as described previously (52). Each dish was transfected with both 20 µg pSX rbGHR and 6 µg pSX mJAK2 along with 20 µg Gab1 WT, Gab1 Y627F, or Gab1 delPH cDNA in pcDNA3.1. Ten micrograms of pRc/CMV ERK2tag were transfected along with each Gab1 construct for ERK activation experiments; 20 µg of pXGen/EGFR was transfected along with each Gab1 construct for EGF stimulation experiments.

For packaging of retrovirus, the GP2-293 cell line (CLONTECH Laboratories, Inc., Palo Alto, CA; an HEK-293 derivative) was used. GP2-293 cells (one 6-cm2 dish per condition) were cotransfected with either of the retroviral plasmids, pMX Gab1 WT/EGFP or pMX Gab1 delPH/EGFP (4 µg), plus an equal amount of pVSVG (CLONTECH Laboratories, Inc.; to express retroviral envelop protein) using Lipofectamine Plus (Invitrogen, San Diego, CA). Supernatants containing retrovirus were harvested 48 h after transfection and filtered through a 0.45-µm pore size sterile filter. An infectate solution was made by mixing the filtered stock with fresh medium at a 1:1 (vol/vol) dilution and was applied to 3T3-F442A cells (one 80% confluent 10-cm2 dish per infection) in the presence of polybrene (4 µg/ml, final concentration). Infected cells were incubated at 37 C for 12 h, split 1:1 by trypsinization, and incubated for an additional 48 h. Cells were then harvested by trypsinization, washed with PBS, diluted in fresh growth medium to a density of 10 x 106/ml, and sorted for green fluorescence by fluorescence-activated cell sorting (FACS) (at the University of Alabama Multipurpose Arthritis Center FACS Core Facility). GFP-positive cells were collected and grown in fresh growth medium for use in translocation experiments (below).

Cell stimulation and protein extraction
Serum starvation of 3T3-F442A cells was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V; Roche) for serum in the culture medium for 16–20 h before experiments. For the experiments with COS-7 cells, the transfected cells were serum-starved 24 h after transfection. Pretreatments and stimulations were carried out at 37 C in binding buffer [consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (wt/vol) BSA, and 1 mM dextrose].

Details of the GH treatment protocol have been described previously (56). Stimulations were terminated by washing the cells once with ice-cold PBS containing 0.4 mM sodium orthovanadate (PBS-vanadate) and then harvesting by scraping in PBS-vanadate; pelleted cells were collected by brief centrifugation. Pelleted cells were solubilized for 15 min at 4 C in fusion lysis buffer [1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, and 5 µg/ml aprotinin] as indicated. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent extracts were subjected to immunoprecipitation or were directly electrophoresed, as indicated below.

Immunopreciptiation, electrophoresis, and immunoblotting
For immunoprecipitation, the antibodies described above were used at the following volumes or amounts per precipitation: anti-Gab1, 3 µg; anti-EGFR, 5 µg; and anti-FLAG M2 affinity gel, 30 µl. Protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ) 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.

Resolution of proteins under reduced conditions by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Pharmacia Biotech, Arlington Heights, IL) with 2% BSA were performed as previously described (52). Immunoblotting with antibodies 4G10 (1:2500), anti-Gab1 (1:1000), anti-EGFR (1:1000), anti-phospho-STAT5 (1:500), anti-STAT5 (1:500), anti-active MAPK (1:5000), anti-MAPK (1:1000), antihemagglutinin (anti-HA; 1:500), anti-Grb2 (1:1000), or anti-SHP-2 (1:1000) with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibodies (1:1500) and enhanced chemiluminescence detection reagents (all from Amersham Pharmacia Biotech) and stripping and reprobing of blots were accomplished according to manufacturers’ suggestions.

Densitometric analysis
Densitometry of enhanced chemiluminescence immunoblots was performed using a solid state video camera (Sony 77, Sony Corp., Tokyo, Japan) 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, Bethesda, MD). Relative ERK2 activation in detergent extracts of cells transfected with Gab1 WT or Gab1 mutants was estimated in each instance by the ratio of densitometrically determined antiactive ERK2 immunoblotting signal (transfected ERK2) to the densitometric signal of the same band after stripping and reprobing with anti-HA (transfected ERK2). The relative ERK2 activation, determined as such, was then compared for Gab1 WT vs. each Gab1 mutant transfection. Relative STAT5 tyrosine phosphorylation in detergent extracts of cells was measured by the ratio of densitometrically determined anti-phospho-STAT5 immunoblotting signal (total tyrosine-phosphorylated STAT5) to the densitometric signal of the same band after stripping and reprobing with anti-STAT5 (total STAT5).

c-Fos and Spi-GLE luciferase trans-activation assays
COS-7 cells (6 x 106/dish) were transfected in 10 ml DMEM in 100 x 20-mm dishes (Falcon, BD Biosciences, Franklin Lakes, NJ) by the calcium phosphate precipitation method as described previously (52). Each dish was transfected with both 10 µg pSX rGHR and 3 µg pSX mJak2 along with 10 µg Gab1 WT, Gab1 Y627F, or Gab1 delPH cDNA in pcDNA3.1. p2FTL or Spi-GLE-luc was added at 8 µg/dish. 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 before the addition of hGH (500 ng/ml) or vehicle for 8 h. Stimulations (performed in triplicate) were terminated by aspiration of the medium and addition of luciferase lysis buffer; luciferase activity was assayed as described previously (52). Aliquots of the same extracts were also run on SDS-PAGE and further immunoblotted with anti-Gab1 antibody to ensure the equivalent expression of Gab1 in each transfection. The GH-induced fold increase for each condition within each experiment was compared with that obtained for Gab1 WT-expressing cells (considered 100%).

Gab1/EGFP chimera translocation experiments
3T3-F442A cells retrovirally infected, as described above, with vectors encoding either Gab1 WT or Gab1 delPH fused to EGFP were selected by FACS analysis. Anti-Gab1 immunoblotting (not shown) verified the presence and appropriate SDS-PAGE migration of each fusion protein. Cells were plated onto chamber slides (Nalgene, Rochester, NY) and grown to 50–70% confluence before overnight serum starvation (as described above). After being washed twice with warm PBS, cells were treated with GH (500 ng/ml) or vehicle for 15 min at 37 C. Stimulations were ended by washing three times with cold PBS, after which cells were fixed in 3% formaldehyde for 15 min and washed with PBS. Coverslips were applied with mounting medium. Images were collected at the University of Alabama Cell Biology Imaging Core Facility with a Leitz Orthoplan microscope (Rockleigh, NJ) with epifluorescence and Hoffman Modulation Contrast optics and a Photometrics CH250 liquid-cooled CCD, high resolution, monochromatic camera (Roper Scientific, Tucson, AZ). IPLab Spectrum image acquisition software (Scanalytics, Fairfax, VA) was used. The images shown in Fig. 6Go are composites of cells in each condition from several experiments and were assembled using Adobe Photoshop 6.0.



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  		Figure 6. GH induces surface translocation of a Gab1 WT/EGFP, but not a Gab1 delPH/EGFP chimera. 3T3-F442A cells were retrovirally infected with either Gab1 WT/EGFP (A and B) or Gab1 delPH/EGFP (C and D), as described in Materials and Methods. Serum-starved cells grown in chamber slides were treated with (B and D) or without (A and C) GH for 15 min and examined by immunofluorescence microscopy (x40). Representative images are shown. Note the GH-dependent fluorescence at the cell periphery in the Gab1 WT/EGFP-expressing cells (B) only.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH promotes tyrosine phosphorylation of Gab1 in 3T3-F442A fibroblast cells and in reconstituted COS-7 cells
The 3T3-F442A cell line is a preadipocyte fibroblast that responds to GH stimulation with robust activation of JAK2, tyrosine phosphorylation of the GHR and a number of other substrates, and activation of signaling systems, including the ERK, STAT5, and PI3K pathways (reviewed in Ref. 5). GH-induced tyrosine phosphorylation of IRS-1, IRS-2, and SHP2 have been previously demonstrated as well in these cells (37, 38, 52, 57). We first determined whether Gab1 was detectable in 3T3-F442A cells by anti-Gab1 immunoprecipitation and immunoblotting (Fig. 1Go). Serum-starved cells were solubilized in a detergent-containing lysis buffer after exposure to vehicle or GH for 10 min at 37 C. Extracted proteins were immunoprecipitated with anti-Gab1 or nonimmune rabbit serum as indicated, and eluates were resolved by SDS-PAGE and immunoblotted with anti-Gab1. For comparison, a fraction of the extract was directly resolved without immunoprecipitation. Gab1 was specifically detected at the expected molecular mass of roughly 115 kDa, independent of GH treatment. Although it specifically precipitated Gab1, the anti-Gab1 antibody was relatively inefficient for immunoprecipitation vs. immunoblotting. Notably, GH caused Gab1 to exhibit retarded migration, which could be detected in both immunoblots of the immunoprecipitates and in direct immunoblots of cell extract (Fig. 1AGo, left panel, and Fig. 1BGo, right panel). In other experiments (not shown), the retarded migration of Gab1 in cell extracts could be observed as early as 5 min into stimulation with 500 ng/ml GH and, although maximal with 500 ng/ml, was detected with GH concentrations as low as 25 ng/ml. To determine whether GH-induced retardation of migration could be accounted for by phosphorylation, the blot in the left panel of Fig. 1AGo was stripped and reprobed with antiphosphotyrosine (anti-pY) antibody (Fig. 1AGo, middle panel). This revealed GH-induced Gab1 tyrosine phosphorylation, indicating that at least part of the GH-induced retardation in migration of Gab1 in these cells may be accounted for by phosphorylation on tyrosine residue(s).



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  		Figure 1. GH promotes Gab1 phosphorylation in 3T3-F442A cells. A and B, Serum-starved 3T3-F442A cells (one confluent 150 x 20-mm dish per sample) were exposed to GH (500 ng/ml; +) or vehicle (-) for 10 min. Detergent extracts were either immunoprecipitated (IP; 90% of sample) with the indicated antibodies or not immunoprecipitated (Extract; 10% of sample), as indicated. Proteins were resolved by SDS-PAGE and immunoblotted sequentially with anti-Gab1, anti-pY, and anti-SHP2 (A) or anti-Grb2 and anti-Gab1 (B). NI, Nonimmune antibody was used for a negative immunoprecipitation control. The arrows indicate the positions of Gab1 (A, left panel; B, right panel), SHP2 (A, right panel), Grb2 (B, left panel), and tyrosine-phosphorylated Gab1 (A, middle panel, lane 4). The positions of prestained molecular weight standards are indicated. Note that the proteins in cell extract migrating around the position of Gab1 on the anti-pY blot (A, middle panel) probably include several other basal and GH-induced tyrosine-phosphorylated proteins. Densitometric analysis of the abundance of total and tyrosine-phosphorylated Gab1 in A (left and middle panels, respectively) indicated a 3.5-fold GH-induced increase in specific Gab1 tyrosine phosphorylation in this experiment. Similar analysis of total and Gab1-associated SHP2 (A, right panel) indicated a 2-fold GH-induced increase in Gab1-SHP2 association in this experiment. Analysis of total and Gab1-associated Grb2 (B, left panel and data not shown) indicated a 1.3-fold GH-induced increase in Gab1-Grb2 association in this experiment. This result is representative of two such experiments.

 
In other systems, inducibly tyrosine-phosphorylated Gab1 may associate with other signaling molecules, such as SHP2. Anti-SHP2 immunoblotting of anti-Gab1 precipitates (Fig. 1AGo, right panel) revealed GH-induced coimmunoprecipitation of SHP2 with Gab1. Previous studies (52, 57) indicated that GH induces SHP2 tyrosine phosphorylation in these cells, although it is unclear whether its phosphorylation per se impacts on SHP2’s signaling relevance. Using available anti-Gab1 reagents, we have been unable to detect tyrosine phosphorylation of the Gab1-associated SHP2 in 3T3-F442A cells, perhaps reflecting the relatively inefficient immunoprecipitation of Gab1 from these cells. Grb2-Gab1 association was also assessed in 3T3-F442A cells. As shown in Fig. 1BGo, specific coimmunoprecipitation of Grb2 with Gab1 was detected even in the absence of GH, as has previously been reported in other cell types (42, 43). Thus, both Grb2 and SHP2 can specifically associate with Gab1 in these cells.

To further investigate the involvement of Gab1 in GH signaling, we asked whether GH-induced Gab1 tyrosine phosphorylation could also be detected in transient reconstitution systems (Figs. 2Go and 3Go). We have previously shown that COS-7 cells transiently transfected with the rbGHR and murine JAK2 can respond acutely to GH treatment with GHR and JAK2 tyrosine phosphorylation and that GH-induced trans-activation of reporter gene constructs can also be monitored in this system (22, 52, 54, 56). Using this system, we coexpressed C-terminally FLAG-tagged versions of WT or mutant Gab1 molecules (diagrammed in Fig. 2AGo). As shown in Fig. 2BGo, transfected Gab1 WT was specifically detected in COS-7 cell detergent extracts by anti-FLAG immunoprecipitation and anti-Gab1 immunoblotting independent of acute treatment with GH before cell lysis. As seen in 3T3-F442A cells, the transfected Gab1 underwent a GH-induced mobility shift (Fig. 2BGo, lane 4 vs. lane 3, and Fig. 3AGo, middle panel, lane 2 vs. lane 1). Further, transfected Gab1 WT underwent inducible tyrosine phosphorylation in response to GH, as determined by anti-pY immunoblotting of anti-FLAG immunoprecipitates (Fig. 3AGo, upper panel, lane 2 vs. lane 1). In some experiments (not shown), a faintly tyrosine-phosphorylated protein migrating just less than 68 kDa was also detected in anti-FLAG precipitates from extracts of GH-treated cells expressing Gab1 WT. Reprobing with anti-SHP2 (Fig. 3AGo, lower panel) reliably demonstrated endogenous SHP2 to be specifically coprecipitated with transfected Gab1 WT in response to GH. SHP2’s migration coincided with the variably coprecipitated tyrosine phosphoprotein, leading us to assume it is probably a tyrosine-phosphorylated form of SHP2. GH-induced tyrosine phosphorylation of transfected Gab1 WT was also observed in HEK293 cells cotransfected with rbGHR and mJAK2 (not shown). These findings suggest that transfected Gab1 WT expressed in these reconstitution systems behaves biochemically similarly to the endogenous Gab1 in 3T3-F442A cells.



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  		Figure 2. FLAG-tagged Gab1 molecules and COS-7 cell expression. A, Diagram of human Gab1 WT and the mutants, Gab1 Y627F and Gab1 delPH. Each molecule has a C-terminal FLAG epitope tag. The positions of the PH (residues 10–116) domain and Y-627 are indicated. B, Expression and specific detection of Gab1 WT in COS-7 cells. COS-7 cells were transfected with expression plasmids encoding rbGHR, mJAK2, and Gab1 WT. After overnight serum starvation, the cells were stimulated with or without GH (500 ng/ml) for 15 min. Detergent extracts were immunoprecipitated with either nonimmune serum (NI) or anti-FLAG antibody (M2)-conjugated beads (one confluent 150 x 20-mm dish per sample). Eluates were resolved by SDS-PAGE and immunoblotted with anti-Gab1. The position of the transfected Gab1 is indicated by the arrow. This experiment is representative of five such experiments.

 


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  		Figure 3. GH- and EGF-induced phosphorylation of Gab1 WT and mutants in COS-7 cells. A and B, COS-7 cells were transfected with cDNAs encoding either rbGHR, JAK2, and Gab1 WT or mutants (A) or EGFR and Gab1 WT or mutants (B), as indicated. The mutants are diagrammed in Fig. 2AGo. As in Fig. 2BGo, cells were starved and stimulated with GH or vehicle (A) or with EGF or vehicle (B), and cell extracts were immunoprecipitated with anti-FLAG. Eluted proteins were resolved by SDS-PAGE and immunoblotted sequentially with anti-Gab1 (middle panels), anti-pY (upper panels), and anti SHP2 (lower panels). Relevant molecular weight standards for each region of the gel shown are indicated. Listed below the anti-pY blot (upper panel) in both A and B is the densitometrically derived specific tyrosine phosphorylation of each of the Gab1 molecules (WT and mutants) in the GH (A)- and EGF (B)-treated samples. This was determined as the ratio of the phosphotyrosine signal (upper panel) divided by the Gab1 abundance signal (middle panel) within each experiment and is expressed relative to Gab1 WT (arbitrarily set at 100%). These results are representative of five and two such experiments for A and B, respectively.

 
Deletion of the PH domain of Gab1 impairs the tyrosine phosphorylation of Gab1 in response to GH in reconstituted COS-7 cells
As mentioned above, among its features, Gab1 contains an N-terminal PH domain and an SHP2 association site at tyrosine 627 (Y627), at least when phosphorylated in response to EGF (42, 49). Both features have been implicated in signaling in various systems (41). To examine the impact of the PH domain and Y627 on GH-induced Gab1 tyrosine phosphorylation and SHP2 association, we expressed the C-terminally FLAG-tagged mutants, Gab1 delPH and Gab1 Y627F in the COS-7 reconstitution system. As shown in Fig. 2AGo, Gab1 delPH lacks residues 10–116, and Gab1 Y627F has a point mutation that changes residue 627 from tyrosine to phenylalanine, ensuring that residue 627 cannot be phosphorylated. Similar expression levels and appropriate SDS-PAGE migration of each mutant in comparison with Gab1 WT were verified by anti-FLAG immunoprecipitation and anti-Gab1 immunoblotting (Fig. 3Go, A and B, middle panels). As shown in Fig. 3AGo, upper panel, lane 4 vs. lane 3, Gab1 Y627F was inducibly tyrosine-phosphorylated in response to GH. This was anticipated, because the molecule retains several other tyrosine residues that undergo phosphorylation in response to other stimuli. Despite this induced tyrosine phosphorylation, no SHP2 was detected coimmunoprecipitating with Gab1 Y627F in response to GH (Fig. 3AGo, lower panel, lane 4 vs. lane 2), verifying that phosphorylation of tyrosine 627 is required for SHP2 association in this system.

In contrast to Gab1 WT and Gab1 Y627F, Gab1 delPH underwent very little detectable GH-induced tyrosine phosphorylation in this reconstitution system (Fig. 3AGo, upper panel, lane 6 vs. lanes 4 and 2). Accordingly, SHP2 was not appreciably coimmunoprecipitated with Gab1 delPH in response to GH (Fig. 3AGo, lower panel, lane 6 vs. lane 2). These findings were made despite similar transfected Gab1 protein expression levels and the maintenance of Y627 and other potential tyrosine phosphorylation sites outside of the PH domain in Gab1 delPH. To control for the integrity of the Gab1 delPH molecule and the reconstitution system, we performed similar experiments in which each of the Gab1 proteins was coexpressed with the EGF receptor, instead of the GHR and JAK2, and EGF was used to acutely stimulate the transfected cells (Fig. 3BGo). In this case, Gab1 WT, Gab1 Y627F, and Gab1 delPH each underwent EGF-inducible tyrosine phosphorylation (Fig. 3BGo, upper panel), and Gab1 delPH inducibly associated with SHP2 (Fig. 3BGo, lower panel). Collectively, the data in Fig. 3Go suggested that the Gab1 delPH protein expressed in this system was intact, and that the PH domain may be required for Gab1 involvement with GH signaling.

Impact of deletion of the Gab1 PH domain on GH-induced ERK activation and c-fos trans-activation in reconstituted COS-7 cells
We used the COS-7 reconstitution system to determine whether the mutant Gab1 molecules affected GH-induced ERK signaling in comparison with Gab1 WT (Fig. 4Go). Cells were transfected with cDNAs encoding Gab1 WT, Gab1 Y627F, or Gab1 delPH along with GHR, JAK2, and, to enhance signal detection, an epitope-tagged ERK2 molecule (22). After stimulation, cell extracts were resolved without immunoprecipitation by SDS-PAGE and sequentially immunoblotted with a state-specific antibody to detect activated ERKs, anti-HA to normalize for the level of transfected ERK2, and anti-Gab1 to verify similarity of expression of the transfected Gab1 molecules. As shown in the representative experiment and quantitation of multiple experiments in Fig. 4Go, A and B, respectively, GH-induced ERK2 activation was significantly lessened in the presence of Gab1 delPH compared with Gab1 WT. Notably, Gab1 Y627F, although unable to associate with SHP2 in response to GH, did not reduce GH-induced ERK activation. To further address this issue, we used the reconstitution system to monitor GH-induced trans-activation of a luciferase reporter gene driven by elements of the c-fos enhancer (53). As stated above, expression of this gene by GH probably reflects at least in part activation of the ERK pathway (30). Previously, we observed a 2- to 3-fold induction in reporter activity in response to GH (500 ng/ml) in this reconstitution system (22, 52). In our current experiments, although coexpression of Gab1 WT did not reliably increase this response (data not shown), the same relative pattern of GH-induced increase in luciferase activity was seen for Gab1 Y627F and Gab1 delPH, compared with Gab1 WT, as was seen for GH-induced ERK activation. Significant impairment was seen only for the PH domain deletion mutant and not for the Y627F mutant (Fig. 4CGo). This same relative pattern was observed over a range of GH concentrations from 5–500 ng/ml (data not shown).



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  		Figure 4. GH-induced ERK phosphorylation and c-fos trans-activation are diminished in COS-7 cells expressing Gab1 delPH. A, COS-7 cells transfected with cDNAs encoding rbGHR, JAK2, HA-tagged ERK2, and Gab1 WT or mutants were serum-starved and treated with or without GH for 15 min. Detergent extracts (25% of one 100 x 20-mm dish per lane) were resolved by SDS-PAGE without immunoprecipitation and sequentially immunoblotted with anti-HA, anti-active ERK, and anti-Gab1 as indicated. B, The GH-induced increase in activated ERK2, relative to that seen in cells expressing Gab1 WT, was densitometrically calculated for cells expressing the Gab1 mutants, as described in Materials and Methods. These data are from three experiments. The GH-induced fold increase in Gab1 WT-expressing cells in these experiments ranged from 2.3–3.0. C, COS-7 cells were transfected with cDNAs encoding rbGHR, JAK2, c-fos-luc reporter, and Gab1 WT, or mutants were serum-starved and treated with or without GH for 16 h. Luciferase activity was measured, as described in Materials and Methods and is expressed for three experiments as the GH-induced increase in activity in cells expressing Gab1 mutants relative to that induced by GH in cells expressing Gab1 WT. The GH-induced fold increase in Gab1 WT-expressing cells in these experiments ranged from 2.1–5.6.

 
To test whether the transfected Gab1 molecules in this reconstitution system influenced specific GH signaling pathways, we also examined the STAT5 pathway (Fig. 5Go). Acute GH-induced STAT5 phosphorylation was detected in extracts of COS-7 cells transfected with GHR, JAK2, WT, or mutant Gab1 molecules and a STAT5b expression plasmid. As shown in Fig. 5Go, A and B, there was no significant difference in GH-induced STAT5 tyrosine phosphorylation between cells expressing Gab1 WT, Gab1 Y627F, and Gab1 delPH. As a parallel to the c-fos trans-activation experiments, the STAT5 activation pathway was further pursued by assessing the GH-induced activation of a STAT5-responsive reporter construct in this system (Fig. 5CGo). For this we used a luciferase reporter driven by six copies of the 15-bp interferon-{gamma}-activated sequence-like element from the rat Spi 2.1 gene (Spi-GLE-luc). This element binds activated STAT5 and has been shown to confer GH-dependent luciferase activation in transfected cells (54). Consistent with the profile of acute STAT5 tyrosine phosphorylation, no significant difference was seen in the GH-induced increase in Spi-GLE-luc activity in cells expressing transfected Gab1 WT, Gab1 Y627F, and Gab1 delPH. These data suggest that the dampening of the ERK and c-fos trans-activation pathways seen in the Gab1 delPH-expressing cells in response to GH probably does not represent a global inhibition of GH signaling, but, rather, may selectively affect those pathways.



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  		Figure 5. Lack of influence of expression of Gab1 delPH on GH-induced STAT5 tyrosine phosphorylation and Spi-GLE trans-activation in COS-7 cells. A, COS-7 cells transfected with cDNAs encoding rbGHR, JAK2, STAT5b, and Gab1 WT or mutants were serum-starved and treated with or without GH for 15 min. Detergent extracts (25% of one 100 x 20-mm dish per lane) were resolved by SDS-PAGE and sequentially immunoblotted with anti-STAT5, anti-phospho-STAT5, and anti-Gab1, as indicated. B, The GH-induced increase in activated STAT5, relative to that seen in cells expressing Gab1 WT was densitometrically calculated for cells expressing the Gab1 mutants, as described in Materials and Methods. These data are from three such experiments as shown in A. C, COS-7 cells were transfected with cDNAs encoding rbGHR, JAK2, Spi-GLE-luciferase reporter, and Gab1 WT, or mutants were serum-starved and treated with or without GH for 16 h. Luciferase activity was measured, as described in Materials and Methods, and is expressed for three experiments as the GH-induced increase in activity in cells expressing Gab1 mutants relative to that induced by GH in cells expressing Gab1 WT.

 
GH induces surface translocation of a Gab1 WT/EGFP, but not a Gab1 delPH/EGFP chimera
Our biochemical and functional data suggested that the PH domain allows Gab1 to influence aspects of GH signaling. In other systems this may occur because the PH domain is required for plasma membrane localization of Gab1, placing it in proximity of signaling complexes and bringing with it associated signaling molecules. To test whether GH promotes such translocation, we expressed chimeric proteins composed of either Gab1 WT or Gab1 delPH fused to EGFP by retroviral gene transfer in 3T3-F442A cells. Fluorescence microscopy revealed a diffuse cytoplasmic green signal in vehicle-treated cells that expressed either the Gab1 WT- or Gab1 delPH-containing chimeras, suggesting a relative lack of cell surface localization of either of the Gab1 molecules in serum-starved cells (Fig. 6Go). Notably, Gab1 WT/EGFP, but not Gab1 delPH/EGFP exhibited GH-dependent translocation to the cell periphery, suggesting that GH promoted surface translocation of the WT, but not the PH domain-deleted, molecule. This GH-induced translocation of Gab1 WT is much like that recently reported in response to EGF by Zhang et al. (57) and as we have also observed in response to EGF (not shown). Similar findings in response to GH were observed in the COS-7 reconstitution system with expression of the same chimeras by transfection (not shown).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Like signaling by other cytokine receptors, GH-induced GHR activation of intracellular signal transduction pathways is believed to begin with triggering of a nonreceptor tyrosine kinase activity (5, 6). In the case of the GHR, substantial evidence suggests that the most proximal tyrosine kinase activated is JAK2, which is constitutively associated with the GHR and probably becomes more tightly associated in response to GH-induced GHR dimerization (3, 4). Of the multiple signals that emanate from the productively engaged GHR, STAT5 activation and the resultant enhanced transcription of certain target genes are critical for several aspects of GH action, but may be mechanistically among the simplest. Only proper GHR/JAK2 assembly and the phosphorylation of any of a set of GHR cytoplasmic tyrosine residues are required for initiation of STAT5 activation (10, 11, 12, 13, 14, 15); its regulation is probably far more complex.

In contrast to the apparently simple requirements for GH-induced STAT5 signal initiation, biochemical analyses in several cell systems have revealed, in response to GH, acute phosphorylation (tyrosine and serine/threonine) of a large number of cellular proteins and the formation of signaling complexes that may include other tyrosine kinases (e.g. focal adhesion kinase and Src family kinases) and docking and signaling molecules (e.g. SHC, IRS proteins, SIRP-{alpha}, SH2B, and SHP2) (32, 35, 36, 37, 38, 39, 52, 58, 59, 60). Also in contrast to STAT5 activation, the induction by GH of other pathways, including the ERK, p38, and JNK pathways, may show substantial cell type specificity (61, 62). The mechanistic and physiological bases for this differential GH-induced activation of non-STAT5 signaling are as yet uncertain.

We and others have been interested in the molecules and pathways required for GH-induced ERK activation. Although most studies conclude that GH-induced ERK activation is Ras dependent, no one pathway has been established as the sole route for its induction, and it is conceivable that several pathways could operate to collectively modulate ERK activation in response to GH. The docking protein Shc is tyrosine phosphorylated and acutely associates with the Grb2 adapter protein in response to GH (32). Sos, a Ras guanine nucleotide exchanger associated with Grb2, can then positively regulate Ras, causing Raf, MEK1, and ERK1/2 activation. This classical pathway is operative in response to GH, and Shc can interact physically with JAK2 (32), perhaps enabling it to be involved in GH signaling. However, other platforms that may serve as sites of association for upstream ERK activators in response to GH have also been implicated. Activated JAK2 can tyrosine phosphorylate the EGFR (34). In particular, EGFR tyrosine 1068 can be phosphorylated in response to GH, thereby associating with Grb2, and stable expression of EGFR in CHO-GHR cells allows augmented GH-induced ERK activation and c-fos trans-activation (34). We have studied whether the IRS-1 docking molecule might also be involved in GH-induced ERK activation. Using IRS-deficient 32D-GHR cells, we observed that IRS proteins are not required for ERK1/2 activation in response to GH; however, stable expression of IRS-1 resulted in enhancement of GH-induced ERK activation (25). Although we do not know the mechanism by which IRS-1 or a molecule(s) associated with it might be responsible for this effect, we have observed by coimmunoprecipitation and in vitro fusion protein pulldown assays that IRS-1 can physically associate via its N-terminal PH and PTB domains with JAK2 (23).

In the current study we explored whether a PH domain-containing member of the IRS family, Gab1, might also be involved in GH signaling. We found that GH stimulation of 3T3-F442A fibroblasts resulted in a shift in the SDS-PAGE mobility of Gab1 at least partly attributable to its tyrosine phosphorylation. Given that inducible serine/threonine phosphorylation of Gab1 can retard its mobility in SDS-PAGE (63), it is possible that Gab1 also undergoes GH-inducible phosphorylation on other than tyrosine residues. In these same cells we observed, as have others in different cell types, substantial constitutive association of Gab1 with Grb2. Further, we also detected GH-induced association of Gab1 with SHP2. These biochemical findings suggest that GH is in some way affecting Gab1’s phosphorylation state and that Gab1 could be involved in GH signaling. In contrast to the findings noted above for Shc and IRS-1, however, we have to date been unable to detect either basal or GH-inducible association of Gab1 with either the GHR or JAK2 (data not shown).

In EGFR signaling, the EGF-induced association of SHP2 with tyrosine-phosphorylated Gab1, which is abrogated in the Gab1 Y627F mutant, is critical for optimal activation of the ERK pathway (49). This finding and our previous data suggesting a positive role for SHP2 catalytic activity in GH-induced c-fos trans-activation (52) initially led us to hypothesize that Gab1’s involvement in GH signaling might also be by virtue of its ability to inducibly associate with SHP2. Our reconstitution experiments, however, demonstrated that although tyrosine 627 was required for GH-induced SHP2 association, expression of the Gab1 Y627F mutant did not affect GH-induced ERK activation or c-fos reporter trans-activation compared with Gab1 WT expression. However, expression of the PH domain deletion mutant, Gab1 delPH, compared with that of Gab1 WT, led to significant reduction in GH-induced ERK activation and c-fos trans-activation. This suggests that the PH domain is important in allowing Gab1 to influence GH signaling via the ERK activation pathway, but that this effect does not require SHP2 association with Gab1. Further, the lack of effect of Gab1 mutations on STAT5 activation in the COS-7 reconstitution system indicates that Gab1’s role in GH signaling is probably pathway specific. Our findings with expression of the Gab1 WT/EGFP vs. Gab1 delPH/EGFP chimeras support an involvement of Gab1 in GH signaling being mediated by the PH domain. The chimera without the PH domain failed to translocate to the plasma membrane in response to GH, whereas the Gab1 WT-containing chimera underwent this translocation.

Although we cannot yet completely understand the mechanism of Gab1’s involvement in GH signaling, we hypothesize that it is largely related to GH’s ability to cause translocation of the adapter protein to the cell surface where it can come into proximity of the GH-induced assembly of a protein complex(es) involved in ERK activation. Our tentative conclusion is that Gab1’s ability to participate in this complex may be mediated by specific association with a complex member(s), although we do not yet know the identity of this protein(s). We note that the ability of Gab1 to strongly interact with Grb2 independent of GH stimulation might allow the translocated Gab1 to bring with it an important player in ERK activation in response to GH stimulation. However, other Gab1 binding partners could be involved in this effect as well. Our data do not rule out that SHP2 affects GH-induced ERK pathway signaling, but, rather, suggest that the Gab1-associated component of SHP2 induced by GH does not appear to affect this pathway. We do not know what role, if any, might be played by the Gab1-associated SHP2, but we do note the findings of Stofega et al., (64) suggesting that binding of SHP2 to the activated GHR may contribute to negative regulation of GH-induced signaling. Further studies will also be needed to determine the degree to which Gab1 is used in GH signaling in other cell types and tissues in mediation of the biological effects of GH.


   Acknowledgments
 
The authors appreciate helpful conversations with Drs. M. Gold, M. Park, and A. J. Wong; the kind provision of reagents by those named in the text; and the generous provision of hGH by Eli Lilly \|[amp ]\| Co. We also acknowledge the expert assistance of Dr. Albert Tousson, University of Alabama-Birmingham Cell Biology Imaging Core Facility.


   Footnotes
 
This work was supported in part by NIH Grants DK-46395 and DK-58259 (to S.J.F.). Parts of this work were presented at the 83rd Annual Meeting of The Endocrine Society, Denver, CO, 2001.

Abbreviations: EGFP, Enhanced green fluorescent protein; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorting; Gab1, Grb-2-associated binder-1; GHR, GH receptor; GLE, GAS-like element; HA, hemagglutinin; HGF, hepatocyte growth factor; IRS-1, insulin receptor substrate-1; JAK2, Janus kinase-2; m, murine; luc, luciferase; MAPK, mitogen-activated protein kinase; PH, pleckstrin homology; PI3K, phosphoinositol 3-kinase; rb, rabbit; SH2, Src homology 2; SHP2, an SH2-containing cytoplasmic tyrosine phosphatase; Spi, serine protease inhibitor; STAT, signal transducer and activator of transcription; WT, wild-type.

Received June 12, 2002.

Accepted for publication August 21, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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