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Growth Hormone-induced Phosphorylation of Epidermal Growth Factor (EGF) Receptor in 3T3-F442A Cells

2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês

10.1074/jbc.m300939200

1083-351X

Yao‐Kuang Huang, Sung-Oh Kim, Jing Jiang, Stuart J. Frank,

Metabolism, Diabetes, and Cancer

Growth hormone (GH) promotes signaling by causing activation of the non-receptor tyrosine kinase, JAK2, which associates with the GH receptor. GH causes phosphorylation of epidermal growth factor receptor (EGFR; ErbB-1) and its family member, ErbB-2. For EGFR, JAK2-mediated GH-induced tyrosine phosphorylation may allow EGFR to serve as a scaffold for GH signaling. For ErbB-2, GH induces serine/threonine phosphorylation that dampens basal and EGF-induced ErbB-2 kinase activation. We now further explore GH-induced EGFR phosphorylation in 3T3-F442A, a preadipocytic fibroblast cell line that expresses endogenous GH receptor, EGFR, and ErbB-2. Using a monoclonal antibody that recognizes ERK consensus site phosphorylation (PTP101), we found that GH caused PTP101-reactive phosphorylation of EGFR. This GH-induced EGFR phosphorylation was prevented by MEK1 inhibitors but not by a protein kinase C inhibitor. Although GH did not discernibly affect EGF-induced EGFR tyrosine phosphorylation, we observed by immunoblotting a substantial decrease of EGF-induced EGFR degradation in the presence of GH. Fluorescence microscopy studies indicated that EGF-induced intracellular redistribution of an EGFR-cyan fluorescent protein chimera was markedly reduced by GH cotreatment, in support of the immunoblotting results. Notably, protection from EGF-induced degradation and inhibition of EGF-induced intracellular redistribution afforded by GH were both prevented by a MEK1 inhibitor, suggesting a role for GH-induced ERK activation in regulating the trafficking itinerary of the EGF-stimulated EGFR. Finally, we observed augmentation of early aspects of EGF signaling (EGF-induced ERK2 activation and EGF-induced Cbl tyrosine phosphorylation) by GH cotreatment; the GH effect on EGF-induced Cbl tyrosine phosphorylation was also prevented by MEK1 inhibition. These data indicate that GH, by activating ERKs, can modulate EGF-induced EGFR trafficking and signaling and expand our understanding of mechanisms of cross-talk between the GH and EGF signaling systems. Growth hormone (GH) promotes signaling by causing activation of the non-receptor tyrosine kinase, JAK2, which associates with the GH receptor. GH causes phosphorylation of epidermal growth factor receptor (EGFR; ErbB-1) and its family member, ErbB-2. For EGFR, JAK2-mediated GH-induced tyrosine phosphorylation may allow EGFR to serve as a scaffold for GH signaling. For ErbB-2, GH induces serine/threonine phosphorylation that dampens basal and EGF-induced ErbB-2 kinase activation. We now further explore GH-induced EGFR phosphorylation in 3T3-F442A, a preadipocytic fibroblast cell line that expresses endogenous GH receptor, EGFR, and ErbB-2. Using a monoclonal antibody that recognizes ERK consensus site phosphorylation (PTP101), we found that GH caused PTP101-reactive phosphorylation of EGFR. This GH-induced EGFR phosphorylation was prevented by MEK1 inhibitors but not by a protein kinase C inhibitor. Although GH did not discernibly affect EGF-induced EGFR tyrosine phosphorylation, we observed by immunoblotting a substantial decrease of EGF-induced EGFR degradation in the presence of GH. Fluorescence microscopy studies indicated that EGF-induced intracellular redistribution of an EGFR-cyan fluorescent protein chimera was markedly reduced by GH cotreatment, in support of the immunoblotting results. Notably, protection from EGF-induced degradation and inhibition of EGF-induced intracellular redistribution afforded by GH were both prevented by a MEK1 inhibitor, suggesting a role for GH-induced ERK activation in regulating the trafficking itinerary of the EGF-stimulated EGFR. Finally, we observed augmentation of early aspects of EGF signaling (EGF-induced ERK2 activation and EGF-induced Cbl tyrosine phosphorylation) by GH cotreatment; the GH effect on EGF-induced Cbl tyrosine phosphorylation was also prevented by MEK1 inhibition. These data indicate that GH, by activating ERKs, can modulate EGF-induced EGFR trafficking and signaling and expand our understanding of mechanisms of cross-talk between the GH and EGF signaling systems. Growth hormone (GH) 1The abbreviations used are: GH, growth hormone; GHR, GH receptor; STAT, signal transducers and activators of transcription; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; CFP, cyan fluorescent protein; PBS, phosphate-buffered saline; IL, interleukin; WB, Western blot; pTyr, phosphotyrosine; IP, immunoprecipitation; JAK, Janus kinase; IRS, insulin receptor substrate; SHC, Src homology/collagen.1The abbreviations used are: GH, growth hormone; GHR, GH receptor; STAT, signal transducers and activators of transcription; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; CFP, cyan fluorescent protein; PBS, phosphate-buffered saline; IL, interleukin; WB, Western blot; pTyr, phosphotyrosine; IP, immunoprecipitation; JAK, Janus kinase; IRS, insulin receptor substrate; SHC, Src homology/collagen. is a four-helix bundle protein that shares structural similarity with a large class of hormones and cytokines, including prolactin and various interleukins and colony stimulating factors. GH exerts its profound somatogenic and metabolic regulatory effects by interacting with the GH receptor (GHR), a cell surface glycoprotein member of the cytokine receptor superfamily (1Leung D.W. Spencer S.A. Cachianes G. Hammonds R.G. Collins C. Henzel W.J. Barnard R. Waters M.J. Wood W.I. Nature. 1987; 330: 537-543Crossref PubMed Scopus (1321) Google Scholar, 2Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1869) Google Scholar, 3Frank S.J. Messina J.L. Oppenheim J.J. Feldman M. Cytokine Reference On-Line. Academic Press, Harcourt, London2002Google Scholar). Like other cytokine receptors, the GHR initiates its signal transduction by physical and functional association with JAK2, a cytoplasmic tyrosine kinase member of the JAK family (4Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. Cell. 1993; 74: 237-244Abstract Full Text PDF PubMed Scopus (818) Google Scholar, 5Ihle J.N. Adv. Immunol. 1995; 60: 1-35Crossref PubMed Google Scholar). GH activates several intracellular signaling pathways. GH-induced STAT5b activation has been particularly intensively studied and is strongly implicated in the regulation of certain GH-responsive hepatic genes (6Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (822) Google Scholar). Other signaling pathways induced by GH include the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways, both also targets of a variety of other growth factors and cytokines.GH induces activation of ERK1 and ERK2 in several model systems, including the murine 3T3-F442A preadipocyte fibroblast (7Anderson N.G. Biochem. J. 1992; 284: 649-652Crossref PubMed Scopus (75) Google Scholar, 8Campbell G.S. Pang L. Miyasaka T. Saltiel A.R. Carter-Su C. J. Biol. Chem. 1992; 267: 6074-6080Abstract Full Text PDF PubMed Google Scholar, 9Moller C. Hansson A. Enberg B. Lobie P.E. Norstedt G. J. Biol. Chem. 1992; 267: 23403-23408Abstract Full Text PDF PubMed Google Scholar, 10Winston L.A. Bertics P.J. J. Biol. Chem. 1992; 267: 4747-4751Abstract Full Text PDF PubMed Google Scholar). In contrast to STAT5 activation, which requires the presence of the receptor cytoplasmic domain in addition to activation of JAK2, the ability of GH to activate ERKs appears to require none of the distal GHR tail and instead corresponds to whether it can promote JAK2 activation (9Moller C. Hansson A. Enberg B. Lobie P.E. Norstedt G. J. Biol. Chem. 1992; 267: 23403-23408Abstract Full Text PDF PubMed Google Scholar, 11Sotiropoulos A. Perrot-Applanat M. Dinerstein H. Pallier A. Postel-Vinay M.-C. Finidori J. Kelly P.A. Endocrinol. 1994; 135: 1292-1298Crossref PubMed Scopus (0) Google Scholar, 12VanderKuur J.A. Wang X. Zhang L Campbell G.S. Allevato G. Billestrup N. Norstedt G. Carter-Su C. J. Biol. Chem. 1994; 269: 21709-21717Abstract Full Text PDF PubMed Google Scholar, 13Frank S.J Yi W. Zhao Y. Goldsmith J.F. Gilliland G. Jiang J. Sakai I. Kraft A.S. J. Biol. Chem. 1995; 270: 14776-14785Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). However, ERK is not activated in all cells in which GH activates JAK2 (14Love D.W. Whatmore A.J. Clayton P.E. Silva C.M. Endocrinol. 1998; 139: 1965-1971Crossref PubMed Google Scholar). Though it remains uncertain what factors determine whether a GHR-expressing cell or tissue will respond to GH with ERK activation, evidence exists among responsive cells for the potential involvement of a number of molecules. These include the Shc-Grb2-Sos-Ras-Raf pathway (15VanderKuur J. Allevato G. Billestrup N. Norstedt G. Carter-Su C. J. Biol. Chem. 1995; 13: 7587-7593Abstract Full Text Full Text PDF Scopus (135) Google Scholar, 16VanderKuur J.A. Butch E.R. Waters S.B. Pessin J.E. Guan K.L. Carter-Su C. Endocrinol. 1997; 138: 4301-4307Crossref PubMed Scopus (0) Google Scholar), the phosphatidylinositol 3-kinase (or a related enzyme) pathway (17Kilgour E. Gout I. Anderson N.G. Biochem. J. 1996; 315: 517-522Crossref PubMed Scopus (64) Google Scholar, 18Hodge C. Liao J. Stofega M. Guan K. Carter-Su C. Schwartz J. J. Biol. Chem. 1998; 273: 31327-31336Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 19Liang L. Jiang J. Frank S.J. Endocrinol. 2000; 141: 3328-3336Crossref PubMed Scopus (43) Google Scholar), IRS-1 (19Liang L. Jiang J. Frank S.J. Endocrinol. 2000; 141: 3328-3336Crossref PubMed Scopus (43) Google Scholar) and the IRS-like Gab-1 adapter molecule (20Kim S.-O. Loesch K. Wang X. Jiang J. Mei L. Cunnick J.M. Wu J. Frank S.J. Endocrinol. 2002; 143: 4856-4867Crossref PubMed Scopus (27) Google Scholar), and c-src (21Zhu T. Ling L. Lobie P.E. J. Biol. Chem. 2002; 277: 45592-45603Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar).Recent studies of the mechanisms and consequences of GH-induced ERK activation have revealed interesting relationships between the GH and epidermal growth factor (EGF). These studies suggest previously unrecognized cross-talk between the GHR and members of the EGFR family, examples of seemingly disparate types of signaling receptors (a cytokine receptor and a family of tyrosine kinase receptors, respectively). The EGFR family of structurally related transmembrane glycoproteins includes the EGFR itself (ErbB-1), ErbB-2 (cneu), ErbB-3, and ErbB-4 (22Alroy I Yarden Y. FEBS Lett. 1997; 410: 83-86Crossref PubMed Scopus (650) Google Scholar, 23Riese II, D.J. Stern D.F. Bioessays. 1998; 20: 41-48Crossref PubMed Scopus (694) Google Scholar). Except for ErbB-3, each has intrinsic tyrosine kinase activity in its cytoplasmic domain. Ligands such as EGF, transforming growth factor-α, and neuregulins induce signaling through these receptors by binding to specific EGFR family members in such a way as to promote particular homo- or heterodimers among the family members. ErbB-2 has no known ligand, but it is the preferred heterodimer partner for other family members when they engage their ligands. EGF, for example, promotes formation of EGFR homodimers or EGFR/ErbB-2 heterodimers and thus causes activation of both of these tyrosine kinases in cells that express them. EGF signaling through EGFR and ErbB-2 has a number of biologically relevant signaling outcomes in both normal and neoplastic cells.Yamauchi et al. (24Yamauchi T. Ueki K. Tobe K. Tamemoto H. Sekine N. Wada M. Honjo M. Takahashi M. Takahashi T. Hirai H. Tushima T. Akanuma Y. Fujita T. Komuro I. Yazaki Y. Kadowaki T. Nature. 1997; 39: 91-96Crossref Scopus (256) Google Scholar) first demonstrated that GH caused tyrosine phosphorylation of the EGFR, both in vivo in the livers of mice and in cell culture. This GH-induced EGFR tyrosine phosphorylation was shown to require JAK2, but not EGFR, kinase activity. Partial mapping by mutagenesis suggested that EGFR Tyr-1068, which when phosphorylated resides in a consensus Grb-2 association motif, was a site of GH-induced phosphorylation and that GH caused enhanced EGFR-Grb-2 association. Further, GH-induced EGFR tyrosine phosphorylation was shown in cell culture to likely contribute to GH-induced ERK activation. Thus, this study suggested that EGFR may be a docking molecule involved in GH-induced, JAK2-dependent ERK activation. Our previous study (25Kim S.-O. Houtman J. Jiang J. Ruppert J.M. Bertics P.J. Frank S.J. J. Biol. Chem. 1999; 274: 36015-36024Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) in 3T3-F442A cells confirmed that GH caused EGFR kinase-independent EGFR tyrosine phosphorylation but also suggested GH effects on ErbB-2. GH caused a decrease in basal and EGF-induced ErbB-2 tyrosine kinase activation and tyrosine phosphorylation that was associated with retardation of the electrophoretic migration of ErbB-2. This retarded migration was shown to be because of serine or threonine, rather than tyrosine, phosphorylation of ErbB-2, and both the GH-induced change in migration and inhibited tyrosine kinase activation were prevented by blocking GH-induced ERK activation. These findings suggested that GH caused an ERK pathway-dependent phosphorylation of ErbB2 that rendered it desensitized to activation in response to EGF.We now explore further the mechanisms and consequences of cross-talk between GH and EGF signaling. In particular, we examine GH-induced phosphorylation of EGFR and ErbB-2. Our findings suggest that GH causes an ERK pathway-dependent threonine phosphorylation of both ErbB-2 and EGFR that is recognized by a state-specific antibody reactive with ERK consensus phosphorylation sites. In contrast to our findings for ErbB-2, we observe that this GH-induced EGFR phosphorylation does not significantly alter the intrinsic tyrosine kinase activation of EGFR, but instead slows the rate of EGF-induced EGFR intracellular redistribution and degradation, thereby potentiating EGF-induced EGFR signaling. These results suggest that GH may affect EGF signaling by multiple mechanisms, including modulation of EGF-induced EGFR trafficking.EXPERIMENTAL PROCEDURESMaterials—Recombinant human GH was kindly provided by Lilly. Recombinant human EGF was purchased from Upstate Biotechnology (Lake Placid, NY), and recombinant human PDGF-BB was from Intergen (Purchase, NY). Murine 125I-EGF (150–200 μCi/μg) was purchased from PerkinElmer Life Sciences. PMA was obtained from Sigma. The PKC inhibitor, GF109203X (Calbiochem), and the MEK1 inhibitors, PD98059 (New England Biolabs, Beverly, MA) and U0126 (Promega, Madison, WI), were purchased commercially.Antibodies—Polyclonal anti-ErbB-2, anti-EGFR, and anti-Cbl-b antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal anti-phospho-threonine-proline antibody PTP101, polyclonal anti-phospho-EGFR antibodies Tyr-845, Tyr-992, Tyr-1045, and Tyr-1068 (Cell Signaling Technology, Beverly, MA), anti-active mitogen-activated protein kinase affinity-purified rabbit antibody (anti-active ERK, recognizing the dually phosphorylated Thr-183 and Tyr-185 residues corresponding to the active forms of ERK1 and ERK2; Promega, Madison, WI), monoclonal anti-phosphotyrosine antibody 4G10, anti-mitogenactivated protein kinase affinity-purified rabbit antibody (recognizing both ERK1 and ERK2), polyclonal anti-PDGFR antibody (recognizing both PDGF type A and B receptors), and monoclonal anti-phospho-EGFR antibody Tyr-1173 (Upstate Biotechnology, Lake Placid, NY) were all purchased commercially.Cell Culture and Transfection—3T3-F442A cells (26Green H. Kehinde O. Cell. 1976; 7: 105-113Abstract Full Text PDF PubMed Scopus (612) Google Scholar), kindly provided by Drs. H. Green (Harvard University, Boston, MA) and C. Carter-Su (University of Michigan, Ann Arbor, MI), were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose (Cellgro, Inc.), supplemented with 10% calf serum, 50 μg/ml gentamicin sulfate, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Biofluids, Rockville, MD). 3T3-L1 cells from American Type Culture Collection (Manassas, VA) were grown in the above medium, supplemented with 10% fetal bovine serum (Biofluids, Rockville, MD) instead of 10% calf serum.To generate stable 3T3-L1 transfectants expressing the CFP-tagged EGFR (EGFR-CFP), cells were seeded in 60-mm dishes and used at 50–80% confluency. The plasmid pCFP/EGFR, kindly provided by Dr. L. Samelson, Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, MD, encodes the human EGFR with the CFP fused to its C terminus (27Yamazaki T. Zaal K. Hailey D. Presley J. Lippincott-Schwartz J. Samelson L.E. J. Cell Sci. 2002; 115: 1791-1802Crossref PubMed Google Scholar). pCFP/EGFR was transfected into 3T3-L1 cells using GenePORTER transfection reagent (Gene Therapy Systems, Inc., San Diego, CA) as described previously (28Boney C.M. Sekimoto H. Gruppuso P.A. Frackelton A.R. Cell Growth Differ. 2001; 12: 379-386PubMed Google Scholar). 3T3-L1 cells expressing EGFR-CFP were selected in 1 mg/ml of G418 (Invitrogen) and cloned. Transfectants were maintained in culture medium containing 200 μg/ml of G418.Cell Starvation, Inhibitor Pretreatment, Cell Stimulation, and Protein Extraction—Serum starvation of 3T3-F442A or 3T3-L1 cells was accomplished by substitution of 0.5% (w/v) bovine serum albumin (fraction V: Roche Molecular Biochemicals) for calf serum or fetal bovine serum in the culture medium for 16–20 h prior to experiments. 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% (w/v) bovine serum albumin, and 1 mm dextrose). Serum-starved cells were pretreated with PD98059 (100 μm), U0126 (10 μm), GF109203X (1 μm), or vehicle (as a control) for 30 or 60 min prior to treatment with GH (500 ng/ml), EGF (1 nm), PMA (1 μg/ml), PDGF (40 ng/ml), or vehicle, as specified in each experiment. Stimulations were terminated by washing the cells once with ice-cold phosphate-buffered saline supplemented with 0.4 mm sodium orthovanadate (PBS-vanadate) and then harvested by scraping in PBS-vanadate. Cells were collected by brief centrifugation, and pelleted cells were solubilized for 15 min at 4 °C in lysis buffer (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 phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 10 mm benzamidine, 5 μg/ml aprotinin, and 5 μg/ml leupeptin). After centrifugation at 15,000 × g for 15 min at 4 °C, the detergent extracts (supernatant) were subjected to immunoprecipitation or were directly electrophoresed and immunoblotted, as indicated below. For examining the abundance of EGFR and ErbB-2, total cell lysates were extracted in the presence of 1% SDS.Immunoprecipitation and Immunoblotting—For immunoprecipitation, cell extracts (500–1000 μg) were mixed with 5 μl of polyclonal anti-ErbB-2, -EGFR, -PDGFR, or -Cbl-b antibody (1 μg) and incubated at 4 °C for 2 h with continuous agitation. Protein A-Sepharose (20 μl) (Amersham Biosciences) was added and incubated at 4 °C for an additional hour. The beads were washed four times with lysis buffer. Laemmli sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated below.Proteins resolved by SDS-PAGE were transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences). The membranes were blocked with TBST buffer (20 mm Tris-HCl (pH 7.6), 150 mm NaCl, and 0.1% (v/v) Tween 20) containing 2% (w/v) bovine serum albumin and incubated with primary antibodies (0.5–1 μg/ml) as specified in each experiment. After three washes with TBST, the membranes were incubated with appropriate secondary antibodies (1:10,000 dilution) and washed. The bound antibodies were detected with SuperSignal chemiluminescent substrate (Pierce). Membrane stripping was performed according to the manufacturer's suggestions (Amersham Biosciences).Densitometric Analysis—Densitometry of immunoblots was performed using a solid state video camera (Sony-77; Sony Corp.) and a 28-mm MicroNikkor lens over a light box 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). Pooled data from several experiments are displayed as mean ± S.E. The significance (p value) of differences of pooled results was estimated using unpaired t tests.To verify the fidelity of this densitometric method, we performed a control experiment in which total protein concentration of 3T3-F442A cell extract was determined using BCA protein assay reagents (Pierce), and serially diluted total protein aliquots (2.5–100 μg) were resolved by SDS-PAGE and immunoblotted with anti-EGFR. Densitometry of the EGFR intensities plotted against the known loaded protein amounts yielded a straight line with a correlation coefficient (R) of 0.97 (not shown). This suggested a high degree of reliability for this densitometric analysis.Fluorescence Microscopy—3T3-L1 transfectants expressing EGFRCFP were grown on Corning glass coverslips precoated with gelatin (Sigma) in 6-well plates for 48 h in culture medium until they reached ∼50% confluency. The cells were starved, pretreated with PD98059 (100 μm) or vehicle for 1 h, and stimulated with GH (500 ng/ml), EGF (1 nm), or vehicle as described above and specified in the experiment. The cells were rinsed with PBS and fixed with 4% formaldehyde solution in PBS for 15 min at room temperature. After rinsing with PBS, the coverslips were mounted on microscope slides (Fisher) in Vectorshield mounting medium for fluorescence (Vector Laboratories Inc., Burlingame, CA). Fluorescence patterns were visualized with an Olympus fluorescence microscope at the University of Alabama Cell Biology Imaging Core Facility. Images were collected and analyzed using IPLab Spectrum software (Scanalytics Inc., Fairfax, VA).125I-EGF Internalization Experiments—3T3-F442A cells were grown in six-well plates in culture medium until they formed monolayers. The cells were starved and pretreated with GH (500 ng/ml) or vehicle in binding medium (Dulbecco's modified Eagle's medium containing 4.5 g/l glucose, supplemented with 20 mm HEPES and 0.5% (w/v) bovine serum albumin) at 37 °C for 10 min. To measure the internalization of 125I-EGF, the cells were incubated with 125I-EGF (1 ng/ml) in binding medium in the continued presence or absence of GH at 37 °C for 0–10 min in duplicate. At the end of incubation, the medium was aspirated, and the monolayers were rapidly washed twice with ice-cold binding medium to remove unbound ligand. The cells were then incubated with 0.2 m sodium acetate (pH 4.5) containing 0.5 m NaCl at 4 °C for 5 min. The acid wash was combined with another short rinse in the same buffer and used to determine the amount of surface-bound 125I-EGF. The cells were finally lysed in 100 mm NaOH containing 0.1% (w/v) SDS to determine the amount of internalized 125I-EGF. Radioactivity was counted with a γ-counter. The acid-inaccessible internalized 125I-EGF was presented as a fraction of total cell-associated radioactivity (the sum of cell surface-bound and internalized 125I-EGF) at each time point. Nonspecific binding was measured in the presence of 100-fold molar excess of unlabeled EGF and was not more than 5% of the total counts.RESULTSGH Acutely Promotes ERK-dependent Phosphorylation of EGFR at Sites Recognized by an ERK Phosphorylation Site Antibody—The murine 3T3-F442A fibroblast expresses EGFR and ErbB-2, as well as the receptor for GH (25Kim S.-O. Houtman J. Jiang J. Ruppert J.M. Bertics P.J. Frank S.J. J. Biol. Chem. 1999; 274: 36015-36024Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We and others (4Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. Cell. 1993; 74: 237-244Abstract Full Text PDF PubMed Scopus (818) Google Scholar, 10Winston L.A. Bertics P.J. J. Biol. Chem. 1992; 267: 4747-4751Abstract Full Text PDF PubMed Google Scholar, 18Hodge C. Liao J. Stofega M. Guan K. Carter-Su C. Schwartz J. J. Biol. Chem. 1998; 273: 31327-31336Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 24Yamauchi T. Ueki K. Tobe K. Tamemoto H. Sekine N. Wada M. Honjo M. Takahashi M. Takahashi T. Hirai H. Tushima T. Akanuma Y. Fujita T. Komuro I. Yazaki Y. Kadowaki T. Nature. 1997; 39: 91-96Crossref Scopus (256) Google Scholar, 25Kim S.-O. Houtman J. Jiang J. Ruppert J.M. Bertics P.J. Frank S.J. J. Biol. Chem. 1999; 274: 36015-36024Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 29Wiepz G.J. Houtman J.C. Cha D. Bertics P.J. J. Cell. Physiol. 1997; 173: 44-53Crossref PubMed Scopus (21) Google Scholar, 30Kim S.-O. Jiang J. Yi W. Feng G.S. Frank S.J. J. Biol. Chem. 1998; 273: 2344-2354Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) have detected biochemical responses to both GH and EGF in this cell line, making it an appealing target for evaluation of potential cross-talk between the GH and EGF signaling pathways. Our previous studies (25Kim S.-O. Houtman J. Jiang J. Ruppert J.M. Bertics P.J. Frank S.J. J. Biol. Chem. 1999; 274: 36015-36024Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) indicated that in 3T3-F442A cells GH treatment causes ErbB-2 to undergo serine/threonine phosphorylation that results in a decrease in its basal activation state and its desensitization to EGF-induced activation. These effects of GH were inhibited by drugs that block activation of the MEK1/ERK pathway.To further characterize such serine/threonine phosphorylation of EGFR family members, we employed a state-specific monoclonal antibody (PTP101) that specifically detects proteins phosphorylated at the consensus site(s) for proline-directed protein kinases, such as the ERKs (31Pearson R.B. Kemp B.E. Methods Enzymol. 1991; 200: 62-81Crossref PubMed Scopus (866) Google Scholar, 32Marinissen M.J. Chiariello M. Gutkind J.S. Genes Dev. 2001; 15: 535-553Crossref PubMed Scopus (148) Google Scholar, 33Hall C. Nelson D.M. Ye X. Baker K. DeCaprio J.A. Seeholzer S. Lipinski M. Adams P.D. Mol. Cell. Biol. 2001; 21: 1854-1865Crossref PubMed Scopus (95) Google Scholar). In the experiment shown in Fig. 1A, serum-starved cells were exposed to GH, EGF, or vehicle (_) for 10 min prior to detergent extraction and immunoprecipitation with anti-ErbB-2 (lanes 1–3) or anti-EGFR (lanes 4–6) antibodies. Eluates were resolved by SDS-PAGE and immunoblotted sequentially with anti-ErbB-2 (upper panel, lanes 1–3), anti-EGFR (upper panel, lanes 4–6), antiphosphotyrosine antibodies (anti-pTyr; middle panel, lanes 1–6), and PTP101 (lower panel, lanes 1–6). Consistent with our previous findings (25Kim S.-O. Houtman J. Jiang J. Ruppert J.M. Bertics P.J. Frank S.J. J. Biol. Chem. 1999; 274: 36015-36024Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), neither GH nor EGF acutely changed the abundance of ErbB2 or EGFR (upper panel, lanes 1–6), but both stimuli caused retardation in the SDS-PAGE migration of ErbB-2 (upper panel, lanes 1 and 3 versus 2). For EGF, this retarded migration was accompanied by an increase in ErbB-2 tyrosine phosphorylation (middle panel, lane 3 versus 2), whereas GH caused a decrease in ErbB-2 tyrosine phosphorylation (middle panel, lane 1 versus 2). As expected (24Yamauchi T. Ueki K. Tobe K. Tamemoto H. Sekine N. Wada M. Honjo M. Takahashi M. Takahashi T. Hirai H. Tushima T. Akanuma Y. Fujita T. Komuro I. Yazaki Y. Kadowaki T. Nature. 1997; 39: 91-96Crossref Scopus (256) Google Scholar, 25Kim S.-O. Houtman J. Jiang J. Ruppert J.M. Bertics P.J. Frank S.J. J. Biol. Chem. 1999; 274: 36015-36024Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), EGFR tyrosine phosphorylation was promoted by both GH and EGF, although EGF was more potent than GH (middle panel, lanes 4 and 6 versus 5). Notably, the immunoblot in the lower panel (lanes 1–6) revealed that GH and EGF promoted the appearance of forms of ErbB-2 and EGFR that were recognized by PTP101, suggesting phosphorylation at ERK consensus sites in those molecules. Indeed, immunoblotting of unfractionated cell extracts from the same cells with anti-active ERK antibodies confirmed that both GH and EGF promoted robust ERK activation in these cells (Fig. 1B, lanes 1 and 3 versus 2). Interestingly, whereas GH promoted less EGFR tyrosine phosphorylation than did EGF, comparison of the GH- and EGF-induced PTP101 signals in Fig. 1A suggests that GH promoted substantially more EGFR ERK consensus site phosphorylation than did EGF.The data in Fig. 1 indicated that both GH and EGF caused phosphorylation of ErbB-2 and EGFR at potential ERK consensus sites, suggesting that this is mediated by activation of the ERK pathway by these stimuli. To further test this proposition, we employed inhibitors of MEK1, the upstream activator of ERK1 and ERK2 (Fig. 2). Two separate inhibitors, PD98059 and the more potent U0126 (34Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar, 35Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R.