Epidermal Growth Factor-stimulated Tyrosine Phosphorylation of Caveolin-1
2000; Elsevier BV; Volume: 275; Issue: 11 Linguagem: Inglês
10.1074/jbc.275.11.7481
ISSN1083-351X
AutoresYong-Nyun Kim, Gregory J. Wiepz, Arturo Guadarrama, Paul J. Bertics,
Tópico(s)Signaling Pathways in Disease
ResumoCaveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nm EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor. Caveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nm EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor. epidermal growth factor platelet-derived growth factor phorbol 12-myristate 13-acetate basic fibroblast growth factor polyacrylamide gel electrophoresis mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Upon ligand binding, the activated epidermal growth factor (EGF)1 receptor mediates a number of important biological responses, including the stimulation of cell proliferation, migration, and differentiation (1.Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4569) Google Scholar, 2.Boonstra J. Rijken P. Humbel B. Cremers F. Verkleij A. van Bergen en Henegouwen P. Cell Biol. Int. 1995; 19: 413-430Crossref PubMed Scopus (257) Google Scholar, 3.Wells A. Gupta K. Chang P. Swindle S. Glading A. Shiraha H. Microscopy Res. Technique. 1998; 43: 395-411Crossref PubMed Scopus (87) Google Scholar). The EGF receptor is a transmembrane glycoprotein that consists of an extracellular EGF binding domain in its amino terminus, a transmembrane spanning region, and a cytoplasmic EGF-stimulated protein-tyrosine kinase domain in its C terminus. The C terminus of the EGF receptor is critical for its kinase activity and substrate specificity. For example, the C terminus of the EGF receptor contains five sites of EGF-dependent tyrosine phosphorylation (tyrosines 992, 1068, 1086, 1148, and 1173) (1.Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4569) Google Scholar, 2.Boonstra J. Rijken P. Humbel B. Cremers F. Verkleij A. van Bergen en Henegouwen P. Cell Biol. Int. 1995; 19: 413-430Crossref PubMed Scopus (257) Google Scholar), and these residues have been proposed to serve an autoinhibitory function that can be attenuated by autophosphorylation or truncation (4.Bertics P.J. Gill G.N. J. Biol. Chem. 1985; 260: 14642-14647Abstract Full Text PDF PubMed Google Scholar, 5.Bertics P.J. Chen W.S. Hubler L. Lazar C.S. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1988; 263: 3610-3617Abstract Full Text PDF PubMed Google Scholar). Indeed, removal of the C-terminal 195 or 213 amino acids from the receptor deletes these autophosphorylation sites but allows for an enhanced and/or altered EGF-stimulated tyrosine phosphorylation of protein substrates in vivo (6.Walton G.M. Chen W.S. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1990; 265: 1750-1754Abstract Full Text PDF PubMed Google Scholar). Furthermore, the transforming protein of the avian erythroblastosis virus, v-erbB, differs from the EGF receptor in that it is constitutively active and lacks most of the extracellular ligand binding domain as well as much of the cytoplasmic autophosphorylation region of the EGF receptor (7.Downward J. Yarden Y. Mayes E. Scrace G. Totty N. Stockwell P. Ullrich A. Schlessinger J. Waterfield M.D. Nature. 1984; 307: 521-527Crossref PubMed Scopus (1705) Google Scholar, 8.Maihle N.J. Kung H.-J. Biochem. Biophys. Acta. 1988; 948: 287-304Google Scholar). Successive truncation at the C terminus of v-erb B increases the capacity of the virus to induce sarcomas and decreases its ability to cause erythroblastosis, suggesting that changes in the C terminus may affect cellular substrate specificity (8.Maihle N.J. Kung H.-J. Biochem. Biophys. Acta. 1988; 948: 287-304Google Scholar, 9.Khazaie K. Dull T.J. Graf T. Schlessinger J. Ullrich A. Beug H. Vennstrom B. EMBO J. 1988; 7: 3061-3071Crossref PubMed Scopus (75) Google Scholar). Autophosphorylation sites in the EGF receptor C terminus have also been reported to be the major binding regions for signal transducers that contain Src homology type 2 domain or phosphotyrosine-binding domain, such as phospholipase C-γ1, Grb2, and Shc, which are effectors that are directly affected by the EGF receptor (1.Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4569) Google Scholar, 2.Boonstra J. Rijken P. Humbel B. Cremers F. Verkleij A. van Bergen en Henegouwen P. Cell Biol. Int. 1995; 19: 413-430Crossref PubMed Scopus (257) Google Scholar, 10.Hsuan J.J. Tan S.K. Int. J. Biochem. Cell Biol. 1997; 29: 415-435Crossref PubMed Scopus (20) Google Scholar). Although the association of phosphorylated wild type EGF receptors with these proteins provides a mechanism for triggering downstream signals, truncated receptors that lack the five C-terminal autophosphorylation sites can also activate gene transcription and induce mitogenesis and cell transformation (11.Gotoh N. Tojo A. Muroya K. Hahsimoto Y. Hattori S. Nakamura S. Takenawa T. Yashio Y. Shibuya M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 167-171Crossref PubMed Scopus (107) Google Scholar, 12.Sasaoka T. Ishihara H. Sawa T. Ishiki M. Morioka H. Imamura T. Usui I. Takata Y. Kobayashi M. J. Biol. Chem. 1996; 271: 20082-20087Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 13.Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Crossref PubMed Scopus (342) Google Scholar, 14.Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C.P. Walton G.M. Der C.J. Wiley H.S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (256) Google Scholar). These studies suggest that truncated receptors have a sufficient or altered specificity for target proteins and thus establish appropriate and/or unique signaling pathways without direct association of Src homology type 2 domain-containing signaling molecules to receptor phosphotyrosines. Caveolae are specialized, Triton X-insoluble microdomains in the plasma membrane that are thought to be present in most cell types (15.Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar). Although they were originally implicated in cellular transport processes, recent evidence suggests that they may participate in signal transduction-related events (16.Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar). Relative to the rest of the plasma membrane, caveolae membrane fractions are enriched in various signaling molecules, receptor and nonreceptor tyrosine kinases, and lipids known to be intermediates in cell signaling (15.Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar, 17.Couet J. Li S.W. Okamoto T. Scherer P.E. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar, 18.Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (493) Google Scholar, 19.Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (7948) Google Scholar). Of specific interest is the observation that caveolae have been identified as sites that are enriched in the EGF receptor and platelet-derived growth factor (PDGF) receptor, and both EGF and PDGF stimulate the recruitment of multiple signaling molecules to caveolae (20.Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 21.Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). This information suggests that caveolae can play an important role in the signal transduction processes induced by receptor protein-tyrosine kinases, such as the EGF receptor. Caveolin-1 is a 21–24-kDa integral protein that acts as the coat protein of caveolae, and both its N-terminal and C-terminal domains are cytoplasmically oriented (16.Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar, 22.Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1842) Google Scholar). Interestingly, a cytosolic N-terminal juxtamembrane domain (residue 82–101) known as the caveolin-1 scaffolding domain can interact directly with certain lipid-modified signaling molecules such as heterotrimeric G-proteins, Src family tyrosine kinases, endothelial nitric-oxide synthase, and Ha-Ras through a common motif found within these signaling molecules (16.Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar). These interactions are believed to sequester the proteins within caveolae and to modulate or suppress their catalytic activities (16.Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar,23.Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Caveolin-1 was first identified as one of the major v-Src substrates in Rous sarcoma virus-transformed cells, and the linkage of caveolin-1 tyrosine phosphorylation with v-Src-induced cell transformation suggests that caveolin-1 tyrosine phosphorylation may be a critical process during cell transformation (24.Glenney Jr., J.R. Zokas L. J. Cell Biol. 1989; 108: 2401-2408Crossref PubMed Scopus (354) Google Scholar, 25.Glenney Jr., J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar). In addition, caveolin-1 expression has been shown to be reduced in human mammary carcinoma cells and in cells transformed by activated oncogenes (26.Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (469) Google Scholar,27.Lee S.W. Reimer C.L. Oh P. Campbell D.B. Schnitzer J.E. Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (397) Google Scholar). Furthermore, re-expression of caveolin-1 in oncogenically transformed cells and in human breast cancer cells has been found to inhibit cell growth, implicating caveolin-1 in the regulation of the proliferation of numerous cell types (27.Lee S.W. Reimer C.L. Oh P. Campbell D.B. Schnitzer J.E. Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (397) Google Scholar, 28.Engelman J.A. Wykoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. J. Biol. Chem. 1997; 272: 16374-16381Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Previous studies in our laboratory have shown that the EGF receptor/kinase population in the Triton X-insoluble fraction of murine B82L fibroblasts and human A431 epidermoid carcinoma cells exhibits an increased EGF-stimulated specific kinase activity, suggesting that the association of the EGF receptor with a Triton X-insoluble component(s) can affect kinase activity (29.Benlimame N. Le P.U. Nabi I.R. Mol. Biol. Cell. 1998; 9: 1773-1786Crossref PubMed Scopus (104) Google Scholar, 30.Gronowski A.M. Bertics P.J. Endocrinology. 1993; 133: 2838-2846Crossref PubMed Scopus (31) Google Scholar, 31.Gronowski A.M. Bertics P.J. Endocrinology. 1995; 136: 2198-2205Crossref PubMed Google Scholar). Caveolin-1 is a marker protein of the Triton X-insoluble plasma membrane and has been shown to interact with the EGF receptor and to inhibit receptor kinase activity in vitro (23.Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). In addition, EGF receptors lacking 164 amino acids at its C terminus have an altered substrate specificity, indicating that C-terminal domain is instrumental in defining the substrate specificity of the EGF receptor tyrosine kinase (32.Li X. Modulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Ph.D. thesis. University of Wisconsin, Madison, WI1997Google Scholar, 33.Bertics P.J. Hubler L. Leventhal P.S. Endocrine Society Abstracts of the 73rd Annual Meeting, Washington, D. C., June, 1991. The Endocrine Society, Bethesda, MD1991: 487Google Scholar). Given that overexpression and/or truncation of the EGF receptor has been found to be oncogenic (34.Dickson R.B. Johnson M.D. el-Ashry D. Shi Y.E. Bano M. Zugmaier G. Ziff B. Lippman M.E. Chrysogelos S. Adv. Exp. Med. Biol. 1993; 330: 119-141Crossref PubMed Scopus (19) Google Scholar) and that a potential role for caveolin-1 in cell transformation has been suggested (25.Glenney Jr., J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar, 26.Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (469) Google Scholar, 27.Lee S.W. Reimer C.L. Oh P. Campbell D.B. Schnitzer J.E. Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (397) Google Scholar, 35.Galbiati F. Volonte D. Gil O. Zanazzi G. Salzer J.L. Sargiacomo M. Scherer P.E. Engelman J.A. Schlegel A. Parenti M. Okamoto T. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10257-10262Crossref PubMed Scopus (159) Google Scholar), the present study focused on ascertaining whether modifications in caveolin-1 are involved in the EGF-induced signal transduction events initiated by oncogenic EGF receptors. Murine B82L parental fibroblasts and those expressing wild type or selected mutant human EGF receptors (WT-L, M721K, ct1022, and ct973*; see "Results") were provided by Dr. Gordon Gill (University of California, San Diego, CA). Murine B82L cells expressing high levels of wild type human EGF receptor (WT-H) and or a truncated receptor (ct973) were generated by polymerase chain reaction-based methods and selected as described previously (5.Bertics P.J. Chen W.S. Hubler L. Lazar C.S. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1988; 263: 3610-3617Abstract Full Text PDF PubMed Google Scholar, 36.Gill G.N. Rosenfeld M.G. Chen W.S. Bertics P.J. Lazar C.S. Adv. Exp. Med. Biol. 1988; 234: 91-103Crossref PubMed Scopus (4) Google Scholar). Dulbecco's modified Eagle's medium was purchased from Life Technologies, Inc., and Cosmic calf serum was obtained from Hyclone Laboratories (Logan, UT). Recombinant human EGF was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Insulin and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma. Porcine PDGF-BB was acquired from R & D Systems (Minneapolis, MN). Basic fibroblast growth factor (bFGF) was a generous gift from Dr. Alan C. Rapraeger (University of Wisconsin, Madison, WI). PD98059 and PP1 were obtained from Calbiochem (La Jolla, CA) and Biomol (Plymouth Meeting, PA), respectively. Polyclonal anti-caveolin-1 antibody (sc-894), polyclonal anti-Src antibodies (SC-18), polyclonal anti-EGF receptor antibodies (SC-03), protein A-agarose, horseradish peroxidase-conjugated goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-EGF receptor antibody LA22 and monoclonal c-Src antibody GD11 were obtained from Upstate Biotechnology Inc., and monoclonal anti-EGF receptor antibody Ab-14 was purchased from Neomarkers (Fremont, CA), respectively, whereas the 528 monoclonal antibody against the EGF receptor was prepared as described earlier (37.Kawamoto T. Sato J.D. Le A. Polikoff J. Sato G.H. Mendelsohn J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1337-1341Crossref PubMed Scopus (684) Google Scholar). Monoclonal anti-phosphotyrosine antibodies 4G10 and PY-20 were purchased from Upstate Biotechnology Inc. and Transduction Laboratories (Lexington, KY), respectively. Polyclonal anti-ERK1/ERK2 antibody, calf intestinal alkaline phosphatase, and UO126 were purchased from Promega (Madison, WI). The R-phycoerythrin anti-mouse IgG conjugate was obtained from Molecular Probes (Eugene, OR). Immobilion-P polyvinylidene difluoride membranes (0.45 μm) were acquired from Millipore (Bedford, MA). Micro BCA protein assay reagents were purchased from Pierce. Chemiluminescent reagents were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). All other reagents were purchased from Sigma. Murine B82L parental fibroblasts were grown on 10-cm plastic tissue culture dishes in Dulbecco's modified Eagle's medium containing 10% Cosmic calf serum. B82L cells transfected with wild type or mutated EGF receptors were cultured in Dulbecco's modified Eagle's medium with 10% Cosmic calf serum and 10 μm methotrexate (because a mutant dihydrofolate reductase gene was used as a selectable marker) (38.Simonsen C.C. Levinson A.D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2485-2499Crossref Scopus (286) Google Scholar). Cells were grown for 3 days to approximately 70% confluence and were serum-starved overnight using 0.1% bovine serum albumin in Dulbecco's modified Eagle's medium before treatment. Cells were trypsinized and suspended in phosphate-buffered saline (pH, 7.4, 10 mmNa2HPO4, 145 mm NaCl, 2.5 mm EDTA, 2.5 mm EGTA) containing 1% Cosmic calf serum. Approximately 5 × 105 cells were stained for 30 min with either monoclonal anti-EGF receptor antibody (528) or IgG isotype control (1 μg of antibody/2–3 × 105cell in 100 μl). The cells were then washed with ice-cold phosphate-buffered saline and incubated for 30 min with anti-mouse IgG-R-phycoerythrin conjugate (1 μg antibody/2 × 105 cell in 100 μl) for 30 min. Cells were subsequently washed with ice-cold phosphate-buffered saline and analyzed on the same day using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). All antibody incubations and washing steps were carried out at 4 °C. For the EGF receptor and caveolin-1 expression assay, 5 × 106 cells expressing various EGF receptor constructs were lysed with 1 ml of 2× SDS-PAGE sample buffer (2% SDS, 20 mm dithiothreitol, 1 mm Na3VO4, 2 mm EDTA, 10% glycerol, 20 mm Tris, pH 8.0). For assays of extracellularly regulated kinase (ERK1/ERK2) activation, caveolin-1 electrophoretic mobility shift, and the tyrosine phosphorylation of total cellular proteins, nearly confluent (70%) serum-starved cells were treated with control buffer (20 mm HEPES, pH 7.4) or with 50 nm EGF, and the cells were lysed with 2× SDS-PAGE sample buffer at the indicated times. The protein level of each sample was determined by the Micro BCA assay (Pierce), and equal amounts of protein (30–50 μg) were loaded on SDS-PAGE gels and subjected to immunoblotting. Serum-starved cells expressing various EGF receptor constructs were treated with control buffer or with EGF (0.1–100 nm), and the cells were then lysed in immunoprecipitation assay (IPA) buffer (10 mm Tris, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.05% SDS, 1 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml aprotinin). The cell lysates were centrifuged at 4 °C for 10 min at 14,000 rpm, and the supernatants were precleared by incubating with protein A-conjugated agarose beads at 4 °C for 1 h, followed by centrifugation at 4 °C for 1 min at 14,000 rpm. The supernatants were then subjected to immunoprecipitation using 2 μg of either polyclonal anti-caveolin-1 antibody, polyclonal anti-Src antibody, polyclonal anti-EGF receptor antibody, nonspecific rabbit IgG, or monoclonal anti-phosphotyrosine antibody (PY-20) and an overnight incubation at 4 °C followed by an incubation with protein A-agarose beads for 2 h at 4 °C. After washing the beads three times with ice-cold IPA buffer with detergent and three times with IPA buffer without detergent, the bound proteins were eluted with SDS-PAGE sample buffer and subjected to electrophoresis and immunoblot analysis. Serum-starved cells expressing various EGF receptor constructs were pretreated with vehicle (0.5% Me2SO) for 15 min, with PD98059 (50 or 100 nm) for 1 h, or with UO126 (10 or 50 nm) for 15 min, or with PP1 (1 or 3 nm) for 15 min at 37 °C. After pretreatment, the cells were stimulated with control buffer or with EGF and were lysed using either 2× SDS-PAGE sample buffer or IPA buffer (for immunoprecipitation experiment). Cell lysates and immunoprecipitates were processed via SDS-PAGE and immunoblotting. In these experiments, caveolin-1 immunoprecipitates from EGF-stimulated cells were prepared as described above and then washed twice with dephosphorylation buffer (10 mm Tris, pH 8.0, 10 mm MgCl2, 50 mm NaCl, 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 10 mm benzamidine). The beads were then resuspended in 0.2 ml of dephosphorylation buffer, and 50 units of calf intestinal alkaline phosphatase were added in the absence or presence of 1 mm Na3VO4for 20 min at 37 °C. Beads were then washed twice with ice-cold IPA buffer and processed for SDS-PAGE. Proteins were separated on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes as described by Bates et al. (39.Bates M.E. Busse W.W. Bertics P.J. Am. J. Respir. Cell Mol. Biol. 1998; 18: 75-83Crossref PubMed Scopus (38) Google Scholar). The membrane was blocked in 0.25% gelatin/TBST overnight at 4 °C and then incubated for 1 h at 37 °C with antibodies against phosphotyrosine (a mixture of 2 μg of 4G10 and 2 μg of PY-20 in 5 ml of 0.25% gelatin/TBST). For immunoblotting analysis using other antibodies, the membrane was blocked with 5% nonfat dried milk/TBST overnight at 4 °C and then incubated with 2 μg of primary antibodies in 5% nonfat dried milk/TBST for 1 h at 37 °C. Subsequently, the membrane was washed three times in TBST, incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies for 1 h at 37 °C, and washed in TBST three times. The labeled proteins were visualized by the enhanced chemiluminescence method. For subsequent probing of the same membrane, the membrane was incubated in a stripping buffer (62.5 mmTris, pH 6.8, 2% SDS, 100 mm dithiothreitol) at 70 °C for 30 min, washed extensively, reblocked with 5% nonfat milk, and reprobed with other antibodies. To investigate the involvement of caveolin-1 in EGF receptor signaling, mouse B82L fibroblast cells (B82L parental) that lack detectable endogenous EGF receptors were transfected with constructs encoding either the wild type human EGF receptor (WT), C-terminally truncated receptors at residues 1022 or 973 (ct1022, ct973, and ct973*), or a kinase-inactive receptor (K721M) as diagrammed in Fig. 1 A. The ct973* EGF receptor differs from ct973 in that a tag, NPVYRRPQSKLGSL, has been fused to the C terminus of ct973, and both ct973 and ct973* receptors have been known to behave similarly in various studies, such as those examining EGF-induced receptor endocytosis (40.Chang C.P. Lazar C.S. Walsh B.J. Komuro M. Collawn J.F. Kuhn L.A. Tainer J.A. Trowbridge I.S. Farquhar M.G. Rosenfeld M.G. Wiley H.S. Gill G.N. J. Biol. Chem. 1993; 268: 19312-19320Abstract Full Text PDF PubMed Google Scholar). Expression levels of wild type and mutant EGF receptors were assessed by anti-EGF receptor immunoblotting using extracts prepared from same number of cells (9 × 104 cells/lane) as shown in Fig. 1 B. Two separate populations of B82L cells expressing the wild type EGF receptors were used in these studies. WT-L cells have been characterized to have 1–2 × 105 EGF receptors/cell (14.Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C.P. Walton G.M. Der C.J. Wiley H.S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (256) Google Scholar), whereas WT-H have at least a 2-fold higher level of EGF receptor expression as determined by immunoblotting analysis (Fig.1 B). To examine the cell surface expression of the various forms of the EGF receptor, fluorescence-activated cell sorting analysis was performed (Fig. 1 D). Similar to the results shown in Fig. 1 B, these experiments revealed that WT-L cells contain lower levels of cell surface EGF receptor than the other transfectants. In addition, the fluorescent intensities for cells expressing either the ct973 or ct973 tag receptors were higher than that for cells containing the ct1022 receptor. It has been reported that the transformation of NIH 3T3 cells by various activated oncogenes and the expression of a temperature sensitive v-Src in Rat-1 cells at the permissive temperature lead to a general reduction of caveolin-1 expression and the number of caveolae (26.Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (469) Google Scholar, 41.Ko Y.G. Liu P.S. Pathak R.K. Craig L.C. Anderson R.G.W. J. Cell. Biohem. 1998; 71: 524-535Crossref PubMed Scopus (36) Google Scholar). To determine whether the expression of the wild type or mutant EGF receptors affects caveolin-1 expression, we evaluated caveolin-1 expression level in each transfectant by immunoblotting with anti-caveolin-1 antibody. As shown in Fig. 1 C, there appeared to be no correlation between caveolin-1 expression and the level of different forms of the EGF receptor. Because the C terminus of the EGF receptor is known to regulate its kinase activity and substrate specificity (6.Walton G.M. Chen W.S. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1990; 265: 1750-1754Abstract Full Text PDF PubMed Google Scholar), we evaluated the ability of wild type and C-terminally truncated EGF receptors to induce the tyrosine phosphorylation of cellular substrates following EGF stimulation. Serum-starved cells were stimulated with or without EGF, and the tyrosine phosphorylation of cellular proteins was assessed by immunoblotting using anti-phosphotyrosine antibodies. Upon EGF stimulation, the overall tyrosine phosphorylation of proteins was increased in cells expressing E
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