Replacement of Tyrosine 1251 in the Carboxyl Terminus of the Insulin-like Growth Factor-I Receptor Disrupts the Actin Cytoskeleton and Inhibits Proliferation and Anchorage-independent Growth
1998; Elsevier BV; Volume: 273; Issue: 29 Linguagem: Inglês
10.1074/jbc.273.29.18411
ISSN1083-351X
AutoresVicky A. Blakesley, Anatolii P. Koval, Bethel Stannard, Angus G. Scrimgeour, Derek LeRoith,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoInsulin-like growth factor (IGF)-I signaling through the IGF-I receptor modulates cellular adhesion and proliferation and the transforming ability of cells overexpressing the IGF-I receptor. Tyrosine phosphorylation of intracellular proteins is essential for this transduction of the IGF-I-induced mitogenic and tumorigenic signals. IGF-I induces specific cytoskeletal structure and the phosphorylation of proteins in the associated focal adhesion complexes. The determination of the exact pathways emanating from the IGF-I receptor that are involved in mediating these signals will contribute greatly to the understanding of IGF-I action. We have previously shown that replacement of tyrosine residues 1250 and 1251 in the carboxyl terminus of the IGF-I receptor abrogates IGF-I-induced cellular proliferation and tumor formation in nude mice. In this study, replacement of either tyrosine 1250 or 1251 similarly reduces the cells ability to grow in an anchorage-independent manner. The actin cytoskeleton and cellular localization of vinculin are disrupted by replacement of tyrosine 1251. Tyrosine residues 1250 and 1251 are not essential for tyrosine phosphorylation of two known substrates; insulin receptor substrate-1 and SHC, nor association of known downstream adaptor proteins to these substrates. In addition, these mutant IGF-I receptors do not affect IGF-I-stimulated p42/p44 mitogen-activated protein kinase activation or phosphatidylinositol (PI) 3′-kinase activity. Thus, it appears that in fibroblasts expressing tyrosine 1250 and 1251 mutant IGF-I receptors, the signal transduction pathways impacting on mitogenesis and tumorigenesis do not occur exclusively through the PI 3′-kinase or mitogen-activated protein kinase pathways. Insulin-like growth factor (IGF)-I signaling through the IGF-I receptor modulates cellular adhesion and proliferation and the transforming ability of cells overexpressing the IGF-I receptor. Tyrosine phosphorylation of intracellular proteins is essential for this transduction of the IGF-I-induced mitogenic and tumorigenic signals. IGF-I induces specific cytoskeletal structure and the phosphorylation of proteins in the associated focal adhesion complexes. The determination of the exact pathways emanating from the IGF-I receptor that are involved in mediating these signals will contribute greatly to the understanding of IGF-I action. We have previously shown that replacement of tyrosine residues 1250 and 1251 in the carboxyl terminus of the IGF-I receptor abrogates IGF-I-induced cellular proliferation and tumor formation in nude mice. In this study, replacement of either tyrosine 1250 or 1251 similarly reduces the cells ability to grow in an anchorage-independent manner. The actin cytoskeleton and cellular localization of vinculin are disrupted by replacement of tyrosine 1251. Tyrosine residues 1250 and 1251 are not essential for tyrosine phosphorylation of two known substrates; insulin receptor substrate-1 and SHC, nor association of known downstream adaptor proteins to these substrates. In addition, these mutant IGF-I receptors do not affect IGF-I-stimulated p42/p44 mitogen-activated protein kinase activation or phosphatidylinositol (PI) 3′-kinase activity. Thus, it appears that in fibroblasts expressing tyrosine 1250 and 1251 mutant IGF-I receptors, the signal transduction pathways impacting on mitogenesis and tumorigenesis do not occur exclusively through the PI 3′-kinase or mitogen-activated protein kinase pathways. Insulin-like growth factor-I (IGF-I) 1The abbreviations used are: IGF, insulin-like growth factor; IRS-1, insulin receptor substrate-1; MAP, mitogen-activated protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; SF-DMEM, serum-free DMEM; PI, phosphatidylinositol; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; ECM, extracellular matrix; PTP1D, protein-tyrosine phosphatase 1D; Grb2, growth factor receptor-bound protein 2; Erk, extracellular signal-regulated kinase; MES, 2-(N-morpholino)ethanesulfonic acid. and -II (IGF-II) are ligands of the type I insulin-like growth factor receptor (IGF-I receptor). Binding of these mitogenic ligands to this transmembrane receptor results in autophosphorylation of tyrosine residues of the β-subunit and activation of its intrinsic tyrosine kinase activity, which leads to the activation of multiple intracellular signaling pathways (1Nissley P. Lopaczynski W. Growth Factors. 1991; 5: 29-49Crossref PubMed Scopus (166) Google Scholar, 2LeRoith D. Werner H. Beitner-Johnson D. Roberts Jr, C.T. Endocrinol. Rev. 1995; 16: 143-163Crossref PubMed Scopus (1251) Google Scholar). The importance of a functional IGF-I receptor in normal mammalian development is highlighted by the abnormal phenotype of knockout mice developed in the laboratory of Efstratiadis (3Liu J.-P. Baker J. Perkins A.S. Robertson E.J. Estratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2585) Google Scholar, 4Baker J. Liu J.-P. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 73-82Abstract Full Text PDF PubMed Scopus (2062) Google Scholar). These mice have poor intrauterine growth and die shortly after birth. The IGF-I receptor has also been shown to be important in conferring a transformed phenotype to cells. Signaling through the IGF-I receptor is involved in several spontaneous malignancies (for a review see Blakesley et al. (5Blakesley V.A. LeRoith D. LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus: A Clinical and Practical Approach. Lippincott-Raven Publishers, Philadelphia, PA1996: 824-831Google Scholar)), presumably via an autocrine/paracrine mechanism. Growth of tumor cells in culture can be inhibited by blocking the expression of the IGF-I receptor using antisense strategies (6Shapiro D.N. Jones B.G. Shapiro L.H. Dias P. Houghton P.J. J. Clin. Invest. 1994; 94: 1235-1242Crossref PubMed Scopus (162) Google Scholar, 7Neuenschwander S. Scwartz A. Wood T.L. Roberts Jr., C.T. Heninghausen L. LeRoith D. J. Clin. Invest. 1996; 97: 2225-2232Crossref PubMed Scopus (178) Google Scholar) or using antibodies that bind to and reduce ligand-stimulated activation of the receptor (8Arteaga C.L. Osborne C.K. Cancer Res. 1989; 49: 6237-6241PubMed Google Scholar, 9Arteaga C.L. Kitten L.J. Coronado E.B. Jacobs S. Kull Jr., F.C. Allred D.C. Osborne C.K. J. Clin. 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A. 1992; 89: 4874-4878Crossref PubMed Scopus (180) Google Scholar, 12Trojan J. Johnson T.R. Rudin S.D. Ilan J. Tykocinski M.L. Ilan J. Science. 1993; 259: 94-97Crossref PubMed Scopus (327) Google Scholar, 13Kalebic T. Tsokos M. Helman L.J. Cancer Res. 1994; 54: 5531-5534PubMed Google Scholar, 14Resnicoff M. Sell C. Rubini M. Coppola D. Ambrose D. Baserga R. Rubin R. Cancer Res. 1994; 54: 2218-2222PubMed Google Scholar, 15Resnicoff M. Coppola D. Sell C. Rubin R. Ferrone S. Baserga R. Cancer Res. 1994; 54: 4848-4850PubMed Google Scholar). Overexpression of the IGF-I receptor in nontransformed NIH-3T3 fibroblasts increases both the IGF-I-stimulated mitogenesis and tumorigenicity of these cells (16Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1993; 268: 2655-2661Abstract Full Text PDF PubMed Google Scholar, 17Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. Mol Endocrinol. 1994; 8: 40-50Crossref PubMed Scopus (109) Google Scholar, 18Blakesley V.A. Kato H. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1995; 270: 2764-2769Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 19Blakesley V.A. Kalebic T. Helman L.J. Stannard B. Faria T.N. Roberts Jr., C.T. LeRoith D. Endocrinology. 1996; 137: 410-417Crossref PubMed Google Scholar). Expression of kinase-deficient and dominant negative mutant IGF-I receptors in NIH-3T3 or Rat-1 fibroblasts, however, abrogated IGF-I-stimulated thymidine incorporation and cellular proliferation and inhibited anchorage-independent growth in soft agar and tumor formation in nude mice (5Blakesley V.A. LeRoith D. LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus: A Clinical and Practical Approach. Lippincott-Raven Publishers, Philadelphia, PA1996: 824-831Google Scholar, 16Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1993; 268: 2655-2661Abstract Full Text PDF PubMed Google Scholar, 20Prager D. Li H.-L. Asa S. Melmed S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2181-2185Crossref PubMed Scopus (212) Google Scholar). The above data are consistent with the hypothesis that a fully functional IGF-I receptor capable of transducing the IGF-I mitogenic signal is essential for normal embryonic development. Furthermore, the receptor is capable of initiating and/or maintaining a transformed phenotype in cells in which it is overexpressed. Mutant IGF-I receptors have been used to investigate the specific signaling pathways emanating from the receptor that are responsible for transducing the mitogenic and tumorigenic signals of IGF-I. In particular, the tyrosine residues in the COOH terminus have been mutated to determine if these residues are important in signaling. We and others have previously shown that replacing specific tyrosine residues (tyrosines 1250 and 1251) in the COOH terminus of the receptor reduced both the mitogenic and tumorigenic potential of the mouse fibroblasts in which the receptor was expressed, while replacement of tyrosine 1316 did not change the mitogenic potential of the cells but did abrogate the transformed phenotype (19Blakesley V.A. Kalebic T. Helman L.J. Stannard B. Faria T.N. Roberts Jr., C.T. LeRoith D. Endocrinology. 1996; 137: 410-417Crossref PubMed Google Scholar, 21Miura M. Surmacz E. Burgaud J.-L. Baserga R. J. Biol. Chem. 1995; 270: 22639-22644Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). It has been shown that IGF-I-stimulated IGF-I receptor autophosphorylation and phosphorylation of insulin receptor substrate-1 (IRS-1) were unaffected by these tyrosine residue substitutions (19Blakesley V.A. Kalebic T. Helman L.J. Stannard B. Faria T.N. Roberts Jr., C.T. LeRoith D. Endocrinology. 1996; 137: 410-417Crossref PubMed Google Scholar). To further investigate the role of the tyrosine residues in the COOH terminus of the IGF-I receptor in mediating mitogenic and tumorigenic signals, we studied NIH-3T3 cells stably expressing IGF-I receptors with substitutions, singly and in combination, of tyrosines 1250, 1251, and 1316. Restriction endonucleases were purchased from New England Biolabs (Beverly, MA), Boehringer Mannheim, and Life Technologies, Inc. Cell culture media and reagents were purchased from Biofluids, Inc. (Rockville, MD) and Advanced Biotechnologies (Columbia, MD). Insulin-free bovine serum albumin (BSA, fraction V) was obtained from Armour (Kankakee, IL). Recombinant human IGF-I, monoclonal anti-phosphotyrosine antibody conjugated to horseradish peroxidase (4G10), and fetal bovine serum were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal anti-growth factor receptor-bound protein 2 (Grb2) antibody, monoclonal and polyclonal anti-SHC antibodies, monoclonal anti-PTP1D/Syp, and recombinant anti-phosphotyrosine RC20H horseradish peroxidase-conjugated antibodies were purchased from Transduction Laboratories (Lexington, KY). Polyclonal anti-extracellular signal-regulated kinase-1 (Erk-1) and polyclonal anti-Erk-2 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibody to IRS-1 was the kind gift of J. Pierce (NCI, National Institutes of Health). Phospho-specific MAP kinase antibody that detects phosphorylation of mouse tyrosine 185 was purchased from New England Biolabs, Inc. Monoclonal anti-Erk-2 and polyclonal anti-MAP kinase antibodies were obtained from Zymed Laboratories (S. San Francisco, CA). Fluorescein-labeled goat anti-mouse antibodies were purchased from Kirkegaard and Perry, Inc. (Gaithersburg, MD). Full-length myelin basic protein was obtained from Life Technologies. Monoiodinated 125I-IGF-I and the enhanced chemoluminescence (ECL) detection kit were purchased from Amersham Pharmacia Biotech. Prestained high molecular weight protein standards and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and 1,4-phenylenediamine were purchased from Sigma-Aldrich. Fibronectin was purchased from Life Technologies, and glutaraldehyde was purchased from Calbiochem. Rhodamine phalloidin was purchased from Molecular Probes, Inc. (Eugene, OR). Biomedia Gel Mount was purchased from Fisher. Other standard biochemical reagents were purchased from Sigma-Aldrich, Calbiochem, and Mallinckrodt (Paris, KY). The wild-type human IGF-I receptor expression vector has been previously described (16Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1993; 268: 2655-2661Abstract Full Text PDF PubMed Google Scholar). Plasmid constructs encoding the cDNA for the mutant receptors with tyrosines 1250 and 1251 mutated to phenylalanine and histidine, respectively (yyFH), or tyrosine 1316 mutated to phenylalanine (yCF) have been previously described (19Blakesley V.A. Kalebic T. Helman L.J. Stannard B. Faria T.N. Roberts Jr., C.T. LeRoith D. Endocrinology. 1996; 137: 410-417Crossref PubMed Google Scholar). The mutant IGF-I receptors yNF (tyrosine 1250 mutated to phenylalanine) and yNH (tyrosine 1251 mutated to histidine) were expressed from pBPV plasmids containing cDNA encoding mutant sequences generated by site-specific mutagenesis using mutant primers in a PCR-based mutagenesis strategy. Briefly, segments of the cDNA encoding the human IGF-I receptor were subcloned from pBluescript II into a second vector. Complimentary primer sequences were chemically synthesized to include the mutated triplet codon of interest. The direct mutant primer for yNF was 5′-TTC CGG GAG GTA AG C TTCTTC TAC AGC GAG GAG AAC AAC-3′, and the complimentary primer was 5′-GTT CTC CTC GCT GTA GAA GAA GCT TAC CTC CCG GAA GCC A-3′. For yNH, the direct mutant primer was 5′-TTC CGG GAG GTA AG C TTC TACC AC AGC GAG GAG AAC AAC-3′, and the complimentary primer was 5′-GTT CTC CTC GCT GTG GTA GAA GCT TAC CTC CCG GAA GCC A-3′. The mutated bases are underlined and the introducedHindIII sites are in italics. The mutated codons are presented in boldface type; in yNF TTC is the codon for phenylalanine, and in yNH CAC is the codon for histidine. After excision of the mutant sequences with restriction endonucleases, the overlapping cDNA fragments were ligated into pBluescript. The sequences encoding the full-length cDNA for the mutated IGF-I receptors in pBluescript II were excised with SalI and NotI and cloned into a bovine papilloma virus-derived mammalian expression vector (pBPV; Amersham Pharmacia Biotech) that had been linearized withXhoI and NotI. All NIH-3T3 cell lines were cultured in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose with added 2 mm glutamine (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. NIH-3T3 cells were co-transfected with 20 μg of mutant expression vector or insertless pBPV plus 1 μg of pMC1Neo (CLONTECH, Palo Alto, CA) in Lipofectin reagent (Life Technologies). Clones stably expressing IGF-I receptors were selected in DMEM supplemented with 500 μg/ml G418 (Geneticin; Life Technologies) as described previously (16Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1993; 268: 2655-2661Abstract Full Text PDF PubMed Google Scholar). Clones overexpressing IGF-I receptors were selected based on results of IGF-I binding assays as described previously (16Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1993; 268: 2655-2661Abstract Full Text PDF PubMed Google Scholar). Stably transfected cells were maintained in DMEM supplemented with 10% FBS, antibiotics, and 500 μg/ml G418 (Geneticin; Life Technologies). Cells were split for each experiment and cultured in serum-supplemented DMEM without G418. Serum-free medium (SF-DMEM) composed of DMEM with 0.1% bovine serum albumin, 20 mm Hepes, pH 7.5, and antibiotics was used in assays of IGF-I receptor binding, cellular proliferation, receptor autophosphorylation, and tyrosine kinase activity as measured by phosphorylation of endogenous substrates, MAP kinase phosphorylation and activity, and PI 3′-kinase activity. The control cell line (pNeo1) and the cell lines overexpressing wild-type IGF-I receptors (NWTb3 and NWTc43) have been described previously (18Blakesley V.A. Kato H. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1995; 270: 2764-2769Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 19Blakesley V.A. Kalebic T. Helman L.J. Stannard B. Faria T.N. Roberts Jr., C.T. LeRoith D. Endocrinology. 1996; 137: 410-417Crossref PubMed Google Scholar). Cell lines expressing the mutant receptor with phenylalanine replacing tyrosine 1250 (yNFa38 and yNFa45) or with histidine replacing tyrosine 1251 (yNHa8 and yNHc5) were selected based on expression of approximately equivalent numbers of IGF-I receptors as compared with NWTb3 and NWTc43 cells. Cell growth was determined by measuring the colorimetric change of 10% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide in media without phenol red after incubation with cells for 4 h at 37 °C, followed by lysis of the cells with isopropyl alcohol (22Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46492) Google Scholar,23Denizot F. Lang R. J. Immunol. Methods. 1986; 89: 271-277Crossref PubMed Scopus (4322) Google Scholar). Each cell line was plated in triplicate for each time point. To determine the effect of IGF-I on cellular proliferation, cells were grown in DMEM with 1% FBS and supplemented with 10 nmIGF-I. Cell number was determined daily from time 0 to 96 h. The medium was replenished at 72 h to maintain exponential growth. Each cell line was tested for cellular proliferation in three separate experiments. Standard curves correlating cell number and absorbance were performed for all cell lines and found to give comparable results; therefore, one standard curve using NWTb3 cells was subsequently performed with each experiment. Transformation was measured by analyzing the ability of cells to grow in soft agar. Briefly, 1 × 103 cells in 0.2% agarose in DMEM supplemented with 10% FBS with or without 10 nm IGF-I were overlaid on a base of 0.8% agarose in DMEM supplemented with 10% FBS in 35-mm tissue culture dishes. All cell lines were plated in triplicate. Cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The number of colonies with a greater than 125-μm diameter was scored at the end of 3 weeks. Fibroblasts, grown to subconfluency, were treated with tryspin (2 mg/ml) and immediately dislodged from the tissue culture flask by brisk shaking. The cells were resuspended in DMEM with 10% FBS and allowed to recover for 20 min at 37 °C in a polypropylene conical centrifuge tube. The cells were washed twice with prewarmed SF-DMEM and then plated onto fibronectin-coated 96-well plates at 2 × 104cells/well. The cells were incubated for 30 min at 37 °C. Unbound cells were removed by aspiration of the medium and aspiration of a subsequent 100-μl PBS wash. Bound fibroblasts were fixed by a 30-min treatment of 5% glutaraldehyde. The fixative was removed, and cells were washed three times with water and then air-dried. Cells were stained with 0.1% (w/v) crystal violet in 20 mm MES (pH 6.0) for 20 min at room temperature, followed by three water washes. The stained cells were solubilized with 100% acetic acid and rapid shaking for 5 min at room temperature. Absorbance at 570 nm was determined by measurement in an enzyme-linked immunosorbent assay reader (Microplate Autoreader; Biotek Instruments, Inc., Winooski, Vermont). For each cell line, in each of five separate experiments, the total number of cells plated in duplicate wells was determined by proceeding with fixation and staining of the cells without aspiration of unbound cells. The relative amount of adherent cells was normalized by expressing absorbance of bound cells divided by absorbance of total plated cells. Fibroblasts were plated on glass coverslips in six-well plates to achieve 30% confluency. Medium was changed to DMEM with 0.5% FBS for 24 h, and cells were treated with 10 nm IGF-I for 0, 8, and 15 min. Cells were fixed in formaldehyde with 5% sucrose in PBS for 15 min at room temperature, permeabilized in 0.4% Triton X-100 for 5 min, and blocked in 10% FBS in PBS. Detection of cytoskeletal proteins or tyrosine-phosphorylated proteins was accomplished by treatment of primary antibody for 1 h at room temperature followed by secondary antibody conjugated with fluorescein for 1 h at room temperature. The primary antibodies used were monoclonal anti-paxillin antibody (5 μg/ml) and monoclonal anti-vinculin antibody (5 μg/ml). Following aspiration of the primary antibody and three washes with PBS, secondary antibody was applied as goat anti-mouse antibody conjugated with fluorescein (6.25 μg/ml) along with rhodamine phalloidin 0.67 unit/ml, which labels F-actin. Coverslips were affixed to slides using Biomedia gel mount with 1 mg/ml 1,4-phenylenediamine. Slides were examined by fluorescence microscopy for staining patterns. Confluent cells in 100-mm plates were serum-starved in SF-DMEM for 20 h, washed twice with SF-DMEM, and then incubated either without or with IGF-I (10 nm) at 37 °C for various times as indicated for each experiment. The cells were washed rapidly with ice-cold PBS. The cells were then lysed in the presence of 350 μl of freshly prepared lysis buffer (50 mm Hepes, pH 7.9, 150 mm NaCl, 10 mm EDTA, 1% Triton X-100, 4 mm sodium pyrophosphate, 2 mm sodium orthovanadate (Na3VO4), 1 mm phenylmethylsulfonyl fluoride, 10 mm sodium fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin). Cell lysates were cleared of Triton-insoluble material by centrifugation. Protein content was determined by the method of Bradford (24Bradford M. Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar) using a protein assay kit (Bio-Rad). Proteins were stacked through a 4% SDS-polyacrylamide gel and then separated by electrophoresis through a 9% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane for 5 h at 0.22 amperes in a Tris-glycine buffer with 20% methanol. The amount of IGF-I receptor present on the nitrocellulose membrane was determined by immunoblotting with AB 53, a polyclonal antibody that detects the triple tyrosine cluster of the IGF-I receptor. Equivalent amounts of IGF-I receptors were then resolved by SDS-PAGE and transferred to nitrocellulose. Tyrosine-phosphorylated proteins were immunoblotted with a monoclonal anti-phosphotyrosine antibody (4G10) conjugated to horseradish peroxidase (1:1000 dilution, Upstate Biotechnology Inc.), and antibody-bound proteins were visualized using ECL according to the manufacturer's specifications. Tyrosine-phosphorylated IRS-1 was detected by immunoblotting with RC20H (1:2500 dilution) followed by treatment of the membrane with the ECL detection system. Erk-1 and Erk-2 were detected by immunoblotting with anti-phosphotyrosine MAP kinase antibody (1:1000 dilution), which specifically binds to Erk-1 and Erk-2, which are phosphorylated on tyrosine 185, followed by a secondary antibody horseradish peroxidase conjugate and detection as described above. Immunoprecipitation studies were done on cleared whole cell lysates prepared from cells grown to confluency, serum-starved overnight, stimulated, and lysed as described above for measurements of tyrosine-phosphorylated proteins. IGF-I stimulation of cells was for 1 min. for IRS-1 studies or 3 min for SHC studies. Immunoprecipitations of 600 μg of cellular protein were carried out overnight with rotation at 4 °C in the same buffer in a total volume of 600 ml with either polyclonal anti-IRS-1 antibody (7 μl) or polyclonal anti-SHC antibody (7 μl). Immunoprecipitants were captured by incubation at 4 °C for 4 h. with 50 μl of 10% (w/v) Protein A-Sepharose beads (Amersham Pharmacia Biotech) in 50 mm Tris-HCl (pH 7.0) followed by three 1-ml washes in ice-cold immunoprecipitation wash buffer (10 mm Tris-HCl (pH 7.0), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.2 mmNa3VO4, 0.2 mm phenylmethylsulfonyl fluoride, 1% Triton X-100). Immunoprecipitants were boiled for 5 min in Laemmli buffer (50 mm Tris-HCl (pH 6.7), 2% SDS, and 2% β-mercaptoethanol) just prior to separation by discontinuous SDS-PAGE (9% polyacrylamide gel) and transfer to nitrocellulose membrane as described above. Tyrosine-phosphorylated IRS-1 and SHC were detected by immunoblotting with RC20H (1:2500 dilution) and the antibody-bound proteins were visualized by ECL. Blots were stripped (30-min incubation at 55 °C in 62.5 mm Tris-HCl (pH 6.8), 2% SDS, and 0.7% β-mercaptoethanol) and reblotted with the appropriate antibody to the protein of interest: polyclonal anti-IRS-1 antibody (1:1000 dilution), monoclonal anti-SHC antibody (1:500 dilution), monoclonal anti-Grb2 antibody (1:1000 dilution), or monoclonal anti-PTP1D/Syp antibody (1:1000 dilution). The complexes were detected by blotting with a secondary antibody conjugated to horseradish peroxidase-anti-rabbit immunoglobulin for the polyclonal antibody or anti-mouse immunoglobulin for the monoclonal antibodies. The immunoblotted proteins were visualized by use of the ECL system. Subconfluent cells in 100-mm plates were serum-starved in SF-DMEM for 16 h, washed twice with SF-DMEM, and then incubated either without or with IGF-I (10 nm) at 37 °C for 8 min. The cells were washed rapidly twice with ice-cold PBS. The cells were then solubilized in the presence of 400 μl of freshly prepared ice-cold lysis buffer (20 mm Tris (pH 7.5), 137 mm NaCl, 10% glycerol, 25 mm β-glycerophosphate, 2 mm EDTA, 1% Triton X-100, 2 mm sodium pyrophosphate, 1 mmNa3VO4, and 1 mmphenylmethylsulfonyl fluoride). Cell lysates were cleared by centrifugation. Protein content was determined as described above. The MAP kinase assay was performed as described by work from the Krebs laboratory (25Seger R. Ahn N.G. Posada J. Munar E.S. Jensen A.M. Cooper J.A. Cobb M.H. Krebs E.G. J. Biol. Chem. 1992; 267: 14373-14381Abstract Full Text PDF PubMed Google Scholar) after immunoprecipitation of Erk-2 with a polyclonal antibody. Proteins were separated by discontinuous reducing SDS-PAGE (15% polyacrylamide gel), and the gels were fixed in acetic acid/isopropyl alcohol (12.5%:25%) prior to a brief exposure of the gel to Kodak X-Omat film to determine the relative positions of the radioactive products. These products were quantified by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA). Phosphorylation of phosphatidylinositol was measured as described previously (26Backer J.M. Schroeder G.G. Kahn C.R. Myers Jr., M.G. Wilden P.A. Cahill D.A. White M.F. J. Biol. Chem. 1992; 267: 1367-1374Abstract Full Text PDF PubMed Google Scholar) with modifications (16Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1993; 268: 2655-2661Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were grown to confluency in 100-mm dishes, washed with PBS, and incubated for 24 h in SF-DMEM. Cells were incubated for 1 min in prewarmed (37 °C) SF-DMEM without or with 10 nm IGF-I. Cells were washed twice with ice-cold PBS and twice with freshly prepared wash buffer (20 mm Tris (pH 7.5), 100 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, and 100 μm Na3VO4). Cells were lysed in 500 μl of wash buffer containing 1% Nonidet P-40, 10% glycerol, and 2 mmphenylmethylsulfonyl fluoride for 10 min on ice. Lysates were centrifuged for 10 min at 13,000 × g for 4 °C, and supernatants were frozen. Protein content was measured by the method of Bradford as indicated above. Extracts (600 μg of protein) were immunoprecipitated overnight at 4 °C with 7 μl of monoclonal anti-phosphotyrosine antibody (clone 4G10) with the addition of 30 μl of protein A-Sepharose for the last 4 h. The immunoprecipitant-Sepharose bead complexes were kept on ice and washed sequentially with ice-cold wash buffers: once with wash buffer A (100 μm Na3VO4 and 1% Nonidet P-40 in PBS), twice with wash buffer B (100 mm Tris (pH 7.5), 500 mm LiCl, and 100 μmNa3VO4), and once with wash buffer C (10 mm Tris (pH 7.5), 100 mm NaCl, 1 mmEDTA, and 100 μm Na3VO4). The immunoprecipitant-Sepharose bead complexes were then resuspended in 40 μl of ice-cold 10 mM Tris, pH 7.5, 100 mmNaCl, 100 μm Na3VO4. Samples were incubated for 10 min with 10 μl of 100 mmMnCl2, 10 μl of 2 μg/μl phosphatidylinositol, and 10 μl of 440 μm ATP containing 40 μCi of [32P]ATP. The reaction was stopped with the addition of 20 μl of 8 n HCl and 160 μl of CHCl3/methanol (1:1). The organic phase was extracted, and chromatography was carried out as described (16Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1993; 268: 2655-266
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