FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1978393771> ?p ?o ?g. }

• W1978393771 endingPage "6709" @default.
• W1978393771 startingPage "6702" @default.
• W1978393771 abstract "The insulin-like growth factor I receptor (IGF-IR) has the ability to confer clonogenic radioresistance following ionizing irradiation. We attempted to determine the downstream pathways involved in IGF-IR-mediated radioresistance and used mouse embryo fibroblasts deficient in endogenous IGF-IR (R−) as recipients for a number of mutant IGF-IRs. Mutational analysis revealed that the tyrosine at residue 950 (Tyr-950) of IGF-IR, as well as the C-terminal domain, are required for radioresistance and that both domains must be mutated to abrogate the phenotype. Furthermore, the contribution of downstream pathways was analyzed by combining the use of wild-type or Tyr-950 and C-terminal mutants with specific inhibitors of phosphatidylinositol 3′-kinase (PI3-K) or mitogen-activated protein extracellular signal-regulated kinase (ERK) kinase (MEK). Radioresistance could be induced by IGF-IR as long as the ability of the receptor to stimulate the MEK/ERK pathway was retained. This was confirmed by the expression of constitutively active MEK in R− cells. The ability to stimulate the PI3-K pathway alone was not sufficient, but PI3-K activation coupled with MEK/ERK pathway-independent signals from the C terminus was able to induce radioresistance. Taken together, these results indicate that the IGF-IR-mediated radioresistant signaling mechanism progresses through redundant downstream pathways. The insulin-like growth factor I receptor (IGF-IR) has the ability to confer clonogenic radioresistance following ionizing irradiation. We attempted to determine the downstream pathways involved in IGF-IR-mediated radioresistance and used mouse embryo fibroblasts deficient in endogenous IGF-IR (R−) as recipients for a number of mutant IGF-IRs. Mutational analysis revealed that the tyrosine at residue 950 (Tyr-950) of IGF-IR, as well as the C-terminal domain, are required for radioresistance and that both domains must be mutated to abrogate the phenotype. Furthermore, the contribution of downstream pathways was analyzed by combining the use of wild-type or Tyr-950 and C-terminal mutants with specific inhibitors of phosphatidylinositol 3′-kinase (PI3-K) or mitogen-activated protein extracellular signal-regulated kinase (ERK) kinase (MEK). Radioresistance could be induced by IGF-IR as long as the ability of the receptor to stimulate the MEK/ERK pathway was retained. This was confirmed by the expression of constitutively active MEK in R− cells. The ability to stimulate the PI3-K pathway alone was not sufficient, but PI3-K activation coupled with MEK/ERK pathway-independent signals from the C terminus was able to induce radioresistance. Taken together, these results indicate that the IGF-IR-mediated radioresistant signaling mechanism progresses through redundant downstream pathways. phosphatidylinositol 3′-kinase mitogen-activated protein kinase epidermal growth factor receptor insulin-like growth factor I receptor insulin receptor ataxia telangiectasia-mutated insulin receptor substrate-1 extracellular signal-regulated kinase mitogen-activated protein/extracellular signal-regulated kinase kinase wild-type horseradish peroxidase hemagglutinin Tris-buffered saline plus Tween 20 phosphatidylinositol gray Intrinsic radiosensitivity is one of the critical factors that determines the probability of successful tumor cure or local control following radiotherapy (1Girinsky T. Lubin R. Pignon J.P. Chavaudra N. Gazeau J. Dubray B. Cosset J.M. Socie G. Fertil B. Int. J. Radiat. Oncol. Biol. Phys. 1992; 25: 3-7Abstract Full Text PDF Scopus (101) Google Scholar, 2West C.M.L. Davidson S.E. Roberts S.A. Hunter R.D. Br. J. Cancer. 1993; 68: 819-823Crossref PubMed Scopus (176) Google Scholar). Activation of oncogenes, includingras, and mutation of tumor suppressors such as p53 are well known to induce radioresistance (3Bernhard E.J. Stanbridge E.J. Gupta S. Gupta A.K. Soto D. Bakanauskas V.J. Cerniglia G.J. Muschel R.J. McKenna W.G. Cancer Res. 2000; 60: 6597-6600PubMed Google Scholar, 4Miller A.C. Kariko K. Myers C.E. Clark E.P. Samid D. Int. J. Cancer. 1993; 53: 302-307Crossref PubMed Scopus (122) Google Scholar, 5Bristow R.G. Benchimol S. Hill R.P. Radiother. Oncol. 1996; 40: 197-223Abstract Full Text PDF PubMed Scopus (186) Google Scholar), and the mechanisms by which this occurs have been investigated from a number of aspects, including cell cycle progression and signal transduction (6McKenna W.G. Bernhard E.J. Markiewicz D.A. Rudoltz M.S. Maity A. Muschel R.J. Oncogene. 1996; 12: 237-245PubMed Google Scholar, 7Gupta A.K. Bernhard E.J. Bakanauskas V.J. Wu J.M. Muschel R.J. McKenna W.G. Radiat. Res. 2000; 154: 64-72Crossref PubMed Scopus (41) Google Scholar, 8Gupta A.K. Bakanauskas V.J. Cerniglia G.J. Cheng Y. Bernhard E.J. Muschel R.J. McKenna W.G. Cancer Res. 2001; 61: 4278-4282PubMed Google Scholar). It has been reported that phosphatidylinositol 3′-kinase (PI3-K),1 but not mitogen-activated protein kinase (MAPK), is important for mutant Ras-induced radioresistance, although both are known to convey potent survival signals (8Gupta A.K. Bakanauskas V.J. Cerniglia G.J. Cheng Y. Bernhard E.J. Muschel R.J. McKenna W.G. Cancer Res. 2001; 61: 4278-4282PubMed Google Scholar). Some of the growth factors, which activate a variety of downstream pathways including Ras, also mediate cell survival functions through their cognate receptors (9Harrington E.A. Bennett M.R. Fanidi A. Evan G.I. EMBO J. 1994; 13: 3286-3295Crossref PubMed Scopus (732) Google Scholar). Of these, epidermal growth factor receptor (EGFR), which is often overexpressed in various tumor types, has been shown to induce radioresistance; specific antibodies for EGFR or expression of dominant negative EGFR significantly radiosensitizes tumor cells both in vitro andin vivo (10Lammering G. Valerie K. Lin P.S. Mikkelsen R.B. Contessa J.N. Feden J.P. Farnsworth J. Dent P. Schmidt-Ullrich R. Clin. Cancer Res. 2001; 7: 682-690PubMed Google Scholar, 11Harari P.M. Huang S.-M. Int. J. Radiat. Oncol. Biol. Phys. 2001; 49: 427-433Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 12Lammering G. Hewit T.H. Hawkins W.T. Contessa J.N. Reardon D.B. Lin P.S. Valerie K. Dent P. Mikkelsen R.B. Schmidt-Ullrich R.K. J. Natl. Cancer Inst. 2001; 93: 921-929Crossref PubMed Scopus (89) Google Scholar). It has been suggested that stimulation of survival signals such as the PI3-K and MAPK pathways following EGFR activation contributes to radioresistance (13Todd D.G. Mikkelsen R.B. Rorrer W.K. Valerie K. Schmidt-Ullrich R.K. J. Recept. Signal Transduct. Res. 1999; 19: 885-908Crossref PubMed Scopus (35) Google Scholar, 14Bowers G. Reardon D. Hewitt T. Dent P. Mikkelsen R.B. Valerie K. Lammering G. Amir C. Schmidt-Ullrich R.K. Oncogene. 2001; 20: 1388-1397Crossref PubMed Scopus (147) Google Scholar). The insulin-like growth factor I receptor (IGF-IR) is a transmembrane tyrosine kinase, the amino acid sequence of which is highly homologous to that of the insulin receptor (IR) (15Ullrich A. Gray A. Tam A.W. Yang-Feng T. Tsubokawa M. Collins C. Henzel W. le Bon T. Kahuria S. Chen E. Jakobs S. Francke U. Ramachandran J. Fujita-Yamaguchi Y. EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1486) Google Scholar). It is a generally held view that IGF-IR activation plays a key role in cell growth, establishment, and maintenance of a transformed phenotype, cell survival, and differentiation (16Sell C. Rubini M. Rubin R. Liu J.P. Efstratiadis A. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11217-11221Crossref PubMed Scopus (539) Google Scholar, 17Sell C. Dumenil G. Deveaud C. Miura M. Coppola D. DeAngelis T. Rubin R. Efstratiadis A. Baserga R. Mol. Cell. Biol. 1994; 14: 3604-3612Crossref PubMed Scopus (498) Google Scholar, 18O'Connor R. Kauffmann-Zeh A. Liu Y. Lehar S. Evan G.I. Baserga R. Blattler W.A. Mol. Cell. Biol. 1997; 17: 427-435Crossref PubMed Scopus (241) Google Scholar, 19Baserga R. Exp. Cell Res. 1997; 236: 1-3Crossref PubMed Scopus (39) Google Scholar, 20Morrione A. Romano G. Navarro M. Reiss K. Valentinis B. Dews M. Eves E. Rosner M.R. Baserga R. Cancer Res. 2000; 60: 2263-2272PubMed Google Scholar). Elevated levels of IGF-IR have been observed in human tumors of breast (21Turner B.C. Haffty B.G. Narayanan L. Yuan J. Havre P.A. Gumbs A.A. Kaplan L. Burgaud J.L. Carter D. Baserga R. Glazer P.M. Cancer Res. 1997; 57: 3079-3083PubMed Google Scholar), brain (22Merrill M.J. Edwards N.A. J. Clin. Endocrinol. Metab. 1990; 70: 199-209Crossref Scopus (59) Google Scholar), and lung and colon (23LeRoith D. Baserga R. Helman L. Roberts Jr., C.T. Ann. Intern. Med. 1995; 122: 54-59Crossref PubMed Scopus (296) Google Scholar) and, when observed, are associated with a poor prognosis (21Turner B.C. Haffty B.G. Narayanan L. Yuan J. Havre P.A. Gumbs A.A. Kaplan L. Burgaud J.L. Carter D. Baserga R. Glazer P.M. Cancer Res. 1997; 57: 3079-3083PubMed Google Scholar). As IGF-IR was found to possess the ability to induce radioresistance (21Turner B.C. Haffty B.G. Narayanan L. Yuan J. Havre P.A. Gumbs A.A. Kaplan L. Burgaud J.L. Carter D. Baserga R. Glazer P.M. Cancer Res. 1997; 57: 3079-3083PubMed Google Scholar,24Nakamura S. Watanabe H. Miura M. Sasaki T. Exp. Cell Res. 1997; 235: 287-294Crossref PubMed Scopus (43) Google Scholar, 25Tezuka M. Watanabe H. Nakamura S. Yu D. Aung W. Sasaki T. Shibuya H. Miura M. Clin. Cancer Res. 2001; 7: 3206-3214PubMed Google Scholar, 26Wen B. Deutsch E. Marangoni E. Frascona V. Maggiorella L. Abdulkarim B. Chavaudra N. Bourhis J. Br. J. Cancer. 2001; 85: 2017-2021Crossref PubMed Scopus (89) Google Scholar), directed study of this receptor is likely to shed light on the downstream pathways leading to this phenomenon. Comprehensive study from such a viewpoint has not been previously conducted, except that antisense targeting of IGF-IR reduces the activity of ataxia telangiectasia-mutated (ATM), a necessary factor for proper double strand break repair, resulting in enhanced radiosensitivity (27Macaulay V.M. Salisbury A.J. Bohula E.A. Playford M.P. Smorodinsky N.I. Shiloh Y. Oncogene. 2001; 20: 4029-4040Crossref PubMed Scopus (115) Google Scholar). Direct connection, however, between the IGF-IR pathway and ATM has not been established to date. Two major pathways are thought to originate from IGF-IR, one through insulin receptor substrate-1 (IRS-1), which activates the PI3-K/Akt pathway, and the other through Shc, which activates the Ras/Raf/MEK/ERK pathway (28Peruzzi F. Prisco M. Dews M. Salomoni P. Grassilli E. Romano G. Calabretta B. Baserga R. Mol. Cell. Biol. 1999; 19: 7203-7215Crossref PubMed Scopus (420) Google Scholar). These two substrates bind to the NPXY950 motif in the juxtamembrane domain, and Tyr-950 plays an important role in binding as revealed by a yeast two-hybrid assay (29Tartare-Deckert S. Sawka-Verhelle D. Murdaca J. Van Obberghen E. J. Biol. Chem. 1995; 270: 23456-23460Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 30Craparo A. O'Neill T.J. Gustafson T.A. J. Biol. Chem. 1995; 270: 15639-15643Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The Raf/MEK/ERK pathway is also activated through 14-3-3 proteins, which bind to the C terminus of IGF-IR, a site not available on the IR (31Dews M. Prisco M. Peruzzi F. Romano G. Morrione A. Baserga R. Endocrinology. 2000; 141: 1289-1300Crossref PubMed Scopus (50) Google Scholar, 32Furlanetto R.W. Dey B.R. Lopaczynski W. Nissley S.P. Biochem. J. 1997; 327: 765-771Crossref PubMed Scopus (77) Google Scholar, 33Craparo A. Freund R. Gustafson T.A. J. Biol. Chem. 1997; 272: 11663-11669Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). In addition to these main pathways, activation of c-Raf kinase by 14-3-3 proteins bound to the IGF-IR also results in its translocation to the mitochondria, where it exerts a survival effect with Nedd4 (28Peruzzi F. Prisco M. Dews M. Salomoni P. Grassilli E. Romano G. Calabretta B. Baserga R. Mol. Cell. Biol. 1999; 19: 7203-7215Crossref PubMed Scopus (420) Google Scholar, 34Peruzzi F. Prisco M. Morrione A. Valentinis B. Baserga R. J. Biol. Chem. 2001; 276: 25990-25996Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). How these downstream pathways of IGF-IR influence radioresistance is not known. In this study, we sought to determine the contributions of the different downstream pathways of IGF-IR to IGF-IR-mediated radioresistance. For this purpose, we used a series of mutant IGF-IRs, potentially relevant to PI3-K and MEK/ERK activation, expressed in R− cells deficient in endogenous IGF-IR (17Sell C. Dumenil G. Deveaud C. Miura M. Coppola D. DeAngelis T. Rubin R. Efstratiadis A. Baserga R. Mol. Cell. Biol. 1994; 14: 3604-3612Crossref PubMed Scopus (498) Google Scholar). Radiosensitivity was further analyzed in combination with specific inhibitors of PI3-K and MEK. Here, we show that IGF-IR mediates clonogenic radioresistance through a number of redundant survival signals of differently weighted relevance, including PI3-K, MEK/ERK, and signals stemming from the C terminus domain, presumably through 14-3-3 proteins. Wortmannin, LY294002, and PD98059 were purchased from Sigma. Antibodies against IGF-IR α- and β-subunits, ERK2, goat IgG conjugated with horseradish peroxidase (HRP), rabbit IgG-HRP, and Protein A/G PLUS-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibody (PY20) was purchased from Transduction Laboratory (Lexington, KY) and anti-phosphokinase B/AKT phosphoserine 473 antibody was purchased fromBIOSOURCE (Camarillo, CA). Anti-ACTIVE™ MAPK antibody was purchased from Promega (Madison, WI), and anti-β-actin antibody from Chemicon International (Temecula, CA). Anti-hemagglutinin (HA) antibody was purchased from Roche (Mannheim, Germany). The ECL Western blotting analysis system,125I-IGF-I, and [γ-32P]ATP were purchased from Amersham Biosciences. Recombinant human IGF-I was purchased from Invitrogen. Phosphatidylinositol was purchased from Avanti (Alabaster, AL). pEGFP-C1 vector was purchased from Clontech (Palo Alto, CA). A plasmid containing constitutively active MEK was kindly provided by Dr. E. Nishida (Kyoto University, Kyoto, Japan). Wild-type, Y950F, Y1316F, and Δ1245 receptor mutants were derived from human IGF-IR cDNA (15Ullrich A. Gray A. Tam A.W. Yang-Feng T. Tsubokawa M. Collins C. Henzel W. le Bon T. Kahuria S. Chen E. Jakobs S. Francke U. Ramachandran J. Fujita-Yamaguchi Y. EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1486) Google Scholar) as described previously (35Miura M. Li S. Baserga R. (1995) Cancer Res. 1995; 55: 663-667PubMed Google Scholar, 36Hongo H. D'Ambrosio C. Miura M. Morrione A. Baserga R. Oncogene. 1996; 12: 1231-1238PubMed Google Scholar). For Y950F/1316F, theHindIII-BamHI fragment of pBluescript SK Y950F (35Miura M. Li S. Baserga R. (1995) Cancer Res. 1995; 55: 663-667PubMed Google Scholar) was replaced by the HindIII-BamHI fragment of pBluescript SK Y1316F (36Hongo H. D'Ambrosio C. Miura M. Morrione A. Baserga R. Oncogene. 1996; 12: 1231-1238PubMed Google Scholar) and was designated pBluescript SK Y950F/Y1316F. The XhoI-NotI fragment of pBPV IGF-IR (17Sell C. Dumenil G. Deveaud C. Miura M. Coppola D. DeAngelis T. Rubin R. Efstratiadis A. Baserga R. Mol. Cell. Biol. 1994; 14: 3604-3612Crossref PubMed Scopus (498) Google Scholar), an expression plasmid for the wild-type (WT) receptor, was then replaced with the XhoI-NotI fragment of pBluescript SK Y950F/Y1316F, including the double mutation. For the Y950F/Δ1245 mutant, the HindIII-BamHI fragment of pBluescript SK Y950F was replaced by the corresponding fragment from pBluescript SK Δ1245 (36Hongo H. D'Ambrosio C. Miura M. Morrione A. Baserga R. Oncogene. 1996; 12: 1231-1238PubMed Google Scholar). Then, the XhoI-NotI fragment of pBPV IGF-IR was replaced by theXhoI-NotI fragment of the Y950F/Δ1245 cDNA in pBluescript SK. For the construction of IGF-IR truncated at residue 950, the XhoI-BamHI fragment of pBPV IGF-IR was transferred into the vector pEGFP-C1. TheScaI-BamHI fragment of the IGF-IR cDNA in this vector was replaced by the following double-stranded oligodeoxynucleotides: 5′-ACTGAGAATTCG and 3′-TGACTCTTAAGCCTAG. The oligodeoxynucleotides were designed to terminate translation at residue 950 followed by the stop codon TGA, and they contained aBamHI restriction overhang for ligation to theBamHI site of the IGF-IR cDNA in pEGFP-C1 and anEcoRI restriction site for confirmation. TheXhoI-BamHI fragment of pBluescript SK IGF-IR was replaced by the corresponding fragment of truncated IGF-IR cDNA in vector pEGFP-C1. The XhoI-NotI fragment of pBPV IGF-IR was then replaced with the corresponding fragment containing Δ950 in pBluescript SK. R− cells were obtained from mouse embryo fibroblasts possessing a null mutation of the IGF-IR gene (17Sell C. Dumenil G. Deveaud C. Miura M. Coppola D. DeAngelis T. Rubin R. Efstratiadis A. Baserga R. Mol. Cell. Biol. 1994; 14: 3604-3612Crossref PubMed Scopus (498) Google Scholar). Plasmids containing WT or mutant IGF-IR cDNAs were stably transfected into R− cells with a pPDV6+ plasmid carrying the puromycin resistance gene (37de la Luna S. Soria I. Pulido D. Ortin J. Jimenez A. Gene (Amst.). 1988; 62: 121-126Crossref PubMed Scopus (135) Google Scholar) by calcium phosphate precipitation. Cells were selected in 4 μg/ml puromycin, and the resultant clones were mixed and sorted as described previously (38Miura 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). Mixed populations or clones were used in the present study. For transient expression of constitutively active MEK in R− cells, a plasmid containing LA-SDSE MAPK kinase cDNA (39Fukuda M. Gotoh I. Adachi M. Gotoh Y. Nishida E. J. Biol. Chem. 1997; 272: 32642-32648Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) was transfected into R− cells, and the cells were prepared for Western blotting and a colony-forming assay 48 h after transfection. Mock-transfected R− cells were treated similarly as a control. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2 in Eagle's minimal essential medium containing 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin supplemented with 10% (v/v) fetal bovine serum. Exponentially growing cells were used for all experiments. Radiosensitivity was determined by colony-forming assay as described previously (25Tezuka M. Watanabe H. Nakamura S. Yu D. Aung W. Sasaki T. Shibuya H. Miura M. Clin. Cancer Res. 2001; 7: 3206-3214PubMed Google Scholar). To assess the effect of exogenously added IGF-I or inhibitors of PI3-K or MEK, cells in plastic flasks grown for roughly 10 h were treated with inhibitors for 1 h and then γ-irradiated. Cells were transferred to a 37 °C incubator and rendered to form colonies. Surviving fraction was calculated based on the plating efficiency determined from the IGF-I- or inhibitor-treated cells. Cell survival was corrected using the equation S = 1 − (1 −f)1/N, where S is the single cell survival rate, f is the measured surviving fraction, and N is the multiplicity determined by the average number of cells per microcolony at the time of irradiation. Multiplicity ranged from 1.1 to 1.2 for all cell lines under the described conditions. Cells were digested in a lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 0.1% SDS, 1 mm EDTA, 100 mm NaF, 1 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin). Equal amounts of cell lysates were separated in SDS-polyacrylamide gel (PAGE), and proteins were transferred to a nitrocellulose membrane in a Tris-glycine buffer containing 20% methanol. The membrane was blocked in 5% nonfat milk in TBST (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.1% Tween 20). Filters were probed with primary antibodies against target proteins for 1 h at room temperature or overnight at 4 °C. Filters were washed three times in TBST, incubated with secondary antibodies conjugated with HRP in TBST for 1 h at room temperature, and then washed three times in TBST. Proteins were visualized using the ECL system. For the detection of activated proteins, cells were incubated in serum-free medium containing 1 μg/ml bovine serum albumin overnight. Serum-starved cells either treated or untreated with indicated concentrations of IGF-I for 10 min were processed as described above. The number of IGF-I binding sites was determined in each cell line as described previously (38Miura 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). Cells grown on six-well cell plates were washed with Hanks' balanced salt solution and incubated for 4 h at 4 °C in binding buffer (Eagle's minimal essential medium plus 25 mm Hepes, pH 7.4, and 1 mg/ml bovine serum albumin) containing 0.5 ng/ml 125I-IGF-I and/or increasing concentrations of unlabeled IGF-I. After washing with cold Hanks' balanced salt solution, cells were lysed with 0.03% SDS, and cell-associated radioactivities were measured by an autowell γ-counter. Specific binding was expressed by subtracting nonspecific binding as determined in the presence of excess unlabeled IGF-I (200 ng/ml). Relative number of specific binding sites in cells incubated in buffer containing 0.5 ng/ml 125I-IGF-I alone was determined in each cell line with values of WT 11 cells normalized to 1.0. Receptor number per cell and dissociation constants (K d) were also estimated in some cell lines by Scatchard analysis as described previously (38Miura 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). Activity of PI3-K was measured as described previously (40Kobayashi M. Nagata S. Iwasaki T. Yanagihara K. Saitoh I. Karouji Y. Ihara S. Fukui Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4874-4879Crossref PubMed Scopus (63) Google Scholar). Briefly, IGF-I-treated or untreated cells were lysed in a buffer containing 20 mmTris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, and 1% Nonidet P-40, and phosphotyrosine-containing proteins were immunoprecipitated with anti-phosphotyrosine antibody (PY20) bound to Protein A/G-agarose. PI3-K activity in the immunoprecipitates was measured in a reaction mixture containing phosphatidylinositol and [γ-32P]ATP. After 10–40 min, the reaction was stopped by the addition of a solution (chloroform:methanol:HCl = 2:1:0.1) and analyzed by thin layer chromatography. γ-Irradiation was performed using a60Co γ-ray therapeutic machine, RCR-120 (Toshiba, Tokyo, Japan), at a dose rate of 1.4–1.6 Gy/min. Statistical comparison of mean values was performed using the Student's t test or one-way analysis of variance followed by Fisher's protected least significant difference. Differences with a p value of <0.05 were considered statistically significant. We previously reported that introduction of IGF-IR into R− cells, which are deficient in endogenous IGF-IR, confers clonogenic radioresistance (25Tezuka M. Watanabe H. Nakamura S. Yu D. Aung W. Sasaki T. Shibuya H. Miura M. Clin. Cancer Res. 2001; 7: 3206-3214PubMed Google Scholar). To confirm this, we newly established several clones and their radiosensitivities were determined by colony-forming assay. Of these, mixed populations (WTmix), clones 9 and 11, which expressed similar levels of IGF-IR, exhibited a significant radioresistance (to a similar extent) compared with R− or R−(puro) cells expressing a marker gene alone (Fig.1, A and B). Because the extent of radioresistance was relatively modest, we attempted to examine whether radioresistance was increased when cells were stimulated with exogenously added IGF-I, although growth medium already contained IGF-I in serum. Addition of 10, 20, and 50 ng/ml IGF-I in growth medium 1 h before irradiation, however, did not confer any further significant increase in survival fractions at 6 Gy in WTmix and WT 11 cells (Fig. 1 C). One may argue that the structure of IGF-IR per se, irrespective of its signaling function, could somehow affect the radiosensitivity of R− cells. We therefore tested R− cells expressing Δ950 IGF-IR, which lack most of the β-subunit including the tyrosine kinase domain. The expression levels of Δ950 clones 5 and 7 are shown in Fig. 1 D. An antibody specific for the α-subunit of IGF-IR detected levels of receptor similar as or even higher than clone WT 9. The proreceptor could rarely be distinguished because of overlapping with the α-subunit, and the observed size of β-subunit was small because of the large deletion. When an antibody specific for the C terminus of the β-subunit was used, only WT receptors were detected. Both Δ950 clones displayed the same radiosensitivity as R− or R−(puro) cells (Fig. 1 E), demonstrating that the IGF-IR-mediated clonogenic radioresistance is attributable to signal transduction via the tyrosine kinase of the receptor. Furthermore, considering that IGF-IR kinase activation was not clearly detectable in the growth medium following a 6-Gy irradiation (data not shown), a very low level of receptor activation should be sufficient to saturate clonogenic radioresistance in WT cells, because no further radioresistance was obtained by exogenously added IGF-I (Fig. 1 C). We next attempted to determine which domains of the IGF-IR β-subunit are necessary for clonogenic radioresistance. For this purpose, we made various mutant receptors with specific mutations potentially relevant to activation of downstream pathways. For clarity, the mutant receptors used in this study are shown in Fig. 2. The tyrosine residue at position 950 is part of the NPXY950motif, a major binding site for IRS-1 and Shc, which is conserved in IR (41White M.F. Livingston J.N. Backer J.M. Lauris V. Dull T.J. Ullrich A. Kahn C.R. Cell. 1988; 54: 641-649Abstract Full Text PDF PubMed Scopus (275) Google Scholar) and interleukin 4 receptor (42Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (282) Google Scholar). IRS-1 activates PI3-K, which phosphorylates phosphatidylinositol (PtdIns) phosphates, converting PtdIns 4,5-P2 to PtdIns 3,4,5-P3. This lipid activates phosphoinositide-dependent kinases 1 and 2, which in turn activate Akt (43Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 8684-8691Abstract Full Text Full Text PDF Scopus (301) Google Scholar). Shc strongly activates the Ras/Raf/MEK/ERK pathway (44Sasaoka T. Draznin B. Leitner J.W. Langlois W.J. Olefsky J.M. J. Biol. Chem. 1994; 269: 10734-107348Abstract Full Text PDF PubMed Google Scholar, 45Sasaoka T. Rose D.W. Jhun B.H. Saltiel A.R. Draznin B. Olefsky J.M. J. Biol. Chem. 1994; 269: 13689-13694Abstract Full Text PDF PubMed Google Scholar). Tyrosine 1316 is a constituent of the Y1316 XXM motif, a binding site for the regulatory subunit p85 of PI3-K, and is able to stimulate its activity (46Seely B.L. Reichart D.R. Staubs P.A. Jhun B.H. Hsu D. Maegawa H. Milarski K.L. Saltiel A.R. Olefsky J.M. J. Biol. Chem. 1995; 270: 19151-19157Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This is also conserved in IR (47van Horn D.J. Myers Jr., M.G. Backer J.M. J. Biol. Chem. 1994; 269: 29-32Abstract Full Text PDF PubMed Google Scholar). Because each tyrosine, Tyr-950 and Tyr-1316, is reported to play a critical role in each binding function (29Tartare-Deckert S. Sawka-Verhelle D. Murdaca J. Van Obberghen E. J. Biol. Chem. 1995; 270: 23456-23460Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 30Craparo A. O'Neill T.J. Gustafson T.A. J. Biol. Chem. 1995; 270: 15639-15643Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 46Seely B.L. Reichart D.R. Staubs P.A. Jhun B.H. Hsu D. Maegawa H. Milarski K.L. Saltiel A.R. Olefsky J.M. J. Biol. Chem. 1995; 270: 19151-19157Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), these residues were mutated to phenylalanines to attenuate the relevant pathways. The C terminus of IGF-IR includes a quartet of serine residues 1280–1283, which is a binding site for 14-3-3 proteins (33Craparo A. Freund R. Gustafson T.A. J. Biol. Chem. 1997; 272: 11663-11669Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) that in turn lead to activation of c-Raf and the MAPK pathway. c-Raf undergoes mitochondrial translocation and exerts a survival effect in cooperation with Nedd4 (34Peruzzi F. Prisco M. Morrione A. Valentinis B. Baserga R. J. Biol. Chem. 2001; 276: 25990-25996Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). We will refer to this pathway as 14-3-3/c-Raf hereafter to differentiate this c-Raf signaling event as separate from its activation of the MAPK pathway. To eliminate the binding site of the 14-3-3 proteins, the receptor was truncated at residue 1245. Double mutation at Tyr-950 and the C terminus was also introduced to exclude both signals. Mutant receptors were expressed in R− cells, and their expression levels were assayed by Western blotting using antibodies specific for the IGF-IR α- or β-subunit (Fig.3 A). Clones expressing levels of receptor almost similar to those of WT 9 or 11 were selected. To assess the number of mature cell-surface receptors,125I-IGF-I-binding assay was also done (Fig.3 B). Although there were some variations in the number of IGF-I binding sites among cell lines, all the mutants possessed levels of binding sites at least more than WT 11 cells. Specific binding was undetectable in R− cells. As an example, 125I-IGF-I binding competition in WT 11 and Y950F/Δ1245 clone 3 cells is shown in Fig. 3 C, exhibiting similar displacement properties (IC50 = ∼1 nm). Scatchard analysis revealed that receptor number per cell and dissociation constant (K d) in" @default.
• W1978393771 created "2016-06-24" @default.
• W1978393771 creator A5002808615 @default.
• W1978393771 creator A5067396850 @default.
• W1978393771 creator A5071346122 @default.
• W1978393771 creator A5089552764 @default.
• W1978393771 date "2003-02-01" @default.
• W1978393771 modified "2023-10-16" @default.
• W1978393771 title "Redundancy of Radioresistant Signaling Pathways Originating from Insulin-like Growth Factor I Receptor" @default.
• W1978393771 cites W1490666791 @default.
• W1978393771 cites W1523684384 @default.
• W1978393771 cites W1559597555 @default.
• W1978393771 cites W1564546569 @default.
• W1978393771 cites W1609778130 @default.
• W1978393771 cites W1931843124 @default.
• W1978393771 cites W1967434204 @default.
• W1978393771 cites W1969882621 @default.
• W1978393771 cites W1971850308 @default.
• W1978393771 cites W1974241409 @default.
• W1978393771 cites W1975566386 @default.
• W1978393771 cites W1980303501 @default.
• W1978393771 cites W1982725837 @default.
• W1978393771 cites W1999012962 @default.
• W1978393771 cites W2000074832 @default.
• W1978393771 cites W2002052645 @default.
• W1978393771 cites W2004327412 @default.
• W1978393771 cites W2005532023 @default.
• W1978393771 cites W2007217993 @default.
• W1978393771 cites W2011259448 @default.
• W1978393771 cites W2011447447 @default.
• W1978393771 cites W2012940389 @default.
• W1978393771 cites W2014564257 @default.
• W1978393771 cites W2015707895 @default.
• W1978393771 cites W2016711993 @default.
• W1978393771 cites W2023676204 @default.
• W1978393771 cites W2033707974 @default.
• W1978393771 cites W2044217649 @default.
• W1978393771 cites W2054891257 @default.
• W1978393771 cites W2067162187 @default.
• W1978393771 cites W2070075260 @default.
• W1978393771 cites W2071387709 @default.
• W1978393771 cites W2082130460 @default.
• W1978393771 cites W2091130756 @default.
• W1978393771 cites W2116189789 @default.
• W1978393771 cites W2124222608 @default.
• W1978393771 cites W2139381527 @default.
• W1978393771 cites W2140884727 @default.
• W1978393771 cites W2142279958 @default.
• W1978393771 cites W2143786004 @default.
• W1978393771 cites W2169083148 @default.
• W1978393771 cites W2173747422 @default.
• W1978393771 cites W2314177115 @default.
• W1978393771 cites W7405620 @default.
• W1978393771 doi "https://doi.org/10.1074/jbc.m209809200" @default.
• W1978393771 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12493743" @default.
• W1978393771 hasPublicationYear "2003" @default.
• W1978393771 type Work @default.
• W1978393771 sameAs 1978393771 @default.
• W1978393771 citedByCount "59" @default.
• W1978393771 countsByYear W19783937712012 @default.
• W1978393771 countsByYear W19783937712013 @default.
• W1978393771 countsByYear W19783937712014 @default.
• W1978393771 countsByYear W19783937712015 @default.
• W1978393771 countsByYear W19783937712016 @default.
• W1978393771 countsByYear W19783937712017 @default.
• W1978393771 countsByYear W19783937712018 @default.
• W1978393771 countsByYear W19783937712019 @default.
• W1978393771 countsByYear W19783937712020 @default.
• W1978393771 countsByYear W19783937712023 @default.
• W1978393771 crossrefType "journal-article" @default.
• W1978393771 hasAuthorship W1978393771A5002808615 @default.
• W1978393771 hasAuthorship W1978393771A5067396850 @default.
• W1978393771 hasAuthorship W1978393771A5071346122 @default.
• W1978393771 hasAuthorship W1978393771A5089552764 @default.
• W1978393771 hasBestOaLocation W19783937711 @default.
• W1978393771 hasConcept C111919701 @default.
• W1978393771 hasConcept C112446052 @default.
• W1978393771 hasConcept C134018914 @default.
• W1978393771 hasConcept C152124472 @default.
• W1978393771 hasConcept C170493617 @default.
• W1978393771 hasConcept C185592680 @default.
• W1978393771 hasConcept C2775960820 @default.
• W1978393771 hasConcept C2777391703 @default.
• W1978393771 hasConcept C2779306644 @default.
• W1978393771 hasConcept C2780689927 @default.
• W1978393771 hasConcept C41008148 @default.
• W1978393771 hasConcept C502942594 @default.
• W1978393771 hasConcept C54355233 @default.
• W1978393771 hasConcept C55493867 @default.
• W1978393771 hasConcept C58962609 @default.
• W1978393771 hasConcept C62478195 @default.
• W1978393771 hasConcept C70721500 @default.
• W1978393771 hasConcept C81885089 @default.
• W1978393771 hasConcept C86803240 @default.
• W1978393771 hasConcept C95444343 @default.
• W1978393771 hasConceptScore W1978393771C111919701 @default.
• W1978393771 hasConceptScore W1978393771C112446052 @default.
• W1978393771 hasConceptScore W1978393771C134018914 @default.

• next