FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034625230> ?p ?o ?g. }

• W2034625230 endingPage "23532" @default.
• W2034625230 startingPage "23525" @default.
• W2034625230 abstract "Insulin-like growth factor I (IGF-I) plays an important role in cell survival, proliferation, and differentiation. Diverse kinases, including AKT/protein kinase B, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), can be activated by IGF-I. Here, we show that the receptor-interacting protein (RIP), a key mediator of tumor necrosis factor-induced NF-κB and JNK activation, plays a key role in IGF-I receptor signaling. IGF-I induced a robust JNK activation in wild type but not RIP null (RIP–/–) mouse embryonic fibroblast cells. Reconstitution of RIP expression in the RIP–/– cells restored the induction of JNK by IGF-I, suggesting that RIP is essential in IGF-I-induced JNK activation. Reconstitution experiments with different RIP mutants further revealed that the death domain and the kinase activity of RIP are not required for IGF-I-induced JNK activation. Interestingly, the AKT and ERK activation by IGF-I was normal in RIP–/– cells. The phosphatidylinositol 3-kinase inhibitor, wortmannin, did not affect IGF-I-induced JNK activation. These results agree with previous studies showing that the IGF-I-induced JNK activation pathway is distinct from that of ERK and AKT activation. Additionally, physical interaction of ectopically expressed RIP and IGF-IRβ was detected by co-immunoprecipitation assays. More importantly, RIP was recruited to the IGF-I receptor complex during IGF-I-induced signaling. Furthermore, we found that IGF-I-induced cell proliferation was impaired in RIP–/– cells. Taken together, our results indicate that RIP, a key factor in tumor necrosis factor signaling, also plays a pivotal role in IGF-I-induced JNK activation and cell proliferation. Insulin-like growth factor I (IGF-I) plays an important role in cell survival, proliferation, and differentiation. Diverse kinases, including AKT/protein kinase B, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), can be activated by IGF-I. Here, we show that the receptor-interacting protein (RIP), a key mediator of tumor necrosis factor-induced NF-κB and JNK activation, plays a key role in IGF-I receptor signaling. IGF-I induced a robust JNK activation in wild type but not RIP null (RIP–/–) mouse embryonic fibroblast cells. Reconstitution of RIP expression in the RIP–/– cells restored the induction of JNK by IGF-I, suggesting that RIP is essential in IGF-I-induced JNK activation. Reconstitution experiments with different RIP mutants further revealed that the death domain and the kinase activity of RIP are not required for IGF-I-induced JNK activation. Interestingly, the AKT and ERK activation by IGF-I was normal in RIP–/– cells. The phosphatidylinositol 3-kinase inhibitor, wortmannin, did not affect IGF-I-induced JNK activation. These results agree with previous studies showing that the IGF-I-induced JNK activation pathway is distinct from that of ERK and AKT activation. Additionally, physical interaction of ectopically expressed RIP and IGF-IRβ was detected by co-immunoprecipitation assays. More importantly, RIP was recruited to the IGF-I receptor complex during IGF-I-induced signaling. Furthermore, we found that IGF-I-induced cell proliferation was impaired in RIP–/– cells. Taken together, our results indicate that RIP, a key factor in tumor necrosis factor signaling, also plays a pivotal role in IGF-I-induced JNK activation and cell proliferation. As a potent mitogen, insulin-like growth factor I (IGF-I) 3The abbreviations used are: IGF-I, insulin-like growth factor I; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; PI, phosphoinositol; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; RIP, receptor-interacting protein; TNF, tumor necrosis factor; NF-κB, nuclear factor-κB; TLR, toll-like receptor; MEF, mouse embryonic fibroblast; IL, interleukin; HA, hemagglutinin; WT, wild type. plays an important role in cell proliferation, differentiation, and survival (1Grimberg A. Cancer Biol. Ther. 2003; 2: 630-635Crossref PubMed Scopus (161) Google Scholar, 2Benito M. Valverde A.M. Lorenzo M. Int. J. Biochem. Cell Biol. 1996; 28: 499-510Crossref PubMed Scopus (105) Google Scholar, 3Baserga R. Peruzzi F. Reiss K. Int. J. Cancer. 2003; 107: 873-877Crossref PubMed Scopus (540) Google Scholar). Accumulating evidence also suggests that IGF-I contributes to carcinogenesis. Epidemiological studies have shown consistently that elevated circulating IGF-I is associated with increased risk for several common cancers (4Krajcik R.A. Borofsky N.D. Massardo S. Orentreich N. Cancer Epidemiol. Biomarkers Prev. 2002; 11: 1566-1573PubMed Google Scholar, 5Renehan A.G. Zwahlen M. Minder C. O'Dwyer S.T. Shalet S.M. Egger M. Lancet. 2004; 363: 1346-1353Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar, 6Zhang P. Ostrander J.H. Faivre E.J. Olsen A. Fitzsimmons D. Lange C.A. J. Biol. Chem. 2005; 280: 1982-1991Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 7Agarwal A. Das K. Lerner N. Sathe S. Cicek M. Casey G. Sizemore N. Oncogene. 2005; 24: 1021-1031Crossref PubMed Scopus (161) Google Scholar). In many cell and tumor systems, IGF-I is involved in cell transformation and maintenance of the malignant phenotype. The combination of mitogenic and anti-apoptotic properties of IGF-I has a profound impact on tumor growth, rendering the IGF-I system a good target for potential adjunct therapy to standard chemotherapy (8Pollak M.N. Schernhammer E.S. Hankinson S.E. Nat. Rev. Cancer. 2004; 4: 505-518Crossref PubMed Scopus (1191) Google Scholar, 9LeRoith D. Helman L. Cancer Cell. 2004; 5: 201-202Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Yee D. Br. J. Cancer. 2006; 94: 465-468Crossref PubMed Scopus (95) Google Scholar). The anti-apoptosis of IGF-I may involve activation of AKT and the regulation of the ratio of Bcl2 and Bax that leads to blockage of the initiation of the apoptotic pathway (11Linseman D.A. Phelps R.A. Bouchard R.J. Le S.S. Laessig T.A. McClure M.L. Heidenreich K.A. J. Neurosci. 2002; 22: 9287-9297Crossref PubMed Google Scholar, 12Sekharam M. Zhao H. Sun M. Fang Q. Zhang Q. Yuan Z. Dan H.C. Boulware D. Cheng J.Q. Coppola D. Cancer Res. 2003; 63: 7708-7716PubMed Google Scholar). The binding of IGF-I to its cell membrane IGF-I receptor (IGF-IR) is regulated by a group of six specific IGF-I binding proteins (IGFBP1–6), which belong to the IGF family (13Fang P. Hwa V. Rosenfeld R. Novartis Foundation Symposium. 2004; 262 (discussion 230–234, 265–268): 215-230Crossref PubMed Google Scholar, 14Adams T.E. Epa V.C. Garrett T.P. Ward C.W. Cell. Mol. Life Sci. 2000; 57: 1050-1093Crossref PubMed Scopus (486) Google Scholar, 15LeRoith D. Roberts Jr., C.T. Cancer Lett. 2003; 195: 127-137Crossref PubMed Scopus (931) Google Scholar). The IGF-IR is a heterotetramer of two identical α- and β-subunits, IGF-IRα and IGF-IRβ, respectively, that are generated by proteolysis and glycosylation of the αβ precursor encoded by a single gene. Binding of IGF-I to the IGF-IR activates the tyrosine kinase of the receptor, which in turn triggers a cascade of interactions among a number of molecules involved in signal transduction (15LeRoith D. Roberts Jr., C.T. Cancer Lett. 2003; 195: 127-137Crossref PubMed Scopus (931) Google Scholar). Distinct signal transduction pathways have been identified for the IGF-IR, and it appears that there is a considerable overlap in the pathways used for the receptor functions (14Adams T.E. Epa V.C. Garrett T.P. Ward C.W. Cell. Mol. Life Sci. 2000; 57: 1050-1093Crossref PubMed Scopus (486) Google Scholar, 15LeRoith D. Roberts Jr., C.T. Cancer Lett. 2003; 195: 127-137Crossref PubMed Scopus (931) Google Scholar). One pathway activated by the IGF-IR is that through the insulin receptor substrate (IRS)-1 or IRS-2, leading to the activation of phosphoinositol (PI) 3-kinase and AKT, which promote suppression of apoptosis via phosphorylation of downstream factors such as caspase-9, Bad, GSK3-β, and the transcription factors FKHRL1 and CREB (16Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2222) Google Scholar). The next pathway activates the extracellular signal-regulated kinase (ERK) through the Ras/Raf/MAP or Ras/Rsk-1/MAP cascade (14Adams T.E. Epa V.C. Garrett T.P. Ward C.W. Cell. Mol. Life Sci. 2000; 57: 1050-1093Crossref PubMed Scopus (486) Google Scholar, 15LeRoith D. Roberts Jr., C.T. Cancer Lett. 2003; 195: 127-137Crossref PubMed Scopus (931) Google Scholar). Additionally, binding of IGF-I to the IGF-IR also induces the transient activation of c-Jun N-terminal kinase (JNK) (17Monno S. Newman M.V. Cook M. Lowe Jr., W.L. Endocrinology. 2000; 141: 544-550Crossref PubMed Scopus (29) Google Scholar). IGF-I-induced JNK activation could be anti-apoptotic (18Krause D. Lyons A. Fennelly C. O'Connor R. J. Biol. Chem. 2001; 276: 19244-19252Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 19Walsh P.T. Smith L.M. O'Connor R. Immunology. 2002; 107: 461-471Crossref PubMed Scopus (59) Google Scholar). The mechanism of the IGF-IR mediated activation of JNK has not been well elucidated. Because the C terminus of the IGF-IR, IRS-1, and IRS-2, which are required for AKT activation, are unnecessary for JNK activation, it appears that signals that activate JNK mediated by the IGF-IR are distinct to those leading to PI 3-kinase and AKT activation (18Krause D. Lyons A. Fennelly C. O'Connor R. J. Biol. Chem. 2001; 276: 19244-19252Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The death domain kinase receptor-interacting protein (RIP) plays a central role in tumor necrosis factor (TNF)-induced nuclear factor-κB (NF-κB) activation, which contributes significantly to cell survival (20Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar, 21Van Antwerp D.J. Martin S.J. Verma I.M. Green D.R. Trends Cell Biol. 1998; 8: 107-111Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 22Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar). RIP also contributes to TNF-induced JNK, ERK, and p38 MAP kinases activation (23Devin A. Lin Y. Liu Z.G. EMBO Rep. 2003; 4: 623-627Crossref PubMed Scopus (112) Google Scholar, 24Lee T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google Scholar). Recent studies in different cell models have revealed that RIP is also critical in NF-κB activation by other agents, including TNF-related apoptosis-inducing ligand, DNA damaging agents, and double-stranded RNA (25Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Crossref PubMed Scopus (189) Google Scholar, 26Wajant H. Vitam. Horm. 2004; 67: 101-132Crossref PubMed Scopus (59) Google Scholar, 27Hur G.M. Lewis J. Yang Q. Lin Y. Nakano H. Nedospasov S. Liu Z.G. Genes Dev. 2003; 17: 873-882Crossref PubMed Scopus (123) Google Scholar, 28Meylan E. Burns K. Hofmann K. Blancheteau V. Martinon F. Kelliher M. Tschopp J. Nat. Immunol. 2004; 5: 503-507Crossref PubMed Scopus (663) Google Scholar). In addition, RIP is crucial for toll-like receptor (TLR)4- and TLR6-mediated AKT activation (29Vivarelli M.S. McDonald D. Miller M. Cusson N. Kelliher M. Geha R.S. J. Exp. Med. 2004; 200: 399-404Crossref PubMed Scopus (62) Google Scholar). Further, it is reported that RIP interacts with the epidermal growth factor receptor, contributing to epidermal growth factor-induced NF-κB activation (30Habib A.A. Chatterjee S. Park S.K. Ratan R.R. Lefebvre S. Vartanian T. J. Biol. Chem. 2001; 276: 8865-8874Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In this study, we address whether RIP is involved in IGF-IR signaling. With RIP–/– mouse embryonic fibroblast (MEF) cells, we demonstrated that RIP is required for IGF-I-induced JNK activation. The results indicate that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in the IGF-IR-mediated activation of JNK. Reagents and Plasmids—Recombinant human IGF-I and wortmannin were purchased from Calbiochem. TNF and interleukin (IL)-1 were from R&D Systems (Minneapolis, MN). Anti-RIP, c-Myc, and JNK1 antibodies were purchased from BD Pharmingen. Anti-IGF-IRβ, -Xpress, and -HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-AKT, -ERK, -phospho-AKT, and -phospho-ERK antibodies were from Upstate Biotechnology (Chicago, IL). Anti-phospho-c-Jun was purchased from Cell Signaling (Beverly, MA). The mammalian expression plasmids for RIP, RIP-(559–671), RIP (K45A), RIP-(324–671), CrmA, and HA-JNK1 are described previously (23Devin A. Lin Y. Liu Z.G. EMBO Rep. 2003; 4: 623-627Crossref PubMed Scopus (112) Google Scholar, 31Lin Y. Devin A. Rodriguez Y. Liu Z.G. Genes Dev. 1999; 13: 2514-2526Crossref PubMed Scopus (684) Google Scholar). Xpress-tagged IGF-IRβ was constructed by inserting the polymerase chain reaction-amplified fragment from the pBPV IGF-IR, a gift from Dr. D. LeRoith (Mt. Sinai School of Medicine, New York), into the BamHI and EcoRI sites of the pcDNA 3.1 vector. The construct was confirmed by DNA sequencing. Cell Culture and Transfection—MEF and HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were transfected with Lipofectamine Plus (Invitrogen) as described previously (31Lin Y. Devin A. Rodriguez Y. Liu Z.G. Genes Dev. 1999; 13: 2514-2526Crossref PubMed Scopus (684) Google Scholar). Western Blot Analysis and Co-immunoprecipitation—After treatment with different reagents as described in the figure legends, cells were collected and lysed in M2 buffer (20 mm Tris, pH 7, 0.5% Nonidet P-40, 250 mm NaCl, 3 mm EDTA, 3 mm EGTA, 2 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 20 mm β-glycerol phosphate, 1 mm sodium vanadate, 1 μg/ml leupeptin). Fifty μg of the cell lysate from each sample were fractionated by SDS-PAGE and analyzed by Western blot. The proteins were visualized by enhanced chemiluminescence according to the manufacturer's (Amersham Biosciences) instructions (32Lin Y. Choksi S. Shen H.M. Yang Q.F. Hur G.M. Kim Y.S. Tran J.H. Nedospasov S.A. Liu Z.G. J. Biol. Chem. 2004; 279: 10822-10828Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). For immunoprecipitation assays of transfected proteins, HEK293 cells were transiently co-transfected with different plasmids and then were lysed in M2 buffer. The expression of each transfected protein was verified by Western blot analysis. The immunoprecipitation experiments were performed with anti-myc or -Xpress and protein A-Sepharose beads by incubation at 4 °C overnight. The beads were washed five times with M2 buffer, and the bound proteins were resolved in 10% SDS-PAGE. The Xpress-IGF-IR and myc-RIP were detected by Western blot analysis with anti-Xpress and myc, respectively. For immunoprecipitation assays of endogenous proteins, 5 × 107 of wild type (WT) cells were serum-starved for 4 h followed by treatment with IGF-I (100 ng/ml) as indicated in the legend of Fig. 5. The cells were then lysed in M2 buffer and precipitated with 1 μg of the anti-IGF-IRβ antibody as described above. RIP was detected by Western blot. All experiments were repeated at least three times, and representative data are shown. JNK Assay—MEF cells (5 × 105) were serum-starved for 4 h and treated with IGF-I as described in the figure legends. Cells were collected in 300 μl of M2 lysis buffer. JNK1 was immunoprecipitated with JNK1 antibody, and JNK kinase activity was determined by using 1 μg of glutathione-S-transferase-c-Jun-(1–79) as a substrate (25Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Crossref PubMed Scopus (189) Google Scholar). Transfected cells were serum-starved for 4 h and treated with IGF-I 24 h after transfection as described (18Krause D. Lyons A. Fennelly C. O'Connor R. J. Biol. Chem. 2001; 276: 19244-19252Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The cells were collected in 300 μl of M2 lysis buffer. HA-JNK1 was immunoprecipitated with an HA antibody, and their kinase activities were determined. All experiments were repeated at least three times, and representative data are shown. Cell Proliferation Assays—MEF cells were cultured overnight in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The medium was then replaced with serum-free Dulbecco's modified Eagle's medium. After continuing the culture for 4 h, the cells were treated with IGF-I. Cells were collected and counted at 1, 2, or 3 days post-treatment. The experiments were repeated at least three times, and data shown (mean ± S.D.) are representative of three independent experiments. Impaired JNK Activation by IGF-I in RIP–/– Cells—RIP plays a pivotal role in activation of NF-κB, JNK, and p38 induced by diverse stimuli (23Devin A. Lin Y. Liu Z.G. EMBO Rep. 2003; 4: 623-627Crossref PubMed Scopus (112) Google Scholar, 24Lee T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google Scholar, 25Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Crossref PubMed Scopus (189) Google Scholar, 26Wajant H. Vitam. Horm. 2004; 67: 101-132Crossref PubMed Scopus (59) Google Scholar, 27Hur G.M. Lewis J. Yang Q. Lin Y. Nakano H. Nedospasov S. Liu Z.G. Genes Dev. 2003; 17: 873-882Crossref PubMed Scopus (123) Google Scholar, 28Meylan E. Burns K. Hofmann K. Blancheteau V. Martinon F. Kelliher M. Tschopp J. Nat. Immunol. 2004; 5: 503-507Crossref PubMed Scopus (663) Google Scholar, 33Kelliher M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar). Using a BLAST search for RIP homolog(s), we found a significantly high homology between the RIP and the IGF-I receptor β subunit (IGF-IRβ) (data not shown). This prompted us to address whether RIP is involved in IGF-I-induced signaling. Based on the fact that RIP is involved in JNK activation by different stimulations, we explored the possible role of RIP in IGF-I-induced JNK activation with RIP–/– MEF cells as a cell model. JNK activity was measured by an in vitro kinase assay with glutathione-S-transferase-c-Jun (1–79 amino acids) as the substrate. In WT MEF cells IGF-I treatment caused a robust activation of JNK, which started at 10 min, peaked at 20–40 min, and lasted up to 60 min post-treatment (Fig. 1A). However, there was no detectable JNK induction by IGF-I in RIP–/– cells (Fig. 1B, top panel). RIP–/– cells were confirmed by Western blot analysis to be null of RIP and defective to TNF-induced JNK activation by an in vitro kinase assay (Fig. 1B and data not shown). Because comparable expression levels of JNK1 and IGF-IR were detected in both RIP–/– and WT cells (Fig. 1B, second and fourth panels), it is unlikely that the decrease of JNK activation in RIP–/– cells resulted from the altered expression of JNK or the IGF-IR. Deficient JNK activation by IGF-I in RIP–/– cells was further confirmed with an anti-phospho-JNK antibody. In this experiment, JNK activation by IGF-I was detected in a dose-dependent manner in WT cells (Fig. 1C, upper panel). The induction of c-Jun, the direct downstream target of JNK, was also impaired in RIP–/– cells (Fig. 1C, middle panel). These results suggest that RIP is required for transducing the IGF-I-induced signal to activate JNK. Defective JNK activation in RIP–/– cells was specific to IGF-I treatment because RIP–/– cells responded to IL-1 and ultraviolet treatment as efficiently as the WT cells did in terms of JNK activation (Fig. 1D and data not shown). IGF-I-induced Normal AKT and ERK Activation in RIP–/– Cells—Upon activation by IGF-I, the IGF-IR transduces signals to activate ERK and AKT through Ras/Raf and PI 3-kinase, respectively. Activation of AKT or ERK is associated with the phosphorylation of AKT or ERK proteins. Because RIP is also involved in AKT and ERK activation under certain situations (23Devin A. Lin Y. Liu Z.G. EMBO Rep. 2003; 4: 623-627Crossref PubMed Scopus (112) Google Scholar, 24Lee T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google Scholar, 28Meylan E. Burns K. Hofmann K. Blancheteau V. Martinon F. Kelliher M. Tschopp J. Nat. Immunol. 2004; 5: 503-507Crossref PubMed Scopus (663) Google Scholar), we addressed whether RIP is also involved in mediating IGF-I-induced AKT and ERK activation. RIP–/– and WT cells were treated with IGF-I and collected at different time points post-treatment, and the activated forms of AKT and ERK were detected with anti-phospho-AKT and anti-phospho-ERK antibodies, respectively. As shown in Fig. 2, IGF-I-induced AKT phosphorylation was comparable in the RIP–/– and WT cells. A similar result was obtained regarding the IGF-I-induced ERK activation. These results suggested that RIP is unnecessary for AKT and ERK activation pathways. Although RIP is a key factor for TNF-induced NF-κB activation, and IGF-I was found to activate NF-κB activity in some cell types such as multiple myeloma cells (34Mitsiades C.S. Mitsiades N. Poulaki V. Schlossman R. Akiyama M. Chauhan D. Hideshima T. Treon S.P. Munshi N.C. Richardson P.G. Anderson K.C. Oncogene. 2002; 21: 5673-5683Crossref PubMed Scopus (447) Google Scholar), we did not detect NF-κB activation by IGF-I in MEF cells, either by Western blot for IκBα degradation or NF-κB-reporter assay for NF-κB activity (Fig. 2 and data not shown). Whether RIP is involved in IGF-IR-mediated activation of NF-κB remains to be determined in other cell models. Restoration of IGF-I-induced JNK Activation in RIP-reconstituted RIP–/– Cells—To rule out the possibility that some other flaws in the signaling pathway of IGF-I-induced JNK activation are present in RIP–/– cells, we tested whether IGF-I-induced JNK activation could be restored when RIP is reconstituted in those cells. To examine the reconstitution of IGF-I-induced JNK activation, the expression vector for myc-RIP was co-transfected with HA-JNK1 into RIP–/– cells. Following treatment with IGF-I, the transfected HA-JNK1 was immunoprecipitated for in vitro JNK activity assay. As shown in Fig. 3, RIP expression restored JNK activation in response to IGF-I treatment in RIP–/– cells (Lane 4). This result indicated that defective IGF-I-induced JNK activation in RIP–/– cells is caused by the absence of RIP. IGF-I-induced JNK Activation Does Not Require the Death Domain and Kinase Activity of RIP—RIP possesses three functional domains: kinase domain (1–300 amino acids), intermediate domain (300–582 amino acids), and death domain (583–653 amino acids). The death domain is essential in TNF receptor signaling as it is a protein-protein interacting motif that is used to recruit RIP to the tumor necrosis factor receptor signaling complex. The intermediate domain is crucial for NF-κB activation. The biological relevance of the kinase domain is not well elucidated, although it may be involved in TNF-induced ERK activation and necrotic cell death (23Devin A. Lin Y. Liu Z.G. EMBO Rep. 2003; 4: 623-627Crossref PubMed Scopus (112) Google Scholar, 35Holler N. Zaru R. Micheau O. Thome M. Attinger A. Valitutti S. Bodmer J.L. Schneider P. Seed B. Tschopp J. Nat. Immunol. 2000; 1: 489-495Crossref PubMed Scopus (1467) Google Scholar). To determine the domain prerequisite for IGF-I-induced JNK activation, the expression vectors for either myc-RIP (K45A), a kinase dead RIP mutant, or myc-RIP-(1–558), a death domain deleted RIP mutant, was co-transfected with HA-JNK1 into RIP–/– cells. WT RIP was used as a positive control. After treatment with IGF-I, HA-JNK1 was immunoprecipitated, and JNK activity was measured by in vitro kinase assay. As shown in Fig. 3, both RIP (K45A) and RIP-(1–558) expression restored the JNK activation in response to IGF-I treatment in RIP–/– cells as efficiently as WT RIP did. The expression levels of RIP (K45A), RIP-(1–558), and RIP WT were comparable (Fig. 3, bottom panel). These results indicated that both the kinase and death domains of RIP are dispensable for IGF-I-induced JNK activation in MEF cells. IGF-I-induced JNK Activation Is Not Mediated by PI 3-Kinase—It was reported that the IGF-I-induced JNK activation pathway is distinct to that of the PI 3-kinase-mediated AKT activation (18Krause D. Lyons A. Fennelly C. O'Connor R. J. Biol. Chem. 2001; 276: 19244-19252Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The results indicating that RIP is unnecessary for IGF-I-induced ERK and AKT activation supported the notion that JNK activation does not involve PI 3-kinase (Fig. 2). To further test this hypothesis, WT cells were pretreated with the PI 3-kinase inhibitor wortmannin followed by IGF-I treatment. The cell extracts were subjected to in vitro JNK assay and Western blot analysis for phospho-AKT. TNF treatment was included as a control. When treated with a moderate concentration of wortmannin (100 nm), which specifically and efficiently suppressed PI 3-kinase-mediated AKT activation by IGF-I, the IGF-I-induced JNK activity was barely affected (Fig. 4A). As expected, pre-incubation of wortmannin did not interfere with TNF-induced JNK activation. (Fig. 4A). This result supported the conclusion that IGF-I-induced JNK activation does not involve the PI 3-kinase pathway. When cells were treated with a high concentration of wortmannin (10 μm), the IGF-I-induced JNK activation was completely blocked, whereas TNF-induced activation was not affected (Fig. 4B). Because high concentrations of wortmannin inhibit other kinases in addition to the PI 3-kinase, the blockage of JNK activation under this condition likely resulted from the inhibition of an unidentified kinase by wortmannin. However, as the TNF-induced JNK activation was not affected under the same condition, the mechanism of IGF-I-induced JNK activation might be different from that of TNF, although both IGF-I-and TNF-induced signaling to JNK activation require RIP. RIP Interacts with the IGF-IR—Because our data suggested that RIP is essential in IGF-I-induced JNK activation, RIP might also interact with the IGF-IR. To test this possibility, we investigated whether RIP interacts with the IGF-IR by ectopic expression of RIP and the IGF-IRβ in HEK293 cells. The Xpress-tagged IGF-IRβ was immunoprecipitated with an anti-Xpress antibody, and the immunoprecipitants were analyzed by Western blot with an anti-myc antibody. As shown in Fig. 5A, myc-tagged RIP was specifically co-precipitated with the Xpress-tagged IGF-IRβ when RIP and the IGF-IRβ were co-expressed. There was no detectable precipitation of RIP when it was expressed alone, although the expression level was similar to that of RIP and IGF-IRβ co-expression (Fig. 5A). Conversely, the IGF-IRβ was specifically co-immunoprecipitated with myc-RIP when the two proteins were co-expressed (Fig. 5B). These results suggested a specific binding between RIP and the IGF-IRβ. With ectopic expression and co-immunoprecipitation, we mapped the IGF-IRβ binding region in RIP. Whereas the death domain (RIP-(559–671)) was unable to bind IGF-IRβ, RIP-(324–671) as well as RIP-(1–558) retained the binding activity (Fig. 5C). Therefore, the minimal IGF-IRβ binding region is mapped to be between 324–558 amino acids, locating in the intermediate domain of RIP. It is noteworthy that the binding of RIP-(324–671) and RIP-(1–558) to IGF-IRβ was greatly reduced compared with that of WT RIP. It is possible that the sequence flanking this minimal binding region may help to stabilize the association between RIP and IGF-IRβ. Nevertheless, these results are consistent with the RIP reconstitution experiment, showing that the death domain and kinase activity of RIP are unnecessary for IFG-I-induced JNK activation (Fig. 3). RIP Is Part of the IGF-IR Signaling Complex—To test whether endogenous RIP is recruited to the IGF-IR complex during IGF-I signaling, we performed co-immunoprecipitation experiments with cell extracts derived from WT cells with or without IGF-I treatment. In these experiments, the endogenous IGF-IRβ was immunoprecipitated with an anti-IGF-IRβ antibody, and the immunoprecipitants were analyzed with an anti-RIP antibody. As shown in Fig. 5D, whereas no detectable RIP was co-precipitated by an anti-IGF-IRβ antibody from the nontreated cell extract, RIP was present in the IGF-IR complex that was immunoprecipitated from the cell extract with IGF-I treatment for 10 min. (Lane 5). Under the same condition, RIP was not precipitated using the anti-JNK1 antibody, suggesting the association of RIP and IGF-IRβ induced by IGF-I is specific (data not shown). These results suggested that IGF-I induces the recruitment of RIP to the IGF-IR. Interestingly, only a trace amount of RIP was detected after 20 min of treatment (Lane 6), suggesting that RIP was released rapidly from the IGF-IR signaling complex after being recruited there. This release may enable a downstream signaling that activates JNK, which is not recruited to IGF-IR (Fig. 5D). RIP Is Required for IGF-I-induced Cell Proliferation—It was suggested that JNK contributes to IGF-I-induced anti-apoptosis (18Krause D. Lyons A. Fennelly C. O'Connor R. J. Biol. Chem. 2001; 276: 19244-19252Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, IGF-I had" @default.
• W2034625230 created "2016-06-24" @default.
• W2034625230 creator A5005424988 @default.
• W2034625230 creator A5049798132 @default.
• W2034625230 creator A5064985084 @default.
• W2034625230 creator A5068691968 @default.
• W2034625230 date "2006-08-01" @default.
• W2034625230 modified "2023-10-17" @default.
• W2034625230 title "The Essential Role of the Death Domain Kinase Receptor-interacting Protein in Insulin Growth Factor-I-induced c-Jun N-terminal Kinase Activation" @default.
• W2034625230 cites W135294775 @default.
• W2034625230 cites W1511528799 @default.
• W2034625230 cites W1523845694 @default.
• W2034625230 cites W1572909157 @default.
• W2034625230 cites W1965391576 @default.
• W2034625230 cites W1966565183 @default.
• W2034625230 cites W1968860872 @default.
• W2034625230 cites W1981207182 @default.
• W2034625230 cites W1984098660 @default.
• W2034625230 cites W1985101923 @default.
• W2034625230 cites W1985871447 @default.
• W2034625230 cites W1986978728 @default.
• W2034625230 cites W1989839815 @default.
• W2034625230 cites W1990239056 @default.
• W2034625230 cites W1990430867 @default.
• W2034625230 cites W1993772406 @default.
• W2034625230 cites W2004205588 @default.
• W2034625230 cites W2007279797 @default.
• W2034625230 cites W2010296481 @default.
• W2034625230 cites W2012197678 @default.
• W2034625230 cites W2024969918 @default.
• W2034625230 cites W2028062775 @default.
• W2034625230 cites W2039478887 @default.
• W2034625230 cites W2048121649 @default.
• W2034625230 cites W2051026238 @default.
• W2034625230 cites W2057418848 @default.
• W2034625230 cites W2060060231 @default.
• W2034625230 cites W2065257477 @default.
• W2034625230 cites W2066211653 @default.
• W2034625230 cites W2078960019 @default.
• W2034625230 cites W2087735238 @default.
• W2034625230 cites W2097800689 @default.
• W2034625230 cites W2098386233 @default.
• W2034625230 cites W2102683312 @default.
• W2034625230 cites W2104473825 @default.
• W2034625230 cites W2107411016 @default.
• W2034625230 cites W2111843196 @default.
• W2034625230 cites W2114727688 @default.
• W2034625230 cites W2122814014 @default.
• W2034625230 cites W2124202816 @default.
• W2034625230 cites W2133984207 @default.
• W2034625230 cites W2147286656 @default.
• W2034625230 cites W2157384083 @default.
• W2034625230 cites W2162824132 @default.
• W2034625230 cites W2170236911 @default.
• W2034625230 cites W2315255667 @default.
• W2034625230 cites W3140002906 @default.
• W2034625230 cites W4212962560 @default.
• W2034625230 doi "https://doi.org/10.1074/jbc.m601487200" @default.
• W2034625230 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16793775" @default.
• W2034625230 hasPublicationYear "2006" @default.
• W2034625230 type Work @default.
• W2034625230 sameAs 2034625230 @default.
• W2034625230 citedByCount "20" @default.
• W2034625230 countsByYear W20346252302012 @default.
• W2034625230 countsByYear W20346252302014 @default.
• W2034625230 countsByYear W20346252302020 @default.
• W2034625230 crossrefType "journal-article" @default.
• W2034625230 hasAuthorship W2034625230A5005424988 @default.
• W2034625230 hasAuthorship W2034625230A5049798132 @default.
• W2034625230 hasAuthorship W2034625230A5064985084 @default.
• W2034625230 hasAuthorship W2034625230A5068691968 @default.
• W2034625230 hasBestOaLocation W20346252301 @default.
• W2034625230 hasConcept C104317684 @default.
• W2034625230 hasConcept C112446052 @default.
• W2034625230 hasConcept C134018914 @default.
• W2034625230 hasConcept C143065580 @default.
• W2034625230 hasConcept C184235292 @default.
• W2034625230 hasConcept C185592680 @default.
• W2034625230 hasConcept C2777391703 @default.
• W2034625230 hasConcept C2779087070 @default.
• W2034625230 hasConcept C2779306644 @default.
• W2034625230 hasConcept C2779664074 @default.
• W2034625230 hasConcept C32619005 @default.
• W2034625230 hasConcept C41008148 @default.
• W2034625230 hasConcept C55493867 @default.
• W2034625230 hasConcept C76155785 @default.
• W2034625230 hasConcept C86339819 @default.
• W2034625230 hasConcept C86803240 @default.
• W2034625230 hasConcept C90934575 @default.
• W2034625230 hasConcept C95444343 @default.
• W2034625230 hasConcept C97029542 @default.
• W2034625230 hasConceptScore W2034625230C104317684 @default.
• W2034625230 hasConceptScore W2034625230C112446052 @default.
• W2034625230 hasConceptScore W2034625230C134018914 @default.
• W2034625230 hasConceptScore W2034625230C143065580 @default.
• W2034625230 hasConceptScore W2034625230C184235292 @default.
• W2034625230 hasConceptScore W2034625230C185592680 @default.
• W2034625230 hasConceptScore W2034625230C2777391703 @default.

• next