Inositol 1,3,4-Trisphosphate 5/6-Kinase Inhibits Tumor Necrosis Factor-induced Apoptosis
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m300674200
ISSN1083-351X
AutoresYoung Joo Sun, Yasuhiro Mochizuki, Philip W. Majerus,
Tópico(s)interferon and immune responses
ResumoTumor necrosis factor receptor 1 (TNF-R1) signaling elicits a wide range of biological responses, including inflammation, proliferation, differentiation, and apoptosis. TNF-R1 activates both caspase-mediated apoptosis and NF-κB transcription of anti-apoptotic factors. We now report a link between the TNF-R1 and inositol phosphate signaling pathways. We observed that overexpression of inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) inhibited apoptosis induced by TNFα. The anti-apoptotic effect by 5/6-kinase is not attributable to NF-κB activation, as no changes were detected in the levels of NF-κB DNA binding, IκBα degradation, or anti-apoptotic factors, such as x-linked inhibitor of apoptosis protein. Decreased expression of 5/6-kinase by RNA interference rendered HeLa cells more susceptible to TNFα-induced apoptosis. Overexpression of 5/6-kinase in human embryonic kidney 293 cells inhibited TNFα-induced activation of caspases-8, -3, and -9, BID, and poly(ADP-ribose) polymerase. However, 5/6-kinase did not protect against Fas-, etoposide-, or cycloheximide-induced apoptosis. Further, 5/6-kinase protected against apoptosis induced by the overexpression of TNF-R1-associated death domain but not Fas-associated death domain. Therefore, we suggest that 5/6-kinase modifies TNFα-induced apoptosis by interfering with the activation of TNF-R1-associated death domain. Tumor necrosis factor receptor 1 (TNF-R1) signaling elicits a wide range of biological responses, including inflammation, proliferation, differentiation, and apoptosis. TNF-R1 activates both caspase-mediated apoptosis and NF-κB transcription of anti-apoptotic factors. We now report a link between the TNF-R1 and inositol phosphate signaling pathways. We observed that overexpression of inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) inhibited apoptosis induced by TNFα. The anti-apoptotic effect by 5/6-kinase is not attributable to NF-κB activation, as no changes were detected in the levels of NF-κB DNA binding, IκBα degradation, or anti-apoptotic factors, such as x-linked inhibitor of apoptosis protein. Decreased expression of 5/6-kinase by RNA interference rendered HeLa cells more susceptible to TNFα-induced apoptosis. Overexpression of 5/6-kinase in human embryonic kidney 293 cells inhibited TNFα-induced activation of caspases-8, -3, and -9, BID, and poly(ADP-ribose) polymerase. However, 5/6-kinase did not protect against Fas-, etoposide-, or cycloheximide-induced apoptosis. Further, 5/6-kinase protected against apoptosis induced by the overexpression of TNF-R1-associated death domain but not Fas-associated death domain. Therefore, we suggest that 5/6-kinase modifies TNFα-induced apoptosis by interfering with the activation of TNF-R1-associated death domain. Inositol phosphate signaling is involved in many different cellular processes, ranging from intracellular calcium regulation to mRNA export from the nucleus (for review see Refs. 1Majerus P.W. Genes Dev. 1996; 10: 1051-1053Crossref PubMed Scopus (50) Google Scholar, 2Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (528) Google Scholar, 3Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (342) Google Scholar). In mammalian cells, activation of phospholipase C generates inositol 1,4,5-trisphosphate, which then becomes phosphorylated and dephosphorylated by a series of inositol phosphate kinases and phosphatases to generate inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate (3Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (342) Google Scholar, 4Verbsky J.W. Wilson M.P. Kisseleva M.V. Majerus P.W. Wente S.R. J. Biol. Chem. 2002; 277: 31857-31862Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 5Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 6Chang S.C. Miller A.L. Feng Y. Wente S.R. Majerus P.W. J. Biol. Chem. 2002; 277: 43836-43843Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) functions at the branch point in this pathway (7Hansen C.A. vom Dahl S. Huddell B. Williamson J.R. FEBS Lett. 1988; 236: 53-56Crossref PubMed Scopus (16) Google Scholar, 8Yang X. Shears S.B. Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (59) Google Scholar). Initially purified from calf brain, 5/6-kinase was identified by its inositol phosphate kinase activity. Using inositol 1,3,4-trisphosphate as a substrate, 5/6-kinase generates two different products, inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate. The latter product is then further phosphorylated to produce inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate (4Verbsky J.W. Wilson M.P. Kisseleva M.V. Majerus P.W. Wente S.R. J. Biol. Chem. 2002; 277: 31857-31862Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 5Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 6Chang S.C. Miller A.L. Feng Y. Wente S.R. Majerus P.W. J. Biol. Chem. 2002; 277: 43836-43843Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In addition, 5/6-kinase has been reported to phosphorylate inositol 3,4,5,6-tetrakisphosphate to inositol 1,3,4,5,6-pentakisphosphate (8Yang X. Shears S.B. Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (59) Google Scholar). Recently, we have reported that 5/6-kinase also exhibits protein kinase activity and associates with the COP9 signalosome complex (9Wilson M.P. Sun Y. Cao L. Majerus P.W. J. Biol. Chem. 2001; 276: 40998-41004Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Sun Y. Wilson M.P. Majerus P.W. J. Biol. Chem. 2002; 277: 45759-45764Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The COP9 signalosome has been implicated in regulation of the cell cycle, proteolysis in the proteasome, and plant development (11Schwechheimer C. Serino G. Callis J. Crosby W.L. Lyapina S. Deshaies R.J. Gray W.M. Estelle M. Deng X.W. Science. 2001; 292: 1379-1382Crossref PubMed Scopus (396) Google Scholar, 12Lyapina S. Cope G. Shevchenko A. Serino G. Tsuge T. Zhou C. Wolf D.A. Wei N. Shevchenko A. Deshaies R.J. Science. 2001; 292: 1382-1385Crossref PubMed Scopus (554) Google Scholar, 13Tomoda K. Kubota Y. Arata Y. Mori S. Maeda M. Tanaka T. Yoshida M. Yoneda-Kato N. Kato J.Y. J. Biol. Chem. 2002; 277: 2302-2310Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). We have shown that 5/6-kinase associates with the CSN1 subunit of the COP9 signalosome and phosphorylates IκBα and c-Jun (9Wilson M.P. Sun Y. Cao L. Majerus P.W. J. Biol. Chem. 2001; 276: 40998-41004Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Sun Y. Wilson M.P. Majerus P.W. J. Biol. Chem. 2002; 277: 45759-45764Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). IκBα phosphorylation is an important regulatory mechanism for TNFα 1The abbreviations used are: TNFα, tumor necrosis factor α; DISC, death inducing signaling complex; FADD, Fas-associated death domain; TNF-R1, TNF receptor 1; TRADD, TNF-R1-associated death domain; PARP, poly(ADP-ribose) polymerase; NF-κB, nuclear factor-κB; IKK, IκB kinase; XIAP, x-linked inhibitor of apoptosis protein; CHX, cycloheximide; EMSA, electrophoretic mobility shift assay; WB, Western blot; HEK, human embryonic kidney; MOPS, 4-morpholinepropanesulfonic acid; RNAi, RNA interference; SODD, silencer of death domain.1The abbreviations used are: TNFα, tumor necrosis factor α; DISC, death inducing signaling complex; FADD, Fas-associated death domain; TNF-R1, TNF receptor 1; TRADD, TNF-R1-associated death domain; PARP, poly(ADP-ribose) polymerase; NF-κB, nuclear factor-κB; IKK, IκB kinase; XIAP, x-linked inhibitor of apoptosis protein; CHX, cycloheximide; EMSA, electrophoretic mobility shift assay; WB, Western blot; HEK, human embryonic kidney; MOPS, 4-morpholinepropanesulfonic acid; RNAi, RNA interference; SODD, silencer of death domain.-induced NF-κB activation. To elucidate the physiological consequence of 5/6-kinase-mediated IκBα phosphorylation, we examined TNFα signaling in vivo.TNFα, a member of a large class of cytokines, elicits a wide spectrum of cellular responses including differentiation, proliferation, inflammation, and cell death. TNFα signaling simultaneously triggers apoptotic and anti-apoptotic pathways (for review see Ref. 14Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2433) Google Scholar). Although caspase activation results in apoptosis, NF-κB mediates transcription of anti-apoptotic factors that can block the caspase cascade; integration of these events determines the cellular response to TNFα stimulation.Upon TNFα binding to TNF-R1, interaction of the receptor with the TNF receptor-associated death domain (TRADD), a central adaptor protein for TNF-R1, leads to the formation of the death-inducing signaling complex (DISC) (15Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1767) Google Scholar). TNFα stimulates both the mitochondrial-dependent (intrinsic) and -independent (extrinsic) apoptotic pathways. In response to extracellular stimuli, the extrinsic pathway of TNFα signaling induces the TRADD-dependent recruitment of Fas-associated death domain (FADD), which recruits and activates procaspase-8 leading to activation of the caspase cascade (16Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2148) Google Scholar). Caspase-8 also stimulates the intrinsic pathway through the cleavage and activation of BID, resulting in cytochrome c release from mitochondria (17Kuida K. Haydar T.F. Kuan C.Y. Gu Y. Taya C. Karasuyama H. Su M.S. Rakic P. Flavell R.A. Cell. 1998; 94: 325-337Abstract Full Text Full Text PDF PubMed Scopus (1446) Google Scholar, 18Yoshida H. Kong Y.Y. Yoshida R. Elia A.J. Hakem A. Hakem R. Penninger J.M. Mak T.W. Cell. 1998; 94: 739-750Abstract Full Text Full Text PDF PubMed Scopus (992) Google Scholar).Binding of TRADD to TNF-R1 is also crucial for anti-apoptotic signaling events. Receptor-interacting protein and TNF receptor-associated factor-2 are recruited to the receptor, and this complex activates the IκB-kinase (IKK) signalosome, which phosphorylates IκBα (for review see Refs. 19Rothwarf D.M. Karin M. Science's STKE. 1999; (http://stke.sciencemag.org/cgi/content/sull/oc_sigtrans;1999/5/re1)PubMed Google Scholar and 20Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3272) Google Scholar). In resting cells, IκBα inhibits NF-κB by preventing translocation of NF-κB subunits into the nucleus. However, upon TNFα stimulation, phosphorylation of IκBα by the IKK signalosome, and subsequent ubiquitination, causes its degradation by the 26 S proteasome. As a result, NF-κB translocates to the nucleus and activates the transcription of many anti-apoptotic factors, such as inhibitor of apoptosis protein 1, x-linked inhibitor of apoptosis protein (XIAP), and Bcl family members, which can inhibit the TNFα-induced apoptotic pathway (21Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2562) Google Scholar, 22Tamatani M. Che Y.H. Matsuzaki H. Ogawa S. Okado H. Miyake S. Mizuno T. Tohyama M. J. Biol. Chem. 1999; 274: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 23Zong W.X. Edelstein L.C. Chen C. Bash J. Gelinas C. Genes Dev. 1999; 13: 382-387Crossref PubMed Scopus (638) Google Scholar).We now report a link between the inositol phosphate and TNFα signaling pathways. We show that 5/6-kinase inhibits TNFα-induced apoptosis. In 5/6-kinase-overexpressing HEK 293 cells, activation of PARP, BID, caspases-8, -3, and -9 are inhibited in a time- and dose-dependent manner. Surprisingly, the protective effect of 5/6-kinase is not because of enhanced NF-κB activation. In addition, 5/6-kinase does not protect against Fas-, etoposide-, or cycloheximide-induced cell death. Therefore, we conclude that 5/6-kinase inhibits apoptosis by blocking the TNFα-induced activation of the caspase cascade.MATERIALS AND METHODSReagents—All chemicals and reagents were obtained from Sigma, unless otherwise specified. Antibodies against PARP, BID, XIAP, caspase-3, and caspase-8 (1C12) were obtained from Cell Signaling. According to the manufacturer, purified anti-PARP antibody preferentially recognizes the cleaved form of PARP. Antibodies against IκBα, p50 (NF-κB), receptor-interacting protein, and α-tubulin were purchased from Santa Cruz Biotechnology, Inc. Anti-caspase-9 antibodies and irreversible caspase inhibitor Z-Glu-Val-Asp-fluoromethyl ketone (20 μm) were obtained from Oncogene, and anti-TRADD antibodies were from BD Biosciences. Antibodies against human 5/6-kinase were described previously (9Wilson M.P. Sun Y. Cao L. Majerus P.W. J. Biol. Chem. 2001; 276: 40998-41004Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). TNFα (Calbiochem) and anti-Fas antibody (BD Biosciences) were used to induce apoptosis. Protein G was purchased from Upstate Biotechnology. [3H]Inositol 1,3,4-trisphosphate was prepared from [3H]inositol 1,3,4,5-tetrakisphosphate as described previously (9Wilson M.P. Sun Y. Cao L. Majerus P.W. J. Biol. Chem. 2001; 276: 40998-41004Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar).DNA Constructs—An expressed sequence tag clone encoding human FADD (GenBank™ accession number BE794964) (ATCC), was used as template for amplifying full-length FADD open reading frame. The PCR reaction used primers 5′-GATCTGAATTCCATGGACCCGTTCCTGGTGCTGCTG-3′ and 5′-AGTCATGGATCCGGACGCTTCGGAGGTAGATGCGTCTGAG-3′. The product of the PCR reaction was digested with EcoRI (5-prime) and BamHI (3-prime) and subcloned into p3XFLAG-CMV-14 expression vector (Sigma). Full-length TRADD was obtained by PCR using a template from the human expressed sequence tag clone (GenBank™ accession number BE563501) (ATCC) with primers 5′-GATCTGAATTCCATGGCAGCTGGGCAAAA TGGGCACG-3′ and 5′-AGTCATGGATCCGGCCAGGCCGCCATTGGGATCGGTC AGGCC-3′. The PCR product was digested with EcoRI and BamHI and inserted into p3XFLAG-CMV-14 expression vector (Sigma) to generate TRADD-FLAG. The sequence of each DNA construct was verified by PCR using ABI PRISM Big Dye Terminators v3.0 Cycle Sequencing Kit and analyzed at the Protein and Nucleic Acid Chemistry Laboratory (Washington University, St. Louis, MO).Generation of 5/6-Kinase RNA Interference Cell Lines—HeLa cells (1 × 106 cells) were transfected with a plasmid containing either pSuper vector (a gift from Dr. Reuven Agami) or pSuper 5/6-kinase using LipofectAMINE2000 (Invitrogen) according to the manufacturer's instructions. Three days post-transfection, HeLa cells were trypsinized and placed in puromycin-containing selection medium (Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine, 10% fetal bovine serum (Invitrogen), and 1 μg/ml puromycin). Isolated clones were harvested after 4 weeks and assayed for inositol 1,3,4 trisphosphate 5/6-kinase activity (5Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and for protein expression by Western blot analysis using an antibody generated against a peptide encoding exons 1–3 of 5/6-kinase. The antibody was affinity purified on a peptide affinity column and eluted with 0.1 m glycine, pH 2.5, then neutralized with 1 m Tris, pH 8.0.Cell Lines—293T-Rex cells (Clontech) stably expressing 5/6-kinase or pcDNA4.0 vector-transfected cells were described previously (9Wilson M.P. Sun Y. Cao L. Majerus P.W. J. Biol. Chem. 2001; 276: 40998-41004Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Tetracycline (0.1 μg/ml) in Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine and 10% fetal bovine serum (Invitrogen) was added to cells 48 h prior to treatments.Apoptosis Assays—Trypan blue staining and APOPercentage Apoptosis Assay (Biocolor, Belfast, Northern Ireland) were used to measure the effect of 5/6-kinase overexpression on apoptosis. For APOPercentage assay, HEK 293 cells (2 × 104 cells) stably transfected with expression plasmids encoding either 5/6-kinase or vector alone were plated into 24-well plates. After 48 h of tetracycline induction (0.1 μg/ml), cell death was induced with either TNFα/CHX (1 ng/ml and 0.5 μg/ml, respectively) or CHX (0.5 μg/ml) alone in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Apoptotic cells were analyzed using APOPercentage Apoptosis Assay according to the manufacturer's instructions. For trypan blue staining, HEK 293 cells overexpressing 5/6-kinase or vector control cells were plated in triplicate in 48-well plates, and protein expression was induced with tetracycline for 48 h. After treatment with either TNFα/CHX (1 ng/ml and 0.5 μg/ml, respectively) or CHX (0.5 μg/ml) alone, cells were trypsinized and counted with a hemocytometer. An average of three independent counts for each sample point was performed.Apoptosis Induction and Detection—HEK 293 cells overexpressing either 5/6-kinase or vector control cells were plated in 12-well plates (5 × 105 cells/well) and induced with tetracycline (0.1 μg/ml) for 48 h. Apoptosis was induced by the addition of TNFα/CHX (1 ng/ml and 0.5 μg/ml, respectively), CHX (0.5 μg/ml), anti-Fas antibody/protein G (10 μg/ml and 2.5 mg/ml, respectively), or etoposide (25 μm) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. After incubation for various times, cells were harvested and washed once in phosphate-buffered saline, pH 7.6, and lysed in buffer (20 mm HEPES, pH 7.6, 140 mm NaCl, 1 mm dithiothreitol, 0.1% Nonidet P-40, 10% (v/v) glycerol, 1 μm microcystin (Sigma), and Complete™ protease inhibitor tablets (Roche Applied Science)). Total lysates were prepared by one freeze-thaw cycle on dry ice followed by brief sonication (3 × 10 s). The insoluble fraction was removed by centrifugation at 15,800 × g, and protein in the supernatant was measured with a Bio-Rad assay. Equal amounts of protein were then separated by SDS-PAGE followed by Western blot analysis. Data from multiple experiments were compared by densitometry of Western blots using an Eastman Kodak Co. Image Station 440 CF, and the data were analyzed using Kodak 1D V.3.5.4 (Scientific Imaging System). The averages ± S.D. are compared for statistical significance using a student t test.Northern Blot Analysis—For Northern analysis, total RNA extracts were prepared from HEK 293 cells (1 × 108) using RNeasy Mini kits (Qiagen) according to the manufacturer's instructions. Agarose gel (1% w/v) was prepared with 0.6 m formaldehyde in 1× MOPS buffer (40 mm MOPS, 10 mm sodium acetate, 1 mm EDTA, pH 7.2). Ten μg of total RNA was denatured in loading buffer (18.5% formaldehyde, 50% formamide, 4% Ficoll400, and bromphenol blue in 1× MOPS buffer) at 55 °C for 15 min. Samples were separated by agarose gel electrophoresis at 50 mA for 3.5 h and transferred to GeneScreen Plus™ hybridization transfer membrane (Nylon; DuPont) overnight at room temperature. Upon completion of the transfer, membranes were cross-linked using Stratalinker (auto cross-link setting, 254 nm; Stratagene). Hybridization was performed using ExpressHyb™ hybridization solution (Clontech) according to the manufacturer's instructions.DNA probes for IκBα were generated from pEGFP-IκBα-FH (XhoI/SacI digest) (kindly provided by Dr. Yunfeng Feng) and labeled in the presence of 100 μCi of [α-32P]dCTP (6000 Ci/mmol; ICN Pharmaceuticals) using Rediprime II random prime labeling system (Amersham Biosciences). 5/6-Kinase probes were prepared from pBAKPAK-5/6-kinase (EcoRI/SmaI). Probes for NF-κB2 and RelA were kindly provided by Arthur Young. β-Actin probes (Clontech) were used as a loading control.Electrophoretic Mobility Shift Assay (EMSA)—NF-κB/c-Rel binding oligonucleotides (NF-κB-GS1, TTTAGTTGAGGGGACTTTCCCAGGC; NF-κB-GS2, TTTGCCTGGGAAAGTCCCCTCAACT) were annealed overnight, purified by polyacrylamide gel (12%) electrophoresis, and labeled with 10 μCi of [32P]dATP (ICN Pharmaceuticals) in the presence of 10 units of Klenow DNA polymerase (Invitrogen) according to the manufacturer's instructions.To prepare nuclear extracts, cells were washed once in phosphate-buffered saline, pH 7.6, and lysed in hypotonic buffer (20 mm HEPES, pH 7.6, 5 mm MgCl2, 15 mm KCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and Complete™ protease inhibitor (Roche Applied Science)). Cells were broken by 20 passages through a 23-gauge needle, and the nuclear fraction was obtained by centrifugation at 800 × g for 5 min. To ensure complete lysis of cells, nuclear fractions were washed again in hypotonic buffer and examined by light microscopy to confirm lysis. Nuclear extracts were prepared in high salt extraction buffer (20 mm HEPES, pH 7.6, 500 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10 mm β-glycerolphosphate, 2 mm MgCl2, 10 mm KCl, 1 mm sodium orthovanadate, 1 mm sodium fluoride, 1 mm dithiothreitol, 0.1% Nonidet P-40, and Complete™ protease inhibitor (Roche Applied Science)) and passed through QiaThredders (Qiagen) at 12,000 rpm for 5 min. Protein concentrations were determined by Bio-Rad assay. For EMSA, 4–8 μg of nuclear protein was incubated in the presence of 0.2 ng of NF-κB probe (10,000 cpm), 0.3 μg of bovine serum albumin, 1 μg of poly(dI-dC), and 1 μg of salmon sperm DNA in binding buffer (25 mm Tris-HCl, pH 8.0, 5% glycerol, 5 mm MgCl2, 10 mm KCl, 1 mm dithiothreitol, and 1 mmEDTA) for 30 min at room temperature. Samples were applied to polyacrylamide gel (6%) in TGE buffer (50 mm Tris, pH 8.5, 400 mmglycine, and 2 mm EDTA) and electrophoresed at 8 mA for 2 h, dried, and subjected to autoradiography.RESULTS5/6-Kinase Overexpression Protects against TNFα-induced Apoptosis—Previously, we reported that 5/6-kinase phosphorylated IκBα in vitro (10Sun Y. Wilson M.P. Majerus P.W. J. Biol. Chem. 2002; 277: 45759-45764Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). IκBα is a key regulator of the NF-κB response to TNFα stimulation (for review see Ref. 19Rothwarf D.M. Karin M. Science's STKE. 1999; (http://stke.sciencemag.org/cgi/content/sull/oc_sigtrans;1999/5/re1)PubMed Google Scholar); therefore we examined the effect of 5/6-kinase overexpression on TNFα signaling. TNFα can induce activation of NF-κB and transcription of target genes that feed back to protect against TNFα-induced cell death. However, if the NF-κB pathway is blocked, cells become susceptible to TNFα-induced apoptosis. Overexpression of 5/6-kinase protected cells against TNFα-induced cell death, as shown by APOPercentage staining (Fig. 1A). Treatment with TNFα alone (without CHX) did not increase cell death in either 5/6-kinase-overexpressing or vector control cells (data not shown). The anti-apoptotic effect of 5/6-kinase was further analyzed using trypan blue staining of cells treated with either TNFα/CHX or CHX alone. Although 80% of vector-transfected cells died within 20 h of TNFα/CHX stimulation, cells expressing 5/6-kinase exhibited 50% cell death. Treatment by CHX alone caused only 20% cell death in either vector or 5/6-kinase-expressing cells (Fig. 1B). Similar results were observed in HeLa cells transiently transfected with an expression plasmid containing 5/6-kinase (data not shown). These results indicate that overexpression of 5/6-kinase inhibits TNFα-induced apoptosis.5/6-kinase Does Not Enhance NF-κB Activation as a Mechanism for Protection—Signaling triggered by TNF-R1 can also cause activation of NF-κB signaling that leads to the expression of anti-apoptotic genes. To determine whether 5/6-kinase mediates its anti-apoptotic effect(s) by inhibiting the TNFα-induced apoptotic pathway and/or by promoting the TNFα-induced anti-apoptotic pathway, the stability of IκBα was measured after TNFα stimulation. Both vector control cells and 5/6-kinase-expressing cells showed IκBα degradation within 10 min (Fig. 2A). Although IκBα stability in response to TNFα signaling is not increased with the overexpression of 5/6-kinase, this does not directly show whether NF-κB activity is modulated in response to 5/6-kinase and TNFα. To determine whether 5/6-kinase directly affects NF-κB activity, the DNA binding activity of NF-κB was measured by EMSA as described under "Materials and Methods." Treatment with TNFα for 30 min resulted in similar DNA binding activity of NF-κB in both vector control cells and 5/6-kinase-expressing cells (Fig. 2B). Furthermore, no significant differences were detected upon supershifting with an anti-p50 (NF-κB) antibody in 5/6-kinase or vector-expressing cells. Therefore, the ability of NF-κB to bind DNA does not seem to be increased by 5/6-kinase in response to TNFα.Fig. 2Effects of 5/6-kinase overexpression on NF-κB activation. A, HEK 293 cells stably transfected with expression plasmids of vector or 5/6-kinase were treated with TNFα (1 ng/ml) for the indicated times. Western blot analysis was performed using anti-IκBα antibody. B, HEK 293 cells overexpressing 5/6-kinase or vector control cells were stimulated with TNFα (1 ng/ml) for 30 min. Nuclear extracts were prepared, and EMSA was performed as described under "Materials and Methods." C, HEK 293 cells overexpressing 5/6-kinase or vector control cells were treated with TNFα for 6 h, and total mRNA extracts were prepared. Northern blot analysis was performed using 32P-labeled probes for IκBα, NF-κB2, RelA, 5/6-kinase, and actin. D, HEK 293 cells overexpressing 5/6-kinase or vector control cells were stimulated with TNFα/CHX (1 ng/ml and 0.5 μg/ml, respectively) for the indicated times. Total lysate (20 μg) was applied to SDS-PAGE, and Western blot analysis was performed using antibodies against XIAP. The results are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that TNFα-induced NF-κB activity is not affected by 5/6-kinase, the expression of NF-κB anti-apoptotic target genes were measured in mRNA extracts from 5/6-kinase- or vector-transfected cells treated with TNFα for 6 h. As shown in Fig. 2C, in the absence of TNFα, mRNA levels of IκBα and NF-κB2 are low, and RelA is undetectable. After TNFα treatment, IκBα, RelA, and NF-κB2 mRNA levels increase significantly and to similar levels in both vector control cells and 5/6-kinase-overexpressing cells. The mRNA levels of β-actin and 5/6-kinase are shown as controls. Activation of NF-κB increases the synthesis of many anti-apoptotic proteins, such as inhibitor of apoptosis protein 1 and XIAP (21Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2562) Google Scholar). However, the levels of XIAP are unchanged in cells expressing 5/6-kinase treated with TNFα/CHX (Fig. 2D). The mRNA levels of other NF-κB targets genes including TNF receptor-associated factor-2 and TNF receptor-associated factor-6 and Bcl family members, such as Bax, Bcl-2, and Bim were also not changed by the overexpression of 5/6-kinase (data not shown). Consistent with the lack of NF-κB up-regulation in response to 5/6-kinase overexpression, we have observed that 5/6-kinase continues to exert a protective effect against TNFα-induced apoptosis in the presence of inhibitors of NF-κB pathway, such as MG132, BAF (28Deshmukh M. Vasilakos J. Deckwerth T.L. Lampe P.A. Shivers B.D. Johnson Jr., E.M. J. Cell Biol. 1996; 135: 1341-1354Crossref PubMed Scopus (205) Google Scholar), and BAY 11–7082 (data not shown). Therefore, these results argue that 5/6-kinase protects against TNFα-induced apoptosis through inhibition of the protease pathway and not through enhanced NF-κB activation and expression of anti-apoptotic genes.5/6-Kinase RNA Interference Renders HeLa Cells More Susceptible to TNFα-induced Cell Death—To further confirm the protective effect(s) of 5/6-kinase against TNFα-induced apoptosis, endogenous levels of 5/6-kinase were reduced using RNA interference (RNAi). HeLa cells stably transfected with an RNA interference plasmid for 5/6-kinase (5/6-kinase RNAi) or a control vector (pSuper) were treated with TNFα/CHX. Two stable cell lines (KD1 and KD2) were isolated that exhibit marked decreases in 5/6-kinase expression as shown by Western blot analysis (Fig. 3A). The activity of 5/6-kinase was reduced by 90–95% in these cell lines (Fig. 3B). To determine the effect of decreased 5/6-kinase expression on TNFα-induced apoptosis, 5/6-kinase RNAi cells (KD1) and control cells were treated with TNFα/CHX. As shown in Fig. 3C, the control cells exhibited proteolysis of apoptotic substrate PARP within 4 h of TNFα treatment but not of BID. In comparison, 5/6-kinase RNAi cells demonstrated decreased levels of full-length BID and increased levels of cleaved PARP in response to TNFα. Similar results were obtained using the KD2 cell line. We also showed that RNAi of 5/6-kinase increased apoptosis as measured by APOPrecentage stain and trypan blue exclusion as shown (Fig. 3, D and E). These results support the hypothesis that 5/6-kinase affects TNFα-induced cell death as the 5/6-kinase RNAi cells are more susceptible to TNFα-induced apoptosis.Fig. 3Effects of 5/6-kinase RNA interference on TNFα-induced apoptosis. A, HeLa RNAi cells from two clones of either vector (pSuper) or 5/6-kinase were analyzed by Western blotting analysis for 5/6-kinase expression with β
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