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• W2155952015 startingPage "7996" @default.
• W2155952015 abstract "Tumor necrosis factor-α (TNF) receptor-associated factor 2 (TRAF2) is one of the major mediators of TNF receptor superfamily transducing TNF signaling to various functional targets, including activation of NF-κB, JNK, and antiapoptosis. We investigated how TRAF2 mediates differentially the distinct downstream signals. We now report a novel mechanism of TRAF2-mediated signal transduction revealed by an association of TRAF2 with sphingosine kinase (SphK), a lipid kinase that is responsible for the production of sphingosine 1-phosphate. We identified a TRAF2-binding motif of SphK that mediated the interaction between TRAF2 and SphK resulting in the activation of the enzyme, which in turn is required for TRAF2-mediated activation of NF-κB but not JNK. In addition, by using a kinase inactive dominant-negative SphK and a mutant SphK that lacks TRAF2-binding motif we show that the interaction of TRAF2 with SphK and subsequent activation of SphK are critical for prevention of apoptosis during TNF stimulation. These findings show a role for SphK in the signal transduction by TRAF2 specifically leading to activation of NF-κB and antiapoptosis. Tumor necrosis factor-α (TNF) receptor-associated factor 2 (TRAF2) is one of the major mediators of TNF receptor superfamily transducing TNF signaling to various functional targets, including activation of NF-κB, JNK, and antiapoptosis. We investigated how TRAF2 mediates differentially the distinct downstream signals. We now report a novel mechanism of TRAF2-mediated signal transduction revealed by an association of TRAF2 with sphingosine kinase (SphK), a lipid kinase that is responsible for the production of sphingosine 1-phosphate. We identified a TRAF2-binding motif of SphK that mediated the interaction between TRAF2 and SphK resulting in the activation of the enzyme, which in turn is required for TRAF2-mediated activation of NF-κB but not JNK. In addition, by using a kinase inactive dominant-negative SphK and a mutant SphK that lacks TRAF2-binding motif we show that the interaction of TRAF2 with SphK and subsequent activation of SphK are critical for prevention of apoptosis during TNF stimulation. These findings show a role for SphK in the signal transduction by TRAF2 specifically leading to activation of NF-κB and antiapoptosis. Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-α signaling.Journal of Biological ChemistryVol. 286Issue 49PreviewVOLUME 277 (2002) PAGES 7996–8003 Full-Text PDF Open AccessSphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-α signaling.Journal of Biological ChemistryVol. 286Issue 11PreviewVOLUME 277 (2002) PAGES 7996–8003 Full-Text PDF Open Access tumor necrosis factor-α TNF receptor-associated factor sphingosine kinase sphingosine 1-phosphate glutathioneS-transferase IκBα kinase NF-κB-inducing kinase mitogen-activated protein kinase human umbilical vein cells hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide c-Jun N-terminal kinase TNF receptor-associated death domain-containing protein Fas-associated death domain-containing protein receptor-interacting protein Tumor necrosis factor-α (TNF)1 is a pleiotropic cytokine that elicits a wide spectrum of physiologic and pathogenic events such as cell activation, proliferation, cell death, and inflammation. The different cellular responses to TNF are signaled through cell surface receptors (p55, TNFR1 and p75, TNFR2), and their adaptor proteins, initiating different signaling pathways. These distinct signals can lead to opposing cellular effects as best exemplified by TNF's proapoptotic and antiapoptotic role (1Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar). TNF-induced apoptosis primarily depends on the recruitment of a complex of adaptor proteins, including TRADD and FADD/MORT1 leading to the further recruitment and activation of various caspases and, subsequently, to programmed cell death (2Tartaglia L.A. Ayres T.M. Wong G.H. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1169) Google Scholar, 3Fesik S.W. Cell. 2000; 103: 273-282Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). On the other hand, the cell activation, inflammatory reaction, and antiapoptotic function of the TNF receptor superfamily are predominantly mediated by another class of adaptor proteins, TNF receptor-associated factors (TRAF) (1Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar,4Arch R.H. Gedrich R.W. Thompson C.B. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (517) Google Scholar, 5Wajant H. Henkler F. Scheurich P. Cell Signal. 2001; 13: 389-400Crossref PubMed Scopus (310) Google Scholar). To date, six members of TRAF proteins have been identified in mammals from TRAF1 to TRAF6. TRAF2 is the prototypical member of TRAF family. It can interact directly or indirectly with various members of TNF receptor superfamily to mediate the signal transduction of these receptors. TRAF2 can also interact with numerous intracellular proteins, such as I-TRAF/TANK, RIP, MAPK kinase kinase, NIK, and the caspase inhibitors cIAPs, and thereby transduces signals required for the activation of the transcription factor NF-κB, the stress-activated protein kinase (SAPK or JNK) and antiapoptosis (6Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (507) Google Scholar, 7Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1056) Google Scholar, 8Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2580) Google Scholar, 9Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar). While structural studies have revealed the complexity and flexibility of TRAF2 (10Park Y.C. Ye H. Hsia C. Segal D. Rich R.L. Liou H.C. Myszka D.G. Wu H. Cell. 2000; 101: 777-787Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) as a signal junction to transduce various signal pathways, it is still not clear how TRAF2 can differentially activate its distinct downstream signals such as NF-κB and JNK, leading to different biological functions. Sphingolipids have recently emerged as signaling molecules that mediate various activities of TNF (11Adam-Klages S. Schwandner R. Adam D. Kreder D. Bernardo K. Kronke M. J. Leukoc. Biol. 1998; 63: 678-682Crossref PubMed Scopus (67) Google Scholar, 12Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (441) Google Scholar). TNF signaling via sphingolipids is exemplified by two distinct pathways: the formation of ceramide resulting from the activation of sphingomyelinase or de novo synthesis and the production of sphingosine 1-phosphate (S1P) upon sphingosine kinase (SphK) activation. While ceramide has been variably implicated as a mediator of TNF-induced apoptosis (13Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1497) Google Scholar), S1P has been emerged as an antiapoptotic and mitogenic factor (14Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (816) Google Scholar, 15Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1352) Google Scholar, 16Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A. Wattenberg B.W. D'andrea R.J. Vadas M.A. Curr. Biol. 2000; 10: 1527-1530Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). We have recently reported that TNF activated SphK independently of its activation of sphingomyelinase activity and that the resulting production of S1P is a potent antagonist of TNF-induced apoptosis (17Xia P. Wang L. Gamble J.R. Vadas M.A. J. Biol. Chem. 1999; 274: 34499-34505Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Thus we investigated whether SphK could mediate a subset of TRAF2 signaling in response to TNF stimulation. We further demonstrated a physical and functional interaction between TRAF2 and SphK that specifically transduces TNF signal to activation of NF-κB and antiapoptosis. HEK 293T were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (Invitrogen), supplemented with 10% fatal calf serum. Human umbilical vein cells (HUVEC) were isolated and maintained as described previously (18Xia P. Vadas M.A. Rye K.A. Barter P.J. Gamble J.R. J. Biol. Chem. 1999; 274: 33143-33147Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Human SphK1 (SphK) cDNA (GenBankTM accession number AF200328) was FLAG epitope-tagged at the 3′ end and subcloned into pcDNA3 vector (Invitrogen) as described previously (19Pitson S.M. D'andrea R.J. Vandeleur L. Moretti P.A. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (166) Google Scholar). For generation of SphK mutants, the FLAG-tagged SphK was cloned into pALTER (Promega) site-directed mutagenic vector. Single-stranded DNA was prepared and used as a template for oligonucleotide-directed mutagenesis as detailed in the manufacturer's protocol. The mutagenic oligonucleotides (5′-TGCCACTGGCGGCGCCAGTGCC-3′ and 5′-CACCGCCAGCGGCGCCCTTAGA-3′) were designed to generate the TB1-SphK and TB2-SphK mutants, repectively, and in combination for TB1/2-SphK. The mutants were sequenced to verify incorporation of the desired modifications and then subcloned into pcDNA3 vector. Generation of SphKG82D was described previously (20Pitson S.M. Moretti P.A. Zebol J.R. Xia P. Gamble J.R. Vadas M.A. D'andrea R.J. Wattenberg B.W. J. Biol. Chem. 2000; 275: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Expression plasmids of pRK5-TRAF2-FLAG and pRK5-TRAF287–501-FLAG were gifts from Dr. V. Dixit (Genentech Inc., South San Francisco). LipofectAMINE 2000 (Invitrogen) was used for transient transfections according to the manufacturer's protocols. Transfected 293T cells from each 10-cm dish were lysed in 1 ml of lysis buffer (50 mm HEPES, 150 mm NaCl, 5 mm EDTA, 0.1% Nonidet P-40/Triton X-100, 20 mm NaF, 1 mm sodium orthovanadate, 10 μg/ml leupeptin and aprotinin). The lysates equalized with the same amount of proteins were immunoprecipitated with anti-FLAG, anti-HA, or control mouse IgG1 monoclonal antibodies (Sigma) for 2 h at 4 °C, respectively. The immune complexes were precipitated by incubation with protein A/G PLUS-agarose beads (Santa Cruz) for another 1 h. The agarose beads were washed twice with 1 ml of lysis buffer, twice with 1 ml of high salt (1 m NaCl) lysis buffer, and twice more with lysis buffer. The immunoprecipitates were separated by 10% SDS-PAGE and transferred to Hybond-P (Amersham Biosciences, Inc.). Subsequent immunoblotting analyses were performed as described elsewhere (17Xia P. Wang L. Gamble J.R. Vadas M.A. J. Biol. Chem. 1999; 274: 34499-34505Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Antibodies against FLAG-epitope (M2, Eastman Kodak Co.), HA-epitope (Sigma), TRAF2, and IκBα (Santa Cruz) were used at a 1:5,000, 1:2,500 and a 1:1,000 dilution, respectively, for immunoblotting assays. The human SphK cDNA was subcloned in-frame into the GST fusion protein expression vector, pGEX-1 (Amersham Biosciences, Inc.). Expression and purification of the derived GST-SphK fusion proteins were performed as described previously (21Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). Cell lysates from each T75 flask of HUVEC or 293T cells overexpressed with TRAF2 or ΔTRAF2 were incubated with 20 μl of a 1:1 slurry of glutathione-Sepharose beads bound to the GST-SphK or GST alone fusion proteins for 2 h at 4 °C. After six extensive washes with lysis buffer, the coprecipitating proteins, along with whole lysates, were analyzed by an immunoblot assay with anti-TRAF2 antibodies. The transfected 293T cells were seeded on a 48-well plate at a density of 2 × 104 cell/well and stimulated with TNF (10 ng/ml) in the presence or absence of cycloheximide (1 μg/ml) for 18 h. Cell viability was assessed by an MTT dye reduction assay and expressed as a proportion of cells maintained in normal culture medium as described previously (17Xia P. Wang L. Gamble J.R. Vadas M.A. J. Biol. Chem. 1999; 274: 34499-34505Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). SphK activity was measured by incubating the cytosolic fraction with 5 μm sphingosine dissolved in 0.1% Triton X-100 and [γ-32P]ATP (1 mm, 0.5 mCi/ml) for 15 min at 37 °C as described previously (18Xia P. Vadas M.A. Rye K.A. Barter P.J. Gamble J.R. J. Biol. Chem. 1999; 274: 33143-33147Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). SphK kinase activity was expressed as picomoles of S1P formed per min per mg of protein. JNK activity was measured by the immune complex kinase assay in anti-HA immunoprecipitates form the cells coexpressed with HA-tagged JNK. The activity of immunoprecipitated complex was determined by incubation with GST-c-Jun(1–79) fusion protein as substrate as described previously (22Xia P. Gamble J.R. Rye K.A. Wang L. Hii C.S. Cockerill P. Khew-Goodall Y. Bert A.G. Barter P.J. Vadas M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14196-14201Crossref PubMed Scopus (358) Google Scholar). 293T cells were cotransfected the desired expression vectors or empty vector. Nuclear extracts were prepared 48 h after transfection followed by TNF stimulation. The double-stranded oligonucleotides used as a probe in these experiments included 5′-GGATGCCATTGGGGATTTCCTCTTTACTGGATGT-3′, which contains a consensus NF-κB binding site in E-selectin promoter that is underlined. Gel mobility shift of a consensus NF-κB oligonucleotide was performed by incubating a 32P-labeled NF-κB probe with 4 μg of nuclear proteins as described previously (22Xia P. Gamble J.R. Rye K.A. Wang L. Hii C.S. Cockerill P. Khew-Goodall Y. Bert A.G. Barter P.J. Vadas M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14196-14201Crossref PubMed Scopus (358) Google Scholar). The specific DNA-protein complexes were completely abolished by addition of a 50-fold molar excess of unlabeled NF-κB oligonucleotides. Stable transfected 293 cells overexpressing SphK, SphKG82D, or empty vector were cotransfected with pRK5-TRAF2 or pRK5 vector together with Ig-κB-luciferase reporter gene plasmid (pTK81-IgK, 200 ng per transfection) andRenilla luciferase control vector (pRL, 20 ng per transfection). Total amounts of transfected DNA were kept constant by supplementing empty vector as needed. Cell extracts were prepared 24 h after transfection, and reporter gene activity was determined by the dual-luciferase assay system (Promega) and normalized relative to Renilla luciferase activity. Our previous findings have suggested that activation of SphK by TNF is required for cell survival and activation during TNF stimulation (17Xia P. Wang L. Gamble J.R. Vadas M.A. J. Biol. Chem. 1999; 274: 34499-34505Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 22Xia P. Gamble J.R. Rye K.A. Wang L. Hii C.S. Cockerill P. Khew-Goodall Y. Bert A.G. Barter P.J. Vadas M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14196-14201Crossref PubMed Scopus (358) Google Scholar). We thus tested whether TNF-induced SphK activation is mediated by TRAF2, which is a well documented transducer for the antiapoptotic signaling (5Wajant H. Henkler F. Scheurich P. Cell Signal. 2001; 13: 389-400Crossref PubMed Scopus (310) Google Scholar). We transiently transfected human embryonic kidney cell line 293T with wild-type TRAF2, a dominant-negative TRAF2 (TRAF287–501, ΔTRAF2), or an empty vector. As shown in Fig.1, overexpression of TRAF2 not only enhanced TNF-induced SphK activity, but was also itself capable of activating SphK by 2-fold compared with control transfectants. Immunoblotting assay showed equivalent expression levels of the transgenes in the presence or absence of TNF stimulation (Fig.1 b). In addition, the TNF-induced SphK activation was blocked by ΔTRAF2 containing a deletion of the N-terminal RING finger that is fundamentally required for TRAF2 mediating downstream signaling and antiapoptosis (6Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (507) Google Scholar, 7Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1056) Google Scholar, 8Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2580) Google Scholar). These data suggested a role of TRAF2 in mediating TNF-induced SphK activation, a novel signaling pathway for cellular response to TNF stimulation. As TRAF2 does not contain intrinsic catalytic activity, protein-protein interactions are essential for TRAF2-mediated activation of downstream signals (5Wajant H. Henkler F. Scheurich P. Cell Signal. 2001; 13: 389-400Crossref PubMed Scopus (310) Google Scholar). We therefore tested the possibility of a physical interaction between TRAF2 and SphK. We initially performed overexpression-based coimmunoprecipitation assays in HEK 293T cell line coexpressed HA-epitope-tagged SphK with FLAG-epitope-tagged TRAF2 or ΔTRAF2. The cell lysates were immunoprecipitated with anti-FLAG monoclonal antibodies, and the coprecipitated HA-tagged SphK was detected by immunoblot assay with anti-HA antibodies. SphK was found to be associated with TRAF2 in the immunoprecipitate complexes from the transfected cells (Fig. 2 a). Conversely, by using anti-HA-epitope antibodies to perform the immunoprecipitation assays, we also found that FLAG-tagged TRAF2 or ΔTRAF2 was coprecipitated with HA-tagged SphK (data not shown). In addition, we examined whether endogenous TRAF2 could also interact with SphK by using GST-SphK fusion protein to pull-down the associated cellular proteins. As shown in Fig. 2 b, GST-SphK fusion protein was capable of interacting with not only the overexpressed TRAF2 in 293T cells, but also the endogenous TRAF2 in HUVEC, confirming a physical interaction of TRAF2 with SphK. The dominant-negative TRAF2 (ΔTRAF2) was also shown to be associated with SphK (Fig. 2), indicating that the N-terminal RING finger of TRAF2 is not required for the interaction. A structure-based sequence alignment of TRAF2 binding sequences in various members of TNF receptor superfamily demonstrated a major consensus motif of (P/S/T/A)X(Q/E)E and a minor motif of PXQXXD (23;24). Analysis of the SphK sequence (human SphK-1) revealed two possible TRAF2-binding motifs in positions 240–243 (PLEE) and 379–382 (PPEE), respectively, providing a potential structural basis for the interaction of SphK and TRAF2. To test whether these two TRAF2-binding motifs are responsible for the binding of SphK to TRAF2, we generated three mutants of SphK, TB1-SphK, TB2-SphK, and TB1/2-SphK, in which the first, second, or both TRAF2-binding motifs were mutated with alanines, i.e.PLEE → PLAA and PPEE → PPAA, respectively (Fig.3 a). We found that expression of either TB2-SphK or TB1/2-SphK (data not shown), but not TB1-SphK, deleted the ability of SphK to coimmunoprecipitated with TRAF2 (Fig.3 b), indicating that the second TRAF2-binding motif is essential for the interaction of these two molecules. The cells enforced expressing TB1-SphK, TB2-SphK, or TB1/2-SphK raised an unstimulated SphK activity to similar levels found with wild-type SphK-transfected cells (Fig.4 a), revealing an undiminished intrinsic enzyme catalytic activity in these SphK mutants. Strikingly, the activity of TB2-SphK, but not TB1-SphK, failed to respond to TNF stimulation (Fig. 4, a and b), suggesting an important role for C-terminal TB2 site of SphK not only in its capacity of interaction with TRAF2, but also in mediating TNF-induced activation of SphK. By contrast, the response of TB-2 SphK to phorbol ester (phorbol 12-myristate 13-acetate), an activator of SphK through protein kinase C activation (15;17), was undiminished (Fig.4 a), suggesting a TNF-specific defect of TB2-SphK. Taken together, these data indicate that SphK interacts with TRAF2 through the binding motif of PPEE379–382 and that this interaction is responsible for mediating TNF-induced SphK activation.Figure 4TB2-SphK and SphKG82D block TNF-induced SphK activation. a, 293T cells were transfected with the indicated expression vectors, and SphK activity was determined after stimulation with TNF (1 ng/ml), phorbol 12-myristate 13-acetate (100 nm), or nil for 10-min post-transfection at 48 h. Data are the mean (±S.D.) of relative activity of three individual experiments. The mean of unstimulated (Nil) levels of SphK activity in the cells transfected with SphK, TB1-SphK, TB2-SphK, and SphKG82D were 42,600, 43,100, 41,800, and 34 pmol/min/mg of protein, respectively. b, SphK activity was assayed in the SphK- or TB2-SphK-transfected 293T cells at the indicated time points of TNF (1 ng/ml) stimulation. Data shown are mean of activity of one representative experiment done in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given the fact that TRAF2 interacted with and subsequently activated SphK and that SphK has been implicated in signaling to regulate cell survival and activation (15Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1352) Google Scholar, 16Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A. Wattenberg B.W. D'andrea R.J. Vadas M.A. Curr. Biol. 2000; 10: 1527-1530Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar), we sought to determine the role of SphK in the TRAF2-transduced signals. In agreement with previous report (7Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1056) Google Scholar), overexpression of TRAF2 was capable of activating NF-κB as determined here by degradation of IκBα (Fig. 5 a) and gel shift assay of NF-κB DNA binding complex (Fig. 5 b). Coexpression of TB2-SphK markedly inhibited IκBα degradation (Fig. 5 a) and decreased NF-κB DNA binding activity (Fig. 5, b andc) induced by either TNF stimulation or overexpression of TRAF2. By contrast, overexpression of wild-type SphK increased NF-κB activity (Fig. 5 b), suggesting a potential effect of SphK on NF-κB activation. To further establish the role of the interaction of SphK with TRAF2 in mediating TNF-induced NF-κB activation, we used a point mutant of SphK (SphKG82D) that reserves intact TRAF2-binding motif but lacks the enzyme catalytic activity (20Pitson S.M. Moretti P.A. Zebol J.R. Xia P. Gamble J.R. Vadas M.A. D'andrea R.J. Wattenberg B.W. J. Biol. Chem. 2000; 275: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). As anticipated, SphKG82D had undiminished binding ability to TRAF2 as determined by coimmunoprecipitation (data not shown) and completely abolished the SphK activity in response to TNF stimulation (Fig. 4 a). Expression of SphKG82D dramatically blocked the degradation of IκBα (Fig. 5 a) and inhibited the NF-κB DNA binding activity in a dose-dependent manner (Fig. 5, b and c). We further performed NF-κB reporter gene assays that confirmed the result from the assays of IκBα degradation and NF-κB DNA binding, showing that overexpression of TRAF2 or SphK increased NF-κB-dependent gene activity, whereas the effect of TNF or TRAF2 on NF-κB activation was blocked by coexpression of SphKG82D (Fig.5 d). Thus, the TRAF2-mediated SphK activation is apparently necessary for TNF-induced NF-κB activation. Since JNK is another well documented major signal pathway mediated by TRAF2 during TNF stimulation (25Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 26Lee S.Y. Reichlin A. Santana A. Sokol K.A. Nussenzweig M.C. Choi Y. Immunity. 1997; 7: 703-713Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), we tested whether the interaction of TRAF2 with SphK could also regulate the TRAF2-dependent JNK activation. Strikingly, neither TNF stimulation nor overexpression of TRAF2-induced JNK activity was affected by expression of TB2-SphK or SphKG82D (Fig.6). In addition, overexpression of wild-type SphK had no significant effect on JNK activation. Hence, in contrast with the effect of SphK on NF-κB, the activation of JNK induced by TNF or TRAF2 is independent of SphK. An essential role of TRAF2 in antiapoptosis has been definitively identified based on the studies with the dominant-negative TRAF2 and deletion of TRAF2 gene in vivo (7Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1056) Google Scholar, 9Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 26Lee S.Y. Reichlin A. Santana A. Sokol K.A. Nussenzweig M.C. Choi Y. Immunity. 1997; 7: 703-713Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 27Yeh W.C. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. de la Pompa J.L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). We further investigated whether the interaction of TRAF2 with SphK is involved in TRAF2-mediated antiapoptotic siganling pathways. Consistent with previous reports (7Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1056) Google Scholar, 26Lee S.Y. Reichlin A. Santana A. Sokol K.A. Nussenzweig M.C. Choi Y. Immunity. 1997; 7: 703-713Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), expression of ΔTRAF2 increased cell sensitivity to killing by TNF (Fig.7), indicating the role of TRAF2 in antiapoptosis. The effect of ΔTRAF2 was completely prevented by overexpression of SphK, even in the presence of an inhibitor of protein synthesis, cycloheximide, suggesting an independent of de novo protein synthesis antiapoptotic pathway promoted by SphK activation (Fig. 7). While overexpression of TRAF2 had a partially protective effect against TNF-induced apoptosis in the presence of cycloheximide, it was substantially enhanced by coexpression with SphK (Fig. 7, right panel). By contrast, the protective effect of TRAF2 against apoptosis was abolished by coexpression of SphKG82D (Fig. 7). Taken together, our findings suggest that SphK activity is essential to determine the antiapoptotic capacity of TRAF2 during TNF stimulation. In this report, we describe an association of TRAF2 with SphK, the first lipid kinase to interact with this signal transducer, which provides a novel mechanism for the specific signaling pathway leading from TRAF2 to the activation of NF-κB and antiapoptosis (Fig.8). We demonstrate the association between TRAF2 and SphK by coimmunoprecipitation assays from the transfected cells and in vitro binding assays, which show that SphK associated with not only the transfected proteins but also endogenous TRAF2. In addition to the physical association, we provide four lines of evidence for a functional role of SphK in TRAF2 mediated TNF signaling: (i) either TNF or overexpression of TRAF2 was capable of activating SphK; (ii) TNF-induced SphK activation was blocked by the dominant-negative TRAF2, ΔTRAF2; (iii) overexpression of SphK potentiated the ability of TRAF2 in activation of NF-kB and antiapoptosis and restored the effect of ΔTRAF2; and (iv) SphK mutants lacking either TRAF2-binding motif or enzyme catalytic activity abrogated the effect of TRAF2. Thus, the interaction of TRAF2 with and subsequent activation of SphK appears critically involved in the process of TRAF2 mediated TNF signal transduction. TRAF2 is a signal-transducing adapter protein that contains a conserved C-terminal TRAF domain and an N-terminal region consist of a RING finger motif and an additional array of zinc finger-like structures (28Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (932) Google Scholar). The TRAF domain is involved in receptor association and homo/hetero-oligomerization of TRAF proteins and serves as a docking site for a number of other signaling proteins (4Arch R.H. Gedrich R.W. Thompson C.B. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (517) Google Scholar, 5Wajant H. Henkler F. Scheurich P. Cell Signal. 2001; 13: 389-400Crossref PubMed Scopus (310) Google Scholar). A structure-based sequence alignment has revealed a consensus motif of (P/S/T/A)X(Q/E)E existing among the TRAF2-binding receptors including TNFR2, CD40, CD30, OX40, 4-1BB, CD27, LTβ-R, and ATAR (23Ye H. Park Y.C. Kreishman M. Kieff E. Wu H. Mol. Cell. 1999; 4: 321-330Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar,24Ye H. Wu H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8961-8966Crossref PubMed Scopus (54) Google Scholar). Several biochemical studies with mutagenesis have also supported the definition of the TRAF2-binding motifs (29Devergne O. Hatzivassiliou E. Izumi K.M. Kaye K.M. Kleijnen M.F. Kieff E. Mosialos G. Mol. Cell. Biol. 1996; 16: 7098-7108Crossref Pu" @default.
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