Tumor Necrosis Factor-α Activation of NF-κB Requires the Phosphorylation of Ser-471 in the Transactivation Domain of c-Rel (2000)

Activation of the transcription factor NF-κB is controlled at two levels in resting T cells: an initial activation induced by the triggering of the TcR·CD3 complex and a second phase controlled by paracrine- or autocrine-secreted TNFα. The initial phase is regulated by p65 (RelA), whereas the second one is mainly dependent on c-Rel. We describe here a mutant clone, D6, derived from the parental T lymphoblastic line Jurkat that fails to activate NF-κB upon TNFα stimulation. This clone had no alteration in tumor necrosis factor-α (TNFα) signaling pathways nor in IκBα, -β, or -ε expression and degradation. However, TNFα induced an exacerbated apoptotic response in this clone compared with Jurkat cells. This mutant clone showed a defect in the intermediate-late translocation of c-Rel to the nucleus promoted by TNFα stimulation, whereas early translocation is not affected. Activation or translocation of p65-containing complexes was not altered in this mutant clone. Sequencing of the c-Rel gene from this clone revealed a mutation of Ser-471 to Asn in the transactivation domain. The mutant S471N transactivation domain fused to the Gal4 DNA binding domain could not be activated by TNFα, unlike the wild type. Moreover, the overexpression of the mutant protein c-Rel S471N into Jurkat cells abolished TNFα-induced NF-κB activity, thus demonstrating that this mutation is responsible for the failure of TNFα stimulation of NF-κB. Moreover, extracts from TNFα-stimulated Jurkat cells phosphorylated in vitro recombinant wild type GST-c-Rel 464–481 but not the GST-c-Rel mutant. Thus, TNFα-induced phosphorylation of Ser-471 seems to be absolutely necessary for TNFα activation of c-Rel. Activation of the transcription factor NF-κB is controlled at two levels in resting T cells: an initial activation induced by the triggering of the TcR·CD3 complex and a second phase controlled by paracrine- or autocrine-secreted TNFα. The initial phase is regulated by p65 (RelA), whereas the second one is mainly dependent on c-Rel. We describe here a mutant clone, D6, derived from the parental T lymphoblastic line Jurkat that fails to activate NF-κB upon TNFα stimulation. This clone had no alteration in tumor necrosis factor-α (TNFα) signaling pathways nor in IκBα, -β, or -ε expression and degradation. However, TNFα induced an exacerbated apoptotic response in this clone compared with Jurkat cells. This mutant clone showed a defect in the intermediate-late translocation of c-Rel to the nucleus promoted by TNFα stimulation, whereas early translocation is not affected. Activation or translocation of p65-containing complexes was not altered in this mutant clone. Sequencing of the c-Rel gene from this clone revealed a mutation of Ser-471 to Asn in the transactivation domain. The mutant S471N transactivation domain fused to the Gal4 DNA binding domain could not be activated by TNFα, unlike the wild type. Moreover, the overexpression of the mutant protein c-Rel S471N into Jurkat cells abolished TNFα-induced NF-κB activity, thus demonstrating that this mutation is responsible for the failure of TNFα stimulation of NF-κB. Moreover, extracts from TNFα-stimulated Jurkat cells phosphorylated in vitro recombinant wild type GST-c-Rel 464–481 but not the GST-c-Rel mutant. Thus, TNFα-induced phosphorylation of Ser-471 seems to be absolutely necessary for TNFα activation of c-Rel. interleukin-2 tumor necrosis factor phosphatidylinositol 3-kinase Jun kinase phorbol myristate acetate polymerase chain reaction polyacrylamide gel electrophoresis whole cell extracts glutathione S-transferase Activation of T cells is a biphasic process (1Pimentel-Muiños F.X. Mazana J. Fresno M. Eur. J. Immunol. 1995; 25: 179-186Crossref PubMed Scopus (39) Google Scholar). Early events following TcR·CD3 ligation trigger the expression of several genes (such as the IL-21 and IL-2 receptor α chain or tumor necrosis factor-α (TNFα) genes) that are essential regulators of the subsequent second phase of T cell proliferation and acquisition of effector functions (2Crabtree G.R. Science. 1989; 243: 355-361Crossref PubMed Scopus (913) Google Scholar, 3Ullman K.S. Northrop J.P. Verweij C.L. Crabtree G.R. Annu. Rev. Immunol. 1990; 8: 421-452Crossref PubMed Scopus (491) Google Scholar, 4Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar). This program of gene expression is controlled by the regulated activation of several transcription factors such as NF-AT, AP-1, and Oct-1 (2Crabtree G.R. Science. 1989; 243: 355-361Crossref PubMed Scopus (913) Google Scholar, 3Ullman K.S. Northrop J.P. Verweij C.L. Crabtree G.R. Annu. Rev. Immunol. 1990; 8: 421-452Crossref PubMed Scopus (491) Google Scholar). Among the most important factors involved in this process of T cell activation are those belonging to the NF-κB family, which regulates several of the most important genes induced during T cell activation (for review see Ref. 5Li C.C. Korner M. Ferris D.K. Chen E. Dai R.M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar). NF-κB is rapidly activated by the TcR·CD3 complex, but at later phases of T cell activation, autocrine- or paracrine-secreted TNFα takes control of NF-κB activation. The initial phase of NF-κB activation depends on p65 translocation, whereas the later TNFα-dependent phase is controlled by c-Rel (1Pimentel-Muiños F.X. Mazana J. Fresno M. Eur. J. Immunol. 1995; 25: 179-186Crossref PubMed Scopus (39) Google Scholar). The NF-κB family of transcription factors is composed of homo- and heterodimers of a family of proteins that includes theDorsal gene of Drosophila and the mammalian genesnfκb1, nfκb2,c-rel, relA (p65), and relB (for review see Ref. 6Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4631) Google Scholar). All members share a conserved 300-amino acid region in their N-terminal portion that includes the dimerization, nuclear localization, and DNA binding regions. c-Rel, RelB, and RelA also have C-terminal transactivation domains that strongly activate transcription from NF-κB sites. NF-κB is regulated at least in part by its subcellular localization. Thus, functional NF-κB complexes are held in the cytoplasm of resting T cells in an inactive state complexed with members of the IκB family. In response to different activators (which include TcR·CD3 and TNFα), IκB is phosphorylated by IκB kinases and subsequently degraded, liberating the active NF-κB complex, which translocates to the nucleus and activates transcription (for review see Ref. 7May M.J. Ghosh S. Immunol. Today. 1998; 19: 80-88Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). Recently a second level of regulation of NF-κB activation that involves the direct activation of transcriptional-competent NF-κB family members has been emerging. Thus, the catalytic subunit of protein kinase A was shown to be bound to inactive NF-κB complexes, and upon IκB degradation, this catalytic subunit phosphorylated p65, resulting in an enhanced transcription-promoting activity (8Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar). Moreover, it was reported that TNFα treatment of cells resulted in phosphorylation of Ser-529 in the transactivation domain of p65, resulting in the activation of the transcriptional activity of the protein (9Wang D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). The small GTP-binding protein Ras enhanced transcriptional activity a that the protein kinase or a kinase Baldwin Jr., A.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google it was not this kinase was involved in NF-κB or a transcriptional The activity of Ras as as the protein kinase has been also shown to be essential for the transcriptional activity of in cells J. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This activation in the phosphorylation of the N-terminal domain and not on the C-terminal transactivation domain. of Ras such as protein kinase kinase small phosphatidylinositol 3-kinase and the protein kinase not involved in this Ras and protein kinase domain p65 the the NF-κB activity has been to be dependent on activity S. G.R. Mol. Cell. Biol. 19: PubMed Google Scholar). The activation of a signaling that to the phosphorylation of This phosphorylation transcription IκB degradation, nuclear translocation of or the of NF-κB to to the of phosphorylation was not In this that a mutation in Ser-471 to Asn the transactivation domain of c-Rel in the of TNFα-induced NF-κB activity in T cells. This the of a second level of regulation of c-Rel activity by TNFα of the activity of the transactivation domain. Jurkat cells in essential with and at in Jurkat cells by Jurkat cells and of those for TNFα from with derived p65 c-Rel and by and to the proteins on All of at a of which was at a Jun kinase and receptor kinase by for kinase TNFα was from myristate acetate and from was from was from The a of the NF-κB binding of the gene of the kinase and the gene J. M. A. J. PubMed Scopus Google Scholar). The of the NF-κB of the and the gene by J. Gal4 c-Rel transactivation domain wild type and S471N by the into of the Gal4 thus the The for (which of with c-Rel in the and S471N of c-Rel from to A was the with as Jurkat or cells with or TNFα in the of for with with and and cell was in a was as the of cells with of of nuclear extracts was in E. 1989; PubMed Scopus Google Scholar). The binding reaction of of nuclear protein and of in a reaction of This was at for which of the NF-κB with activity was A control reaction was in which a of NF-κB was to the of the binding in an for the reaction on the to cell extracts as the with and and extracts as in the of whole cell extracts or of nuclear extracts on a and to a p65, c-Rel, IκBα, or as was as the and was as the The enhanced was to the from cells in with of on protein and in a and subsequently to a by from Jurkat or cells was following the of was for the following The for c-Rel was and the was also for in an of c-Rel the whole transactivation the was the as whereas the was The was also for Jurkat cells and at in with the in the of the in with the The at in a for with essential and for The cells and at the essential with or of TNFα or was to of cell which the for The cells with cell at for at and of was to activity in a The as in to the with the control with to protein by the acid c-Rel transactivation domain from to as wild type or was into of to recombinant GST-c-Rel recombinant proteins from to the of as of an in vitro phosphorylation reaction in which whole cell extracts from or Jurkat cells from Jurkat cells in as The reaction and of activity at the reaction was by with protein was in of and in by and receptor kinase activity the kinase was as from and the was to an in protein and as Jurkat cells with for of cells of or with TNFα for that of as and resulting extracts with as in a and by TNFα is one of the most activators of NF-κB in T cells (for see Ref. 7May M.J. Ghosh S. Immunol. Today. 1998; 19: 80-88Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). TNFα NF-κB activation on T cells (1Pimentel-Muiños F.X. Mazana J. Fresno M. Eur. J. Immunol. 1995; 25: 179-186Crossref PubMed Scopus (39) Google Scholar). Thus, as TNFα-induced NF-κB activity in Jurkat T cells of the of the NF-κB of the for gene for We to mutant Jurkat T cell by of a parental Jurkat in NF-κB response to TNFα. We to one clone, D6, that had NF-κB with TNFα This failure of TNFα was not to expression of clone the at levels with Jurkat cells not Moreover, stimulation with a T cell such as to NF-κB activity in clone cells the of the defect to TNFα stimulation. We this of to TNFα was for NF-κB activation or it also to The clone showed an enhanced apoptotic response to TNFα, as compared with parental Jurkat cells of a failure of TNFα to activate NF-κB. We this of was to a defect in the TNFα signaling not in the NF-κB is transcription factor activated by TNFα, the activity of in with as a TNFα activated activity in Jurkat and clone cells. a control receptor kinase from the extracts and its kinase activity as We receptor kinase as a control this kinase is in T but it is activated by not by TNFα Annu. Rev. Biochem. 1998; PubMed Scopus Google Scholar). activity was not induced by TNFα, whereas Jurkat and clone cells showed a activity the TNFα of NF-κB was in clone D6, pathways this of activity resulted from the of TNFα to nuclear translocation of NF-κB. shown in with a NF-κB nuclear extracts from Jurkat cells showed the of two NF-κB binding complexes, and from to and at was also of NF-κB binding complexes in clone cells by TNFα stimulation, this was that in Jurkat at but to levels the of nuclear translocation of NF-κB promoted by treatment was in Jurkat and clone cells the of the two complexes and nuclear extracts with different NF-κB family members the of the complexes and the extracts with and also and not that complexes from heterodimers of and with revealed the of complexes and with revealed the of p65 complexes and Thus, complex whereas complex was composed of heterodimers of this showed a in the of NF-κB complexes Jurkat and clone In control Jurkat the of c-Rel in the DNA binding complexes with of TNFα treatment the in However, this was not in cells and We the of c-Rel and p65 proteins in nuclear extracts from TNFα-stimulated cells by We that in Jurkat and clone TNFα induced translocation of p65 with the translocation was at with to levels at c-Rel was also to the nucleus at but a at and this at least for stimulation in Jurkat cells. In clone this translocation also at but it was not in to levels by are of a failure in clone to activated c-Rel TNFα stimulation. with the DNA binding complexes, the of translocation of p65 and c-Rel was in cell The a defect in c-Rel activation by TNFα in clone was a of c-Rel NF-κB complexes with different members of the IκB family, in such a that c-Rel complexes in clone cells could not be activated by TNFα as in Jurkat cells. this IκBα, and from Jurkat and clone cells and with and to the of the complexes bound to IκBα, and no Jurkat cells and clone and bound to c-Rel and p65, was bound to p65 not the that the of IκB Jurkat and clone cells with TNFα for to in the of to the of IκB proteins was of TNFα stimulation in Jurkat and clone whereas and not at no clone and Jurkat with to IκB upon TNFα stimulation. those also that no in the of c-Rel, p65, or IκB proteins cell This has been by of extracts not Thus, the to IκB in clone D6, the TNFα-induced translocation of NF-κB complexes but not the of that could for the defect in c-Rel activation by TNFα in clone cells was a defect in c-Rel this the c-Rel gene in Jurkat and clone We from cell and it as for a for c-Rel to c-Rel and the for a mutation at to A in c-Rel from cells. This resulted in a of to the C-terminal region of c-Rel, which to the c-Rel transactivation domain the the c-Rel the whole transactivation domain the of the c-Rel gene E. 1989; Google a for that to from DNA from clone cells. The the mutation that resulted in the of Ser-471 to Asn in clone c-Rel Moreover, DNA cells A at that mutant. We that the S471N mutation could be responsible of the defect in TNFα-induced activation of c-Rel in the clone. this the of wild type the S471N mutant the Gal4 for the of transcription activation regions. this the region of to of the Gal4 DNA binding domain the into Jurkat and for Gal4 This has the that the proteins are nuclear and are regulated of wild type and S471N showed activity by whereas Gal4 or Gal4 c-Rel (which the of c-Rel transactivation inactive However, the wild type but not the S471N mutant activated transcription of the the cells with TNFα In the S471N mutant as as the wild type. with the defect in clone upon TNFα stimulation NF-κB and that this defect was to the mutant c-Rel. the Gal4 c-Rel wild type and mutant S471N into clone and for TNFα or stimulation of and mutant induced a activity, as in Jurkat cells. TNFα stimulation in clone cells with the was as the in Jurkat whereas Jurkat cells with S471N mutant showed no upon TNFα stimulation This that the signaling involved in c-Rel activation by TNFα in clone D6, at Ser-471 as a in c-Rel, responsible for TNFα activation. the and the S471N mutant mutant S471N transactivation domain was not activated by TNFα stimulation, this mutation was responsible for the in cells. c-Rel mutant S471N into Jurkat cells with a NF-κB and the activity induced by TNFα stimulation. in NF-κB activity was not altered by mutant or TNFα stimulation not NF-κB activity in cells with c-Rel mutant in to cells. overexpression of c-Rel mutant S471N could the in cells. Moreover, overexpression of c-Rel in cells could TNFα-induced NF-κB activity in thus the The of could that S471N protein was as a mutant. on and c-Rel C.C. Korner M. Ferris D.K. Chen E. Dai R.M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar, H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar, D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google showed that c-Rel is that a phosphorylation in Ser-471 was in c-Rel activation by TNFα. of Jurkat cells with showed that c-Rel is a and that its phosphorylation was by TNFα treatment the induced phosphorylation by TNFα takes in the c-Rel transactivation GST-c-Rel (which includes the c-Rel transactivation and M. in wild type and mutant S471N proteins as for a kinase phosphorylation cell extracts from Jurkat cells and clone cells or not with TNFα or for for this phosphorylation the wild type and mutant S471N c-Rel proteins strongly phosphorylated by one or kinase in Jurkat cells. This phosphorylation was in extracts from TNFα or cells in wild type and mutant S471N not demonstrating that TNFα as stimulation the phosphorylation of c-Rel transactivation domain. This that Ser-471 not be the that is phosphorylated upon TNFα activation, functional its in TNFα activation of c-Rel. Thus, a to to the by sites. The GST-c-Rel 464–481 recombinant protein was as a for a phase in vitro phosphorylation shown in the recombinant protein was phosphorylated by the from and this phosphorylation was strongly by extracts TNFα or stimulation. In phosphorylation in the mutant Ser-471 c-Rel was not upon stimulation of that the at is involved in the phosphorylation of c-Rel in a TNFα-dependent NF-κB activation in resting T cells upon activation has been shown to be controlled in a biphasic (1Pimentel-Muiños F.X. Mazana J. Fresno M. Eur. J. Immunol. 1995; 25: 179-186Crossref PubMed Scopus (39) Google Scholar). Thus, nuclear translocation of and heterodimers by the TcR·CD3 complex stimulation was at early whereas at intermediate-late the active NF-κB complexes are mainly composed of those intermediate-late events are controlled by or of TNFα (1Pimentel-Muiños F.X. Mazana J. Fresno M. Eur. J. Immunol. 1995; 25: 179-186Crossref PubMed Scopus (39) Google Scholar). c-Rel as the NF-κB family by TNFα in the of activation of resting T cells. This biphasic of NF-κB activation is in with the in the c-Rel which are in T cell activation A. D. D. S. 1995; PubMed Scopus Google J. S. Immunol. PubMed Scopus Google Scholar). c-Rel to but be by such as IL-2 to the effector thus demonstrating that c-Rel regulates the phase of T cell activation. We here the of c-Rel for NF-κB activity TNFα stimulation. Thus, have a mutant Jurkat T cell clone, clone D6, that is in activity cells activated by TNFα but not by This not of or it a activation in response to TNFα stimulation cells showed an enhanced response to TNFα-induced which the defect to NF-κB activation and with the for NF-κB M.J. A. S. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Chen J. PubMed Scopus Google Scholar). Moreover, as is and c-Rel is upon TNFα stimulation with the in Jurkat and clone D6, an defect in c-Rel Sequencing of the c-Rel gene in clone was that clone a mutant of c-Rel, by a of at to an Asn This the transactivation domain of c-Rel Mol. Cell. Biol. 1990; PubMed Scopus Google Scholar, H. M. S. 8: Google that this mutant of c-Rel be to TNFα-dependent The with proteins that this mutation is responsible for the c-Rel transcriptional Moreover, the response of wild type into mutant or wild type Jurkat cells that c-Rel activation was in clone of c-Rel into cells the whereas overexpression of the c-Rel mutant S471N in Jurkat cells abolished TNFα-induced Thus, this alteration in c-Rel was responsible for the of TNFα response in clone DNA binding of NF-κB complexes was in the nucleus in clone as as in Jurkat T cells upon stimulation by TNFα to the of the active NF-κB complexes bound to the NF-κB In wild type the NF-κB complexes composed of or heterodimers as revealed by of the complexes in the nucleus as as TNFα stimulation, but at the factors bound to the NF-κB composed mainly of c-Rel in wild type Jurkat cells The by TNFα of this complex at was in clone D6, a failure of the later c-Rel upon TNFα stimulation. stimulation the activation of NF-κB binding complexes in the nucleus of Jurkat and clone cells the defect to TNFα stimulation. the biphasic of NF-κB activation by TNFα in T cells (1Pimentel-Muiños F.X. Mazana J. Fresno M. Eur. J. Immunol. 1995; 25: 179-186Crossref PubMed Scopus (39) Google in which was an nuclear translocation of p65 and c-Rel. p65 but to levels in c-Rel was also induced by but was in the a at that was at least stimulation. This nuclear of c-Rel was in clone D6, whereas early p65 was in Jurkat cells. has been that different members of the IκB family to NF-κB and the NF-κB A. J. 16: PubMed Scopus Google Scholar). However, the failure to c-Rel in the nucleus in clone D6, activated with TNFα, could not be to a of c-Rel with several IκB family members in Jurkat and clone cells. In with A. J. 16: PubMed Scopus Google p65 and c-Rel bound to and with also was with the in Jurkat and clone D6, with no of and are that a c-Rel mutation seems to be responsible for TNFα However, be that c-Rel but not p65 gene transcription a TNFα-dependent NF-κB activation (1Pimentel-Muiños F.X. Mazana J. Fresno M. Eur. J. Immunol. 1995; 25: 179-186Crossref PubMed Scopus (39) Google Scholar). that c-Rel was to the but it is it was or from the transcription is strongly in the no c-Rel is the is that response This the TNFα-induced NF-κB activity, in the of T cell activation, mainly in c-Rel activation. This activation is controlled by an TNFα activation of c-Rel in the transactivation of its to c-Rel as a of TNFα-induced Thus, an of c-Rel this and in a of transcription in NF-κB activation by TNFα involves the induced of the activation of the IκB kinase H. A. J. M. M. A. A. Science. PubMed Scopus Google Scholar, A. A. H. M. Mol. Cell. Biol. 19: PubMed Google Scholar, E. M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, E. Chen M. Science. 1998; PubMed Google Scholar). However, a second level of regulation of the transcriptional activity has been for NF-κB family for p65 (9Wang D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google S. G.R. Mol. Cell. Biol. 19: PubMed Google Scholar). This second level of regulation of p65 activity involves the phosphorylation of Ser-529 the transactivation domain upon TNFα stimulation, which its transcriptional The functional of c-Rel mutation that c-Rel is to a regulation by TNFα. Thus, c-Rel activation at a level involves IκB and subsequent translocation to the whereas a second level involves the phosphorylation of the transactivation as is that the at is necessary for TNFα activation of c-Rel but for activation, as cells to as as Jurkat cells. the is activated by of it the that the activation of c-Rel on the phosphorylation of or that the phosphorylation defect is with The that induced a phosphorylation of the wild type the The kinase involved in this TNFα-induced phosphorylation of Ser-471 is NF-κB family IκBα, p65, and c-Rel, have been shown to be a of phosphorylation in functional Thus, nuclear of Drosophila protein is regulated by phosphorylation PubMed Scopus Google Scholar). Moreover, that p65 transcriptional activity is by a TNFα-induced phosphorylation of Ser-529 (9Wang D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). of p65 also regulates its with transcriptional (8Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar). the the Ras and protein kinase phosphorylation of p65 in the N-terminal domain was to be essential for its transcriptional activity J. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, protein kinase have a in NF-κB activation, as it has been also to be a IκB kinase M.J. J. Mol. Cell. Biol. 19: PubMed Google Scholar). A for activation of p65 that activity of activation has been also Baldwin Jr., A.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the p65 phosphorylation and subsequent activation induced by has been shown to be dependent on activation S. G.R. Mol. Cell. Biol. 19: PubMed Google Scholar). Moreover, the kinase as protein kinase activated of activity, has been in the regulation of NF-κB in Jurkat cells D. A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). However, it is that p65, IκB kinase activity and subsequent of IκBα, it is not the activity that the c-Rel was to be phosphorylated in C.C. Korner M. Ferris D.K. Chen E. Dai R.M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar). The has been shown to be in NF-κB activity, and has been to be to c-Rel in D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google not phosphorylated c-Rel in a different of regulation and as a direct We have shown that c-Rel is phosphorylated in Jurkat but TNFα its phosphorylation phase GST-c-Rel as the for phosphorylation by a activity in cells. However, phosphorylation of c-Rel was TNFα a recombinant protein the to was levels of phosphorylation could be but a induced phosphorylation upon TNFα or stimulation of the wild type protein was This TNFα-induced phosphorylation not the mutant protein was thus an essential of Ser-471 phosphorylation for activation of c-Rel Ser-471 could be not the that it could be necessary for the binding of a kinase stimulation also induced a phosphorylation of the 464–481 that it was also to activate the or a different kinase of However, as the phosphorylation not in mutant c-Rel, c-Rel was active in cells. Ser-471 is essential for TNFα-induced c-Rel activity and seems to be have TNFα-induced phosphorylation in the transactivation domain of the S471N mutant This that TNFα activates one or several kinases that c-Rel transactivation domain in vitro at different not in We have not in the of the involved in c-Rel phosphorylation by kinase not which that the kinase involved is not active in this of the it has been that c-Rel and with the transcriptional factors protein and transcription factor their transactivation domains H. E. Mol. Cell. Biol. PubMed Scopus Google Scholar). However, that on the of c-Rel, the domain M.J. PubMed Scopus Google Scholar). S471N a phosphorylation in the transactivation domain that be necessary for the for TNFα activation of c-Rel. be of to Ser-471 is in the of c-Rel with the transcriptional to the of TNFα activation of c-Rel. are in to this We for of the and essential We J. M. for and for