Activation of Stress-activated Protein Kinase/c-Jun N-terminal Kinase, but Not NF-κB, by the Tumor Necrosis Factor (TNF) Receptor 1 through a TNF Receptor-associated Factor 2- and Germinal Center Kinase Related-dependent Pathway (1997)

A key step by which tumor necrosis factor (TNF) signals the activation of nuclear factor-κB (NF-κB) and the stress-activated protein kinase (SAPK, also called c-Jun N-terminal kinase or JNK) is the recruitment to the TNF receptor of TNF receptor-associated factor 2 (TRAF2). However, the subsequent steps in TRAF2-induced SAPK and NF-κB activation remain unresolved. Here we report the identification of a TNF-responsive serine/threonine protein kinase termed GCK related (GCKR) that likely signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase 1 (MEKK1) to activate the SAPK pathway. TNF, TRAF2, and ultraviolet (UV) light, which in part uses the TNF receptor signaling pathway, all increased GCKR activity. A TRAF2 mutant, which inhibits both TRAF2-induced NF-κB and SAPK activation, blocked TNF-induced GCKR activation. Finally, interference with GCKR expression impeded TRAF2- and TNF-induced SAPK activation but not that of NF-κB. This suggests a divergence in the TNF signaling pathway that leads to SAPK and NF-κB activation, which is located downstream of TRAF2 but upstream of GCKR. A key step by which tumor necrosis factor (TNF) signals the activation of nuclear factor-κB (NF-κB) and the stress-activated protein kinase (SAPK, also called c-Jun N-terminal kinase or JNK) is the recruitment to the TNF receptor of TNF receptor-associated factor 2 (TRAF2). However, the subsequent steps in TRAF2-induced SAPK and NF-κB activation remain unresolved. Here we report the identification of a TNF-responsive serine/threonine protein kinase termed GCK related (GCKR) that likely signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase 1 (MEKK1) to activate the SAPK pathway. TNF, TRAF2, and ultraviolet (UV) light, which in part uses the TNF receptor signaling pathway, all increased GCKR activity. A TRAF2 mutant, which inhibits both TRAF2-induced NF-κB and SAPK activation, blocked TNF-induced GCKR activation. Finally, interference with GCKR expression impeded TRAF2- and TNF-induced SAPK activation but not that of NF-κB. This suggests a divergence in the TNF signaling pathway that leads to SAPK and NF-κB activation, which is located downstream of TRAF2 but upstream of GCKR. Tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; TRADD, TNFR1-associated death domain protein; NF-κB, nuclear factor κB; SAPK, stress-activated protein kinase; TRAF2, TNF receptor-associated factor 2; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEKK, MAPK/ERK kinase kinase; HA, hemagglutinin antigen; GCKR, germinal center kinase-related; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; MBP, myelin basic protein; EGF, epidermal growth factor; IL-1, interleukin-1; GST, glutathione S-transferase. 1The abbreviations used are: TNF, tumor necrosis factor; TRADD, TNFR1-associated death domain protein; NF-κB, nuclear factor κB; SAPK, stress-activated protein kinase; TRAF2, TNF receptor-associated factor 2; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEKK, MAPK/ERK kinase kinase; HA, hemagglutinin antigen; GCKR, germinal center kinase-related; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; MBP, myelin basic protein; EGF, epidermal growth factor; IL-1, interleukin-1; GST, glutathione S-transferase. is a pleiotropic cytokine, which plays a major role inflammation (reviewed in Ref. 1Tracey K.J. Cerami A. Annu. Rev. Cell Biol. 1993; 9: 317-343Crossref PubMed Scopus (761) Google Scholar). TNF binds two distinct TNF receptors of 55 kilodaltons (TNFR1) and 75 kilodaltons (TNFR2) (reviewed in Ref. 2Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (1002) Google Scholar). TNF-induced trimerization of TNFR1 triggers an association with TNFR1-associated death domain protein (TRADD), which recruits Fas-associated death domain (FADD, also known as MORT1) protein and TRAF2 (3Hsu H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1728) Google Scholar, 4Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2154) Google Scholar). FADD participates in an apoptosis pathway and TRAF2 signals the activation of the NF-κB activation by both TNFR1 and the B lymphocyte co-receptor CD40 (3Hsu H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1728) Google Scholar, 4Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2154) Google Scholar, 5Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (975) Google Scholar). TRAF2 contains a conserved C-terminal homology region termed the TRAF domain, which interacts with TRADD, and an N-terminal ring finger required for signaling the activation of NF-κB and SAPK (5Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (975) Google Scholar, 6Liu Z. Shu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1779) Google Scholar, 7Natoli G. Costanzo A. Ianni A. Templeton D.J. Woodgett J.R. Balsano C. Levrero M. Science. 1997; 275: 200-203Crossref PubMed Scopus (415) Google Scholar). The SAPK pathway, similar to other extracellular signal-regulated kinase (ERK) pathways, consists of a 3-tiered core of protein kinases in which a mitogen-activated protein kinase (MAPK/ERK) kinase kinase (MEKK) activates a MAPK/ERK kinase (MEK) which in turn activates SAPK (8Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar, 9Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2411) Google Scholar). MEKK1, which activates the SAPK pathway via a MEK termed SEK1 (10Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar, 11Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar, 12Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar, 13Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 28: 286-290Crossref Scopus (708) Google Scholar), has been implicated in TNF-induced SAPK activation (6Liu Z. Shu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1779) Google Scholar). The mixed lineage kinase MLK-3 (14Gallo K.A. Mark M.R. Scadden D.T. Wang Z. Gu Q. Godowski P.J. J. Biol. Chem. 1994; 269: 15092-15100Abstract Full Text PDF PubMed Google Scholar, 15Ezoe K. Lee S.T. Strunk K.M. Spritz R.A. Oncogene. 1994; 9: 935-938PubMed Google Scholar, 16Ing Y.L. Leung I.W. Heng H.H. Tsui L.C. Lassam N.J. Oncogene. 1994; 9: 1745-1750PubMed Google Scholar) also signals the SAPK pathway through SEK1 (17Tibbles L.A. Ing Y.L. Kiefer F. Chan J. Iscove N. Woodgett J.R. Lassam N. EMBO J. 1996; 24: 7026-7035Crossref Scopus (280) Google Scholar). Two other MEKKs, both of which are implicated in TNF-signaling, are NIK, which interacts with TRAF2 and signals NF-κB activation (18Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1160) Google Scholar), and ASK1, which is TNF-inducible and activates the SAPK pathway and the related p38 pathway (19Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazona K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2006) Google Scholar). Above the level of the three-tiered core in the SAPK pathway are two related protein kinases, GCK (20Katz P. Whalen G. Kehrl J.H. J. Biol. Chem. 1994; 269: 16802-16809Abstract Full Text PDF PubMed Google Scholar, 21Pombo C. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar) and HPK1 (22Hu M.C. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (192) Google Scholar, 23Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (196) Google Scholar), which likely activate the SAPK pathway via MEKK1 and/or MLK3; however, they are unlikely to account for TNF-induced SAPK activation. HPK1 has a limited range of tissue expression (22Hu M.C. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (192) Google Scholar, 23Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (196) Google Scholar), inconsistent with the widespread expression of TNFR1, and although widely expressed, GCK is only marginally affected by TNF (21Pombo C. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar). However, here we report the identification of a third family member termed GCK related (GCKR) that is widely distributed, TNF-responsive, and a major mediator of TNF-induced SAPK activation. The PC-12, COS, HeLa, Jurkat, K562, 293, and MOLT-4 cells were obtained from the American Type Culture Collection. The 293T cell line was obtained from Dr. O. Witte (UCLA) following permission from Dr. D. Baltimore (Massachusetts Institute of Technology). The CA46 and MC116 cells were obtained from Dr. I. Magrath (National Institutes of Health), and the NG108 cells were from Dr. W. Klee (National Institutes of Health). The pMT3-HA-SAPK-p46 plasmid was provided by Dr. J. Kyriakis (Harvard Medical School). The pMT2T-TRAF2 and pcDNA3-MEKK1 (K432M) plasmids were provided by Antonio Leonardi and Heidrun Ellinger (National Institutes of Health). The pCR3-TRAF2 (87–501) was created by PCR subcloning the appropriate fragment amplified from pMT2-TRAF2 into pCR3. The pcDNA3-ASK1 (K709R) was provided by Dr. E. Nishida (Kyoto University). The anti-HA (12CA), anti-FLAG, and anti-phosphotyrosine monoclonal antibodies were purchased from Boehringer Mannheim, Eastman Kodak Co., and Upstate Biotechnology. The GCKR polyclonal antiserum was generated in rabbits by immunizing with a peptide (RKETEARDEMC) coupled to Keyhole limpet hemocyanin. The GCK polyclonal antiserum has been previously described (20Katz P. Whalen G. Kehrl J.H. J. Biol. Chem. 1994; 269: 16802-16809Abstract Full Text PDF PubMed Google Scholar). A human spleen cDNA library (CLONTECH) was screened with a PCR product amplified by reverse transcription PCR from human tonsil RNA using primers based on the Z25426 reported nucleotide sequence. Four overlapping cDNA clones were identified, and a composite cDNA of 2746 base pairs was obtained. 2The GenbankTM accession number is pending. A GCKR clone that encompassed the open reading frame was subcloned into pCRIII (Invitrogen) in both orientations to create pCR3-GCKR and pCR3-GCKR(AS). The nucleotide sequences of the cDNA clones and constructs were determined using an automated DNA sequencer. The inserts for plasmids pcDNA3-HA-GCKR and pFLAG-CMV2-GCKR were generated by PCR with the appropriate restriction sites incorporated into the primers and using pCR3-GCKR as a template. pCR3-GCKR-178A was created from overlapping PCR products, one of which was amplified with a mutating primer using pCR3-GCKR as a template. The two products served as a new template to generate a PCR product that spanned the coding region and contained the mutation, which was subcloned into pCR3 and pFLAG-CMV2 (Kodak). Multiple tissue Northern blots were purchased from CLONTECH and processed according to manufacturer recommendations. 293T cells were exposed to TNF (Endogen) or UV light source (Stratagene) as indicated. GCKR and GCK immunoprecipitates were assayed for in vitro kinase activity as described (21Pombo C. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar). For the transfection studies, either a calcium phosphate method (12Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar) or Superfect was used (Qiagen). 10-cm plates of 293T or 293 cells containing approximately 2 × 106 were transfected with various expression vectors or control vectors as indicated, and the total amount of DNA transfected in any one experiment was held constant among the different experimental conditions. After 48 h, cells were treated with anisomycin (10 μg/ml for 15 min), TNF, or UV light. Cell lysis, anti-HA immunoprecipitation, and SAPK immune complex assays were performed as described (21Pombo C. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar). The GCKR (1:300 dilution), HA, FLAG, and phosphotyrosine immunoblots were performed using standard methodology with an additional amplification step using a biotinylated secondary antibody. The signals were detected by enhanced chemiluminescence (ECL, Amersham Corp.). The transfections for the NF-κB CAT assays were performed as above. Two days after the transfection, the cells were harvested and lysed, the protein concentrations were normalized, and CAT activity was measured by scintillation counting. The reporter construct contained two NF-κB sites linked to the CAT gene and was provided by Dr. Keith Brown (National Institutes of Health). The transfection efficiency was monitored by co-transfection of a β-galactosidase reporter gene whose activity varied less than 10% between samples. Using the nucleotide sequence of a PCR product, Z25426, that had been amplified from a gene clearly related to GCK, we isolated a cDNA that encompassed an open reading frame predicted to encode an 846 amino acid protein kinase with a molecular mass of 95 kilodaltons, which we termed GCKR (Fig.1 A). GCKR possesses an amino-terminal catalytic domain 73% identical with that of GCK and related to those of the yeast protein kinases SPS1 and STE20 (24Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (344) Google Scholar, 25Freisen H. Lunz R. Doyle S. Segall J. Genes Dev. 1994; 8: 2162-2175Crossref PubMed Scopus (107) Google Scholar), the human and mouse PAK protein kinases (26Manser E. Leung T. Salihuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1297) Google Scholar), and HPK1 (22Hu M.C. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (192) Google Scholar, 23Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (196) Google Scholar). The catalytic domain of HPK1 shares approximately 66% amino acid identity with GCK and GCKR, but overall HPK1 shares less identity with GCKR than does GCK (40 versus 54%, Fig. 1 A). GCKR and GCK also contain three conserved regions in their putative regulatory domains of approximately 80 amino acids each (GCKR amino acids 485–568, 604–689, and 762–842 share 66%, 61%, and 72% identity with GCK amino acids 459–543, 579–665, and 735–815, respectively), whereas over similar regions, GCKR and human HPK1 share 47%, 51%, and 34% identity. While HPK1 has four potential SH3 binding sites (P1-P4) (23Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (196) Google Scholar), GCKR shares only the P2 site, and it lacks the P3 and P4 sites, which bind the SH3 domain of MLK-3. To determine the range of GCKR mRNA expression, we analyzed GCKR mRNA transcripts in a variety of tissues. We found a 4.4-kilobase GCKR mRNA transcript in all tissues examined (Fig. 1 B). In addition, we generated a rabbit anti-GCKR antiserum that immunoblotted a 97-kDa band in all cell lysates (Fig. 1 C) examined although we detected relatively low levels in the B cell lines CA46, MC116, NALM-6, and RAMOS (bands detected on a longer exposure). Similar to GCK and HPK1, GCKR activated the SAPK pathway. 293T cells co-expressing a hemagglutinin (HA)-tagged SAPK and either GCKR, GCK, or both showed a 3.5-, 5.5-, or 4.5-fold increase in HA-SAPK activity, respectively (Fig. 2 A). These levels compared favorably with those achieved by anisomycin, an established SAPK activator. The increase in SAPK activity depended upon the catalytic activity of GCKR since a GCKR kinase-deficient mutant, GCKR (T178A), contains a threonine to alanine substitution at amino acid position 178 and did not activate the pathway (Fig.2 B). Like GCK, GCKR phosphorylated the proline-rich myelin basic protein (MBP), while GCKR (T178A) did not (Fig.2 C). Also similar to previous studies with GCK (21Pombo C. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar), GCKR did not efficiently activate either an epitope-tagged MAPK or an epitope-tagged p38 kinase, and a catalytically inactive form of SEK1 inhibited GCKR-mediated SAPK activation (data not shown). To determine whether GCKR may be involved in TNF-induced SAPK activation, we exposed 293T cells to 50 or 100 ng of TNF for varying duration times, immunoprecipitated GCKR, and performed GCKR immune complex kinase assays. Consistent with TNF-induced GCKR activation, GCKR immune complexes from TNF-treated cells contained a marked elevation in in vitro protein kinase activity (Fig.3 A). UV light, which in part utilizes TNF receptor signaling in its induction of SAPK (27Roselte C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (941) Google Scholar), also increased GCKR activity. Exposing 293T cells to increasing amounts of UV light resulted in a dose-dependent activation of GCKR (Fig. 3 B). In contrast, UV light did not significantly activate endogenous GCK (Fig. 3 C). Phosphotyrosine immunoblotting the GCKR immunoprecipitates that had been subjected to an in vitro kinase assay revealed a 97-kDa UV light-inducible band consistent with UV light-induced GCKR tyrosine phosphorylation. Autoradiography of the same immunoblot revealed phosphorylation of a 97-kDa band (Fig. 3 UV light utilizes other cell receptors the TNF receptor in SAPK in cells (27Roselte C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (941) Google Scholar), we examined the of concentrations of epidermal growth factor and TNF on GCKR in those and had on GCKR however, their with TNF resulted in a marked increase in activity, that achieved with UV light (Fig.3 TNF-induced SAPK activation TRAF2 (6Liu Z. Shu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1779) Google Scholar, 7Natoli G. Costanzo A. Ianni A. Templeton D.J. Woodgett J.R. Balsano C. Levrero M. Science. 1997; 275: 200-203Crossref PubMed Scopus (415) Google Scholar), we examined the of TRAF2 on GCKR activity by TRAF2 with into 293T In we used only 1 of expression to its level of activity. The of TRAF2 increased activity by (Fig.3 In addition, co-transfection of with the TRAF2 which TNF-induced SAPK activation G. Costanzo A. Ianni A. Templeton D.J. Woodgett J.R. Balsano C. Levrero M. Science. 1997; 275: 200-203Crossref PubMed Scopus (415) Google Scholar), blocked TNF-induced GCKR activation (Fig. 3 These that GCKR is a downstream of TRAF2 in TNF receptor however, they not its in TNF-induced SAPK activation. To whether GCKR TNF-induced SAPK activation required endogenous GCKR. We used two one to endogenous levels by using a GCKR plasmid and the to downstream GCKR signaling by using the catalytically inactive The GCKR plasmid inhibited activation and protein levels (data not shown). transfection of the GCKR construct endogenous GCKR protein levels by approximately a in GCKR levels in the transfected cells (Fig. A). we the GCKR construct with HA-SAPK into 293T The but not a control UV light-induced HA-SAPK activity by in cells exposed to 80 of and by in cells exposed to (Fig. the construct SAPK activation at The GCKR construct also inhibited TRAF2-induced SAPK activation. of the construct with TRAF2 and HA-SAPK into 293T cells SAPK activity in the as compared with a control construct (Fig. In contrast, it had on SAPK activation (Fig. In addition, co-transfection of the GCKR or with HA-SAPK into 293T cells the TNF-induced SAPK activity in immunoprecipitates by and respectively (Fig. C). Finally, we determined whether the catalytically inactive form of GCKR TRAF2-induced SAPK activation. of 293 cells with HA-SAPK and TRAF2 in the of increasing concentrations of TRAF2-induced SAPK activation (Fig. These that in 293 UV light and TNF SAPK activation by GCKR. on the previous that a catalytically inactive form of MEKK1 inhibited TNF-induced SAPK activation (6Liu Z. Shu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1779) Google Scholar), we examined whether a similar construct inhibited SAPK activation. We 293T cells with GCKR, and either (19Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazona K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2006) Google Scholar) or MEKK1 The catalytically inactive form of MEKK1 significantly SAPK activation, whereas the catalytically inactive form of ASK1, which inhibits TNF-induced apoptosis (19Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazona K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2006) Google Scholar), had (Fig. We also that MEKK1 (K432M) TNF-induced SAPK activation but did Finally, since both MEKK1 and TRAF2 been reported to activate NF-κB (6Liu Z. Shu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1779) Google Scholar, 7Natoli G. Costanzo A. Ianni A. Templeton D.J. Woodgett J.R. Balsano C. Levrero M. Science. 1997; 275: 200-203Crossref PubMed Scopus (415) Google Scholar, J. Cell. 1997; Full Text Full Text PDF PubMed Scopus (658) Google Scholar), we whether GCKR Cell lysates from 293T cells transfected with GCKR and an NF-κB reporter construct were assayed for reporter gene activity GCKR did not NF-κB reporter gene activity although TRAF2 and TNF the GCKR construct did not TNF-induced activation of the NF-κB reporter The of TNF-induced reporter gene expression by GCKR, but not GCKR (T178A), was although of GCKR is a of the SAPK pathway and is to UV light, and We that GCKR, and GCK and HPK1, to upstream signaling to the SAPK pathway a However, the upstream signals GCK and HPK1 remain GCK to TNF and UV light and not at all to of the known SAPK (20Katz P. Whalen G. Kehrl J.H. J. Biol. Chem. 1994; 269: 16802-16809Abstract Full Text PDF PubMed Google Scholar). The limited tissue of HPK1 suggests that it may be linked to a receptor (22Hu M.C. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (192) Google Scholar, 23Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (196) Google Scholar). While TNF-induced GCKR activation required TRAF2, we not determined the that TRAF2 to GCKR activation. a serine/threonine protein kinase and a of the molecular mass of GCKR immunoprecipitates with TNFR1 following TNF signaling C.F. J. Immunol. 1994; Google Scholar), we examined whether GCKR be into the TNFR1 signaling complex via an with However, in we been to TRAF2 with GCKR using 293T cells transfected with expression vectors that encode for epitope-tagged of TRAF2 and GCKR. are in The downstream kinases involved in SAPK remain to be is an as it is also however, it is involved in TNF-induced apoptosis and TRAF2-induced SAPK activation through a (19Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazona K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2006) Google Scholar). Also ASK1, a catalytically inactive form of did not SAPK activation, did it TNF-induced SAPK is MEKK1, since a catalytically inactive form of MEKK1 inhibited TNF-induced SAPK activation (6Liu Z. Shu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1779) Google Scholar). However, the studies with MEKK1 (K432M) been to the role of MEKK1 in NF-κB activation (6Liu Z. Shu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1779) Google Scholar, J. Cell. 1997; Full Text Full Text PDF PubMed Scopus (658) Google Scholar). While MEKK1 (K432M) inhibited SAPK activation, additional studies be to that GCKR signals via MLK-3 is a downstream of both GCK and HPK1 (23Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (196) Google Scholar), we are also to determine whether GCKR may also MLK-3 to the SAPK pathway. and less likely to GCKR to the SAPK pathway as they activate both the SAPK and MAPK pathways, whereas GCKR activates the SAPK pathway P. S. Johnson G.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Brown K. K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, a MEKK, which activates the SAPK pathway, is potential of GCKR. However, it to be a downstream mediator of and since a form of blocked and SAPK activation P. Johnson G.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). While it is that GCKR is a mediator of TNF-induced SAPK activation, we that GCKR is involved in TNF-induced NF-κB activation. GCKR to activate NF-κB as by the of an NF-κB reporter and the GCKR, which inhibited TNF-induced SAPK activation, to or TRAF2-induced NF-κB activation. GCK, whose activation of the SAPK pathway is also inhibited by MEKK1 (K432M) also to activate NF-κB in co-transfection either the TNF receptor signaling pathway to SAPK and NF-κB the level of MEKK1 or MEKK1 be activated or into signaling that it activates the kinase complex and In the of studies an identical kinase to GCKR was isolated by two and termed and Similar to and were to activate the SAPK pathway J. Oncogene. 1997; PubMed Scopus Google K. Wang C. Meyer C.F. G. M. Tan Z. S. A. 1997; PubMed Scopus Google Scholar). However, studies significantly those to that GCKR is a major mediator of and SAPK activation in cell studies are at TRAF2 activates GCKR and whether MEKK1 is the major downstream of GCKR. We J. Kyriakis for plasmids and N. for additional H. for the of M. for and A. S. for and