Mitogen-activated Protein Kinase/ERK Kinase Kinases 2 and 3 Activate Nuclear Factor-κB through IκB Kinase-α and IκB Kinase-β

Recent evidence indicates that nuclear factor-κB (NF-κB), a transcription factor critically important for immune and inflammatory responses, is activated by a protein kinase cascade. The essential features of this cascade are that a mitogen-activated protein kinase kinase kinase (MAP3K) activates an IκB kinase (IKK) that site-specifically phosphorylates IκB. The IκB protein, which ordinarily sequesters NF-κB in the cytoplasm, is subsequently degraded by the ubiquitin-proteasome pathway, thereby allowing the nuclear translocation of NF-κB. Thus far, only two MAP3Ks, NIK and MEKK1, have been identified that can activate this pathway. We now show that MEKK2 and MEKK3 can in vivoactivate IKK-α and IKK-β, induce site-specific IκBα phosphorylation, and, relatively modestly, activate an NF-κB reporter gene. In addition, dominant negative versions of either IKK-α or IKK-β abolish NF-κB activation induced by MEKK2 or MEKK3, thereby providing evidence that these IKKs mediate the NF-κB-inducing activities of these MEKKs. In contrast, other MAP3Ks, including MEKK4, ASK1, and MLK3, fail to show evidence of activation of the NF-κB pathway. We conclude that a distinct subset of MAP3Ks can activate NF-κB. Recent evidence indicates that nuclear factor-κB (NF-κB), a transcription factor critically important for immune and inflammatory responses, is activated by a protein kinase cascade. The essential features of this cascade are that a mitogen-activated protein kinase kinase kinase (MAP3K) activates an IκB kinase (IKK) that site-specifically phosphorylates IκB. The IκB protein, which ordinarily sequesters NF-κB in the cytoplasm, is subsequently degraded by the ubiquitin-proteasome pathway, thereby allowing the nuclear translocation of NF-κB. Thus far, only two MAP3Ks, NIK and MEKK1, have been identified that can activate this pathway. We now show that MEKK2 and MEKK3 can in vivoactivate IKK-α and IKK-β, induce site-specific IκBα phosphorylation, and, relatively modestly, activate an NF-κB reporter gene. In addition, dominant negative versions of either IKK-α or IKK-β abolish NF-κB activation induced by MEKK2 or MEKK3, thereby providing evidence that these IKKs mediate the NF-κB-inducing activities of these MEKKs. In contrast, other MAP3Ks, including MEKK4, ASK1, and MLK3, fail to show evidence of activation of the NF-κB pathway. We conclude that a distinct subset of MAP3Ks can activate NF-κB. The transcription factor nuclear factor-κB (NF-κB) 1The abbreviations used are:NF-κB, nuclear factor-κB; ASK1, apoptosis signal-regulating kinase 1; CAT, chloramphenicol acetyl transferase; GST, glutathioneS-transferase; HTLV-I, human T-cell leukemia virus, type I; IKK, IκB kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAP2K, mitogen-activated protein kinase kinase; MAP3K, mitogen-activated protein kinase kinase kinase; MEKK, mitogen-activated protein kinase/ERK kinase kinase; MLK3, mixed-lineage kinase 3; NIK, NF-κB inducing kinase; PAGE, polyacrylamide gel electrophoresis; TNF-α, tumor necrosis factor α. plays a critical role in immune and inflammatory responses (1Maniatis T. Science. 1997; 278: 818-819Crossref PubMed Scopus (234) Google Scholar, 2May M.J. Ghosh S. Immunol. Today. 1998; 19: 80-88Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar). NF-κB, prototypically a heterodimer of p50 and p65 subunits, is sequestered in the cytoplasm of most cell types by virtue of its association with a family of inhibitor molecules, the IκBs. Upon exposure to a wide variety of agents, including the proinflammatory cytokine TNF-α, lipopolysaccharide, oxidative stress, and the HTLV-I Tax protein, the IκB protein is phosphorylated at its N terminus. In the case of IκBα, the most extensively studied IκB isoform, this phosphorylation occurs at Ser-32 and Ser-36 (3Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1491Crossref PubMed Scopus (1326) Google Scholar, 4Brockman J.A. Scherer D.C. Hall S.M. McKinsey T.A. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). This phosphorylation event targets IκB for degradation by the ubiquitin-proteasome pathway (5Chen Z.J. Hagler J. Palombella V. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1177) Google Scholar), allowing the subsequent nuclear translocation of NF-κB. An IκB kinase (IKK) complex with a native molecular mass of 700 kDa was originally identified in cytoplasmic extracts of HeLa cells and shown to perform the site-specific phosphorylation of IκBα (6Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar, 7Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar). A significant advance was the subsequent cloning of the cDNAs for the catalytic, protein kinase subunits of this complex, IKK-α and IKK-β (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1930) Google Scholar, 9Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar, 10Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 11Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1073) Google Scholar, 12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar). Several lines of evidence now indicate that IKK-α and IKK-β can be regulated by phosphorylation. The initial indications were that the IKK complex can be activated in vitro by the MAP3K MEKK1 (MAPK/ERK kinase kinase 1) (7Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar) and that the complex, activated either in vitro by MEKK1 or in vivo by exposure of cells to TNF-α can be inactivated by phosphatase treatment (7Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1930) Google Scholar). Additional work then demonstrated that (i) mutation of potential phosphoacceptor residues to alanine in the activation loop of IKK-α or IKK-β abrogated activity (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar), (ii) mutation of these same residues in IKK-β to the phosphoresidue mimetic glutamic acid results in its constitutive activation (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar), (iii) both IKK-α and IKK-β can be activated in vivo when overexpressed with MEKK1 or the related MAP3K NF-κB-inducing kinase (NIK) (10Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 11Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1073) Google Scholar, 13Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (474) Google Scholar, 14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar, 15Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M. Gaynor R. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), and (iv) immunoprecipitated NIK can phosphorylate immunoprecipitated IKK-α (16Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (454) Google Scholar). Therefore, an important conceptual advance in our understanding of NF-κB regulation is that it can be activated by protein kinase cascade, the core elements of this cascade being a MAP3K and an IKK (7Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 10Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 17Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1173) Google Scholar). These findings have already begun to provide a framework for understanding how NF-κB can be activated by diverse stimuli. For example, compelling evidence has been presented to show that NIK mediates the NF-κB-inducing activity of TNF-α (17Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1173) Google Scholar, 18Song H.Y. Regnier C.H. Kirschning C.J. Ayres T.M. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (508) Google Scholar), whereas MEKK1 mediates the NF-κB-inducing activity of Tax (15Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M. Gaynor R. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Thus, different stimuli can activate NF-κB by targeting different MAP3Ks. These findings moreover raise the possibility that yet other MAP3Ks might activate NF-κB. MAP3Ks were originally identified as components of signaling cascades in which a MAP3K phosphorylates and activates a MAP2K, which in turn phosphorylates and activates a MAPK; the latter include the mitogen-activated ERK and the stress-activated c-Jun N-terminal kinase (JNK, also known as stress-activated protein kinase) and p38 families (19Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1392) Google Scholar). Here we show that MEKK2 and MEKK3, but not certain other MAP3Ks, can activate NF-κB, and show that this activation occurs by their activation of IKK-α and IKK-β. pCMV5-HA-MEKK2 (20Blank J.L. Gerwins P. Elliot E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), pCMV5-HA-MEKK3 (20Blank J.L. Gerwins P. Elliot E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), and pCMV5-ΔMEKK4 (21Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) were gifts of Dr. Gary Johnson (National Jewish Medical and Research Center). pcDNA3-ASK1 (22Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2059) Google Scholar) was a gift of Dr. Hidenori Ichijo (The Cancer Institute, Tokyo). pcDNA3-MLK3 was constructed by subcloning into the BamHI (blunt)/EcoRI site of pcDNA3 the 2.7-kilobase pairNcoI (blunt)/EcoRI coding sequence fragment of pPTK1–3.2 (23Ezoe K. Lee S.-T. Strunk K.M. Spritz R.A. Oncogene. 1994; 9: 935-938PubMed Google Scholar); the latter was a gift of Dr. Richard Spritz (University of Wisconsin-Madison). (PRDII)3E1bCAT, a reporter gene that contains three copies of the NF-κB binding site from the interferon-β enhancer, an E1b promoter, and the CAT gene, was a gift of Dr. Tom Maniatis (Harvard University). The sources of (PRDII)2CAT, which contains two copies of the NF-κB binding site from the interferon-β enhancer, pCMV5-MEKK1, which encodes for a C-terminal 672-residue fragment of MEKK1 (24Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (925) Google Scholar), and all other plasmids have been described (7Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar). HeLa cells were maintained as described (7Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar). Transfections performed in 3.5-cm-diameter wells were conducted by calcium phosphate precipitation (25Thanos D. Maniatis T. Cell. 1992; 71: 777-789Abstract Full Text PDF PubMed Scopus (579) Google Scholar) or by using Fugene 6 according to the manufacturer's instructions (Boehringer Mannheim). CAT and protein measurements were performed as described (7Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.60-16.62Google Scholar). Cells were washed once with Dulbecco's phosphate-buffered saline containing 1 mm EDTA and then lysed by the addition of 1 ml of buffer B (14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar) containing 10 μg/ml leupeptin and 1 mm phenylmethylsulfonyl fluoride. After centrifugation of the whole cell lysate at 16,000 ×g for 10 min at 4 °C, the supernatant was incubated with 10 μl of M2-agarose with end over end rotation for 1 h at 4 °C. The resin was then washed three times with buffer B and eluted by the addition of 20 μl of 2× SDS-PAGE loading buffer. Immunoprecipates were subjected to SDS-PAGE and then transferred to Immobilon-P membranes (Millipore). Membranes were blocked and then incubated with anti-IκBα (C-21, Santa Cruz Biotechnology), anti-phospho-Ser-32 IκBα (New England Biolabs), anti-Flag (D-8, Santa Cruz Biotechnology), or anti-JNK1 (C-17, Santa Cruz Biotechnology) polyclonal rabbit antibodies. After washing, the membranes were incubated with anti-rabbit IgG-horseradish peroxidase conjugates, washed, and then developed using SuperSignal substrate (Pierce). Immunocomplex kinase assays for IKK and JNK were performed essentially as described (14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar), except that 10 μCi of [γ-32P]ATP was employed per assay, 1 μg of GST-IκBα (5–55) was used in the IKK assays, and the ATP concentration employed to initiate the IKK reactions was 50 μm instead of 200 μm. Kinase activities were quantitated using a Molecular Dynamics Storm 860 PhosphorImager. To examine the possibility that MAP3Ks other than MEKK1 or NIK might activate NF-κB HeLa cells were cotransfected with a reporter gene that contains two NF-κB binding sites and expression constructs for a series of MAP3Ks, including MEKK2 (20Blank J.L. Gerwins P. Elliot E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), MEKK3 (20Blank J.L. Gerwins P. Elliot E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), the catalytic domain of MEKK4 (ΔMEKK4) (21Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), apoptosis signal-regulating kinase 1 (ASK1) (22Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2059) Google Scholar), and mixed-lineage kinase 3 (MLK3, also known as protein-tyrosine kinase 1 or SH3 domain-containing proline-rich kinase (23Ezoe K. Lee S.-T. Strunk K.M. Spritz R.A. Oncogene. 1994; 9: 935-938PubMed Google Scholar, 27Ing Y.L. Leung I.W.L. Heng H.H.Q. Tsui L.-C. Lassam N.J. Oncogene. 1994; 9: 1745-1750PubMed Google Scholar, 28Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). All can activate the JNK pathway (20Blank J.L. Gerwins P. Elliot E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 21Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 22Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2059) Google Scholar, 28Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). In addition, MEKK3, MEKK4, and ASK1 can activate the p38 pathway (22Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2059) Google Scholar, 29Deacon K. Blank J.L. J. Biol. Chem. 1997; 272: 14489-14496Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 30Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar), whereas MEKK2 and MEKK3 can activate the ERK pathway (20Blank J.L. Gerwins P. Elliot E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). As shown in Fig.1 A, under conditions where overexpression of the positive controls MEKK1 and NIK induces activation of the NF-κB reporter gene, overexpression of MEKK3 (as reported previously; Ref. 31Ellinger-Ziegelbauer H. Brown K. Kelly K. Siebenlist U. J. Biol. Chem. 1997; 272: 2668-2674Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and, to a lesser extent, MEKK2 induces activation as well. ΔMEKK4, ASK1, and MLK3 did not induce activation in either these cells (Fig. 1 A) or the murine fibroblast cell line L929 (data not shown) but as expected did induce robust activation of coexpressed JNK1 in HeLa cells (data not shown). NIK and MEKK1 activate NF-κB by inducing the site-specific phosphorylation of IκB. In the case of IκBα, this phosphorylation, which occurs at Ser-32 and Ser-36, is manifested by slower mobility when IκBα is examined by SDS-PAGE (3Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1491Crossref PubMed Scopus (1326) Google Scholar, 4Brockman J.A. Scherer D.C. Hall S.M. McKinsey T.A. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). To examine whether MEKK2 and MEKK3 might act through the same mechanism, HeLa cells were cotransfected with expression vectors for Flag-tagged wild-type or phosphorylation-defective (S32A/S36A) IκBα and expression vectors for MEKK2, MEKK3, or empty expression vector. The Flag-tagged IκBα was then immunoprecipitated with anti-Flag antibodies and examined by Western blotting with anti-IκBα antibodies. As shown in Fig. 1 B, MEKK2 and MEKK3 both induce the appearance of a more slowly migrating IκBα species (top panel, lanes 3 and 5, upper bands) that is abolished when an S32A/S36A IκBα mutant is examined (lanes 4 and 6), consistent with this species being N-terminally phosphorylated IκBα. ΔMEKK4, ASK1, and MLK3 did not induce the appearance of this more slowly migrating species (data not shown). This IκBα species was examined further by reprobing this blot with antibodies specific for phospho-Ser-32 IκBα. As shown in Fig. 1 B (bottom panel,lanes 3 and 5), the slower migrating IκBα species induced by MEKK2 or MEKK3 is immunoreactive with these antibodies. We conclude that MEKK2 and MEKK3 can induce site-specific, N-terminal phosphorylation of IκBα in vivo. Both MEKK1 and NIK induce site-specific phosphorylation of IκB by activating IKK-α and IKK-β. To examine whether MEKK2 or MEKK3 acts by the same mechanism, HeLa cells were cotransfected with expression constructs for Flag-tagged IKK-α, IKK-β, or JNK1 and expression constructs for MEKK1, MEKK2, MEKK3, NIK, or MLK3. The IKK or JNK was then immunoprecipitated with anti-Flag antibodies, and the kinase activities of the immunoprecipitated proteins were measured by using as substrates GST fused to the N terminii of IκBα (residues 5–55) or c-Jun (residues 1–79), respectively, in the presence of [γ-32P]ATP. As shown in Fig. 2, (A andB, top panels), not only do MEKK1 (lane 2) and NIK (lane 5) activate IKK-α and IKK-β activity, as reported previously (10Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 11Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1073) Google Scholar, 13Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (474) Google Scholar, 14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar, 15Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M. Gaynor R. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), but MEKK2 and MEKK3 do so as well (lanes 3 and 4). Immunoblot analysis reveals comparable IKK expression levels (Fig. 2, Aand B, lower panels). Furthermore, the MEKK2- or MEKK3-activated IKK-α and IKK-β display the expected substrate specificity, because a S32A/S36A double mutation in the IκBα substrate abolishes phosphorylation of the latter (data not shown). As a negative control, MLK3 does not significantly activate either IKK but does activate JNK (Fig. 2, A, B, andC, upper panels, lane 6) as expected (28Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). ASK1 and ΔMEKK4 likewise activate JNK but neither IKK-α nor IKK-β (data not shown). NIK, in contrast, activates both IKKs but not JNK (Fig. 2, A, B, and C, upper panels, lane 5) as reported previously (14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar, 18Song H.Y. Regnier C.H. Kirschning C.J. Ayres T.M. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (508) Google Scholar). The potencies of MEKK1, MEKK2, MEKK3, and NIK in activating IKK-α, IKK-β, or an NF-κB reporter gene were analyzed in more detail (Fig.3). All four MAP3Ks induce dose-dependent increases in the activities of coexpressed IKK-α or IKK-β. In the case of coexpressed IKK-α, the dose response curves are roughly comparable (Fig. 3 A; see also Fig. 2 A). In the case of coexpressed IKK-β, NIK is a somewhat less potent activator than the other MAP3Ks (Fig.3 B). In contrast, NIK is a substantially more potent activator of an NF-κB reporter gene than the other three MAP3Ks (Fig.3 C). For example, the NF-κB reporter gene activity induced by 40 ng of NIK expression vector is comparable or even greater than that induced by 4000 ng of expression vector for either MEKK1, MEKK2, or MEKK3. The experiments described above indicate that MEKK2 and MEKK3 can activate both IKK-α and IKK-β in vivo. To examine whether this activation is functionally significant, HeLa cells were cotransfected with expression constructs for MEKK1, MEKK2, MEKK3, or empty expression vector, expression constructs for dominant negative, catalytically inactive IKK-α (K44A), IKK-β (K44A), or empty expression vector, and an NF-κB reporter gene. As shown in Fig.4, under conditions where activation of the NF-κB reporter gene induced by MEKK1 is almost completely inhibited by dominant negative IKK-α or dominant negative IKK-β (13Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (474) Google Scholar, 14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar, 32Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar), that induced by either MEKK2 and MEKK3 is completely abolished. This therefore provides evidence that IKK-α and IKK-β mediate the NF-κB inducing activity of MEKK2 and MEKK3. Here we identify two additional, previously cloned MAP3Ks: MEKK2 and MEKK3, which now join NIK and MEKK1 as activators of IKK and NF-κB, thereby enlarging our framework for understanding NF-κB activation. Such knowledge is essential to providing a foundation for understanding how NF-κB can be activated by such a wide variety of stimuli. That a distinct subset of NF-κB-inducing MAP3Ks exists is highlighted by the fact that other MAP3Ks identified as activators of the JNK and/or p38 pathways, such as MEKK4, ASK1, and MLK3, fail to activate NF-κB. Titration experiments reveal that MEKK2 and MEKK3 are comparable in potency to NIK in activating coexpressed IKK-α or IKK-β but are substantially less potent than NIK in activating an NF-κB reporter gene (Fig. 3). Possible factors that might contribute to this apparent discrepancy include the following: (i) MEKK2 and MEKK3 might activate intracellular pathways that inhibit the NF-κB pathway and therefore could be relatively less effective in activating an NF-κB reporter gene; (ii) NIK might activate other components of the NF-κB pathway besides IKK and thus could be relatively more potent in activating an NF-κB reporter gene; and (iii) overexpressed IKK might respond less sensitively than endogenous IKK to coexpressed MAP3K and therefore might not accurately reflect activation of the NF-κB pathway (33Karin M. Delhase M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9067-9069Crossref PubMed Scopus (206) Google Scholar). In any case, relative potencies in transient overexpression assays cannot be used as the sole criterion for assessing physiologic significance. A particularly pertinent example is provided by the fact the HTLV-I protein Tax activates NF-κB through MEKK1 (15Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M. Gaynor R. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) despite the fact that this MAP3K, like MEKK2 or MEKK3, is substantially less potent than NIK in activating an NF-κB reporter gene (14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar, 18Song H.Y. Regnier C.H. Kirschning C.J. Ayres T.M. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (508) Google Scholar). MEKK2 and MEKK3, like other MAP3Ks, contain both catalytic and noncatalytic domains. The catalytic domains of MEKK2 and MEKK3 share 96% homology, consistent with the fact that both can activate NF-κB, whereas their noncatalytic domains are 65% homologous (20Blank J.L. Gerwins P. Elliot E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). MEKK2 is activated by treatment of cells by epidermal growth factor (34Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (256) Google Scholar). In addition, MEKK2 and MEKK3 bind 14-3-3 proteins, an interaction that is mediated at least in part through their catalytic domains but that does not modulate their JNK-inducing activities (35Fanger G.R. Widmann C. Porter A.C. Sather S. Johnson G.L. J. Biol. Chem. 1998; 273: 3476-3483Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Further experimentation will be required to determine the detailed mechanism by which MEKK2 and MEKK3 activate IKK-α and IKK-β. NIK activates IKK-α by inducing phosphorylation of Ser-176 in the activation loop of the latter (16Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (454) Google Scholar). Phosphorylation of Ser-177 and/or Ser-181 in IKK-β is essential for its activity, because a double S176A/S181A mutation abolishes activity (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar). Therefore, MEKK2 and MEKK3 might directly phosphorylate these IKK residues, particularly because these residues are components of canonical MAP2K activation loop motifs (SXXXS) (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar) that might be predicted to be substrates for MAP3Ks. It is worth noting, however, that definitive experimental evidence that either NIK or MEKK1 directly induce IKK-α or IKK-β catalytic activity has yet to be reported. The fact that many MAP3Ks have the capacity to activate distinct pathways now raises the problem of how specificity in signaling pathways is achieved. One example of this is provided by the observation that Tax activates MEKK1 and induces potent NF-κB activity (15Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M. Gaynor R. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) but only modest JNK activity (15Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M. Gaynor R. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 36Jin D.Y. Teramoto H. Giam C.Z. Chun R.F. Gutkind J.S. Jeang K.T. J. Biol. Chem. 1997; 272: 25816-25823Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), despite the fact that MEKK1 overexpression coordinately activates both (7Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar). Thus, the relative capacities of a MAP3K to activate distinct signaling pathways may be modulated in a manner that is stimulus-specific. We are grateful to Drs. Gary Johnson, Hidenori Ichijo, Richard Spritz, Roger Davis, David Wallach, David Goeddel, Dean Ballard, and Tom Maniatis for gifts of plasmids. We thank Drs. Mark Tykocinski and Leonard Jarett for support and encouragement.