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• W2085685524 abstract "The processing of the nfκb2 gene product p100 to generate p52 is a regulated event, which is important for the instrumental function of NF-κB. We previously demonstrated that this tightly controlled event is regulated positively by NF-κB-inducing kinase (NIK) and its downstream kinase, IκB kinase α (IKKα). However, the precise mechanisms by which NIK and IKKα induce p100 processing remain unclear. Here, we show that, besides activating IKKα, NIK also serves as a docking molecule recruiting IKKα to p100. This novel function of NIK requires two specific amino acid residues, serine 866 and serine 870, of p100 that are known to be essential for inducible processing of p100. We also show that, after being recruited into p100 complex, activated IKKα phosphorylates specific serines located in both N- and C-terminal regions of p100 (serines 99, 108, 115, 123, and 872). The phosphorylation of these specific serines is the prerequisite for ubiquitination and subsequent processing of p100 mediated by the β-TrCP ubiquitin ligase and 26 S proteasome, respectively. These results highlight the critical but different roles of NIK and IKKα in regulating p100 processing and shed light on the mechanisms mediating the tight control of p100 processing. These data also provide the first evidence for explaining why overexpression of IKKα or its activation by many other stimuli such as tumor necrosis factor and mitogens fails to induce p100 processing. The processing of the nfκb2 gene product p100 to generate p52 is a regulated event, which is important for the instrumental function of NF-κB. We previously demonstrated that this tightly controlled event is regulated positively by NF-κB-inducing kinase (NIK) and its downstream kinase, IκB kinase α (IKKα). However, the precise mechanisms by which NIK and IKKα induce p100 processing remain unclear. Here, we show that, besides activating IKKα, NIK also serves as a docking molecule recruiting IKKα to p100. This novel function of NIK requires two specific amino acid residues, serine 866 and serine 870, of p100 that are known to be essential for inducible processing of p100. We also show that, after being recruited into p100 complex, activated IKKα phosphorylates specific serines located in both N- and C-terminal regions of p100 (serines 99, 108, 115, 123, and 872). The phosphorylation of these specific serines is the prerequisite for ubiquitination and subsequent processing of p100 mediated by the β-TrCP ubiquitin ligase and 26 S proteasome, respectively. These results highlight the critical but different roles of NIK and IKKα in regulating p100 processing and shed light on the mechanisms mediating the tight control of p100 processing. These data also provide the first evidence for explaining why overexpression of IKKα or its activation by many other stimuli such as tumor necrosis factor and mitogens fails to induce p100 processing. The transcription factor NF-κB plays a central role in the regulation of diverse biological processes including immune response, development, cell growth, and survival (1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4631) Google Scholar, 3Silverman N. Maniatis T. Genes Dev. 2001; 15: 2321-2342Crossref PubMed Scopus (777) Google Scholar, 4Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). Consistently, deregulated function of NF-κB contributes to the development of various cell malignancies (5Rayet B. Gelinas C. Oncogene. 1999; 18: 6938-6947Crossref PubMed Scopus (1010) Google Scholar, 6Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2265) Google Scholar, 7Sun S.-C. Xiao G. Cancer Metastasis Rev. 2003; 22: 405-422Crossref PubMed Scopus (73) Google Scholar). NF-κB represents a family of related DNA-binding proteins, which in mammals includes five members: RelA (p65); RelB; c-Rel; p50; and p52 (8Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar). The NF-κB proteins primarily form p50/Rel or p52/Rel heterodimers, although they also may function as various other homodimers and heterodimers (8Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar). The NF-κB dimers are sequestered normally in the cytoplasm by ankyrin repeat-containing inhibitors called IκB proteins (1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar). The canonical pathway of NF-κB activation involves inducible IκB degradation, which can be stimulated by various cellular stimuli, such as T-cell mitogens, proinflammatory cytokines, and antigens. These stimuli trigger an IκB kinase (IKK) 1The abbreviations used are: IKK, IκB kinase; NIK, NF-κB-inducing kinase; GST, glutathione S-transferase; ARD, ankyrin repeat domain; HRP, horseradish peroxidase; HA, hemagglutinin; IB, immunoblotting; co-IP, co-immunoprecipitation; IP, immunoprecipitation; KA, kinase-inactive; β-TrCP, β-transducin repeat-containing protein. complex, which consists of IκB kinase α (IKKα), IKKβ (two catalytic subunits), and IKKγ (regulatory subunit, also named NEMO), to phosphorylate specific serines within the IκB sequence. The phosphorylated IκB then is targeted for ubiquitination and proteasome-mediated degradation, allowing the NF-κB dimers to move to the nucleus and transactivate target genes (4Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar, 7Sun S.-C. Xiao G. Cancer Metastasis Rev. 2003; 22: 405-422Crossref PubMed Scopus (73) Google Scholar). Unlike the Rel proteins, p50 and p52 are synthesized as large precursors such as NF-κB1 p105 and NF-κB2 p100, respectively (8Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar, 9Fan C.M. Maniatis T. Nature. 1991; 354: 395-398Crossref PubMed Scopus (239) Google Scholar, 10Betts J.C. Nabel G.J. Mol. Cell. Biol. 1996; 16: 6363-6371Crossref PubMed Google Scholar). These precursor proteins generate the mature p50 and p52 NF-κB subunits through proteasome-mediated processing, which involves selective degradation of their C-terminal portions (3Silverman N. Maniatis T. Genes Dev. 2001; 15: 2321-2342Crossref PubMed Scopus (777) Google Scholar, 13Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). Interestingly, the C-terminal regions of both p105 and p100 contain ankyrin repeats and function as IκB-like inhibitors of NF-κB (11Rice N.R. MacKichan M.L. Israel A. Cell. 1992; 71: 243-253Abstract Full Text PDF PubMed Scopus (343) Google Scholar, 12Mercurio F. DiDonato J.A. Rosette C. Karin M. Genes Dev. 1993; 7: 705-718Crossref PubMed Scopus (252) Google Scholar). Thus, the processing of p105 and p100 not only serves to generate p50 and p52 but also plays a role in liberating specific NF-κB complexes such as the RelB-containing complexes. Whereas the processing of p105 is constitutive and largely cotranslational (14Lin L. DeMartino G.N. Greene W.C. Cell. 1998; 92: 819-828Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), the processing of p100 is tightly regulated through its inducible phosphorylation and polyubiquitination (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). The induction of p100 phosphorylation and subsequent processing are mediated by the NF-κB-inducing kinase (NIK) and its downstream kinase IKKα (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 16Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar). Interestingly, neither IKKβ nor IKKγ is required for this non-canonical NF-κB pathway, although both IKKβ and IKKγ are essential for the canonical NF-κB activation (16Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar, 17Xiao G. Civijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.-C. The EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). Consistently, the NIK/IKKα-specific NF-κB pathway cannot be stimulated by most of the classical NF-κB inducers but rather respond to signals involved in B-cell maturation and lymphoid organogenesis including those triggered by lymphotoxin β (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 18Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar), B-cell-activating factor (19Claudio E. Brown K. Park S. Wang H. Siebenlist U. Nat. Immunol. 2002; 3: 958-965Crossref PubMed Scopus (579) Google Scholar, 20Kayagaki N. Yan M. Seshasayee D. Wang H. Lee W. French D.M. Grewal I.S. Cochran A.G. Gordon N.C. Yin J. Starovasnik M.A. Dixit V.M. Immunity. 2002; 17: 515-524Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar), and CD40 ligand (21Coope H.J. Atkinson P.G. Huhse B. Belich M. Janzen J. Holman M.J. Klaus G.G. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar). The tightly regulated p100 processing may be important for proper regulation of NF-κB function in cell growth and survival. Recent gene targeting studies indicated that the germ line knock-out of nfκb2 gene with no expression of both p100 and p52 results in severe defects in B-cell function and impairment in the formation of proper architecture in peripheral lymphoid organs (22Caamano J.H. Rizzo C.A. Durham S.K. Barton D.S. Raventos-Suarez C. Snapper C.M. Bravo R. J. Exp. Med. 1998; 187: 185-196Crossref PubMed Scopus (328) Google Scholar, 23Franzoso G. Carlson L. Poljak L. Shores E.W. Epstein S. Leonardi A. Grinberg A. Tran T. Scharton-Kersten T. Anver M. Love P. Brown K. Siebenlist U. J. Exp. Med. 1998; 187: 147-159Crossref PubMed Scopus (370) Google Scholar), a phenotype not observed in nfκb1-deficient mice (24Sha W.C. Liou H.C. Tuomanen E.I. Baltimore D. Cell. 1995; 80: 321-330Abstract Full Text PDF PubMed Scopus (1062) Google Scholar). Similarly, mice having defects in p100 processing, such as the alymphoplasia mice (carrying nik gene mutation) (for details see Ref. 25Miyawaki S. Nakamura Y. Suzuka H. Koba M. Yasumizu R. Ikehara S. Shibata Y. Eur. J. Immunol. 1994; 24: 429-434Crossref PubMed Scopus (300) Google Scholar) and the A/WySnJ mice (carrying mutations in the B-cell-activating factor receptor gene) (for details see Ref. 26Thompson J.S. Bixler S.A. Qian F. Vora K. Scott M.L. Cachero T.G. Hession C. Schneider P. Sizing I.D. Mullen C. Strauch K. Zafari M. Benjamin C.D. Tschopp J. Browning J.L. Ambrose C. Science. 2001; 293: 2108-2111Crossref PubMed Scopus (777) Google Scholar), also show similar phenotypes. Conversely, overexpression of p52 in the absence of p100 in p100 knock-in mice leads to lymphocyte hyperplasia and transformation (27Ishikawa H. Carrasco D. Claudio E. Ryseck R.P. Bravo R. J. Exp. Med. 1997; 186: 999-1014Crossref PubMed Scopus (160) Google Scholar). In humans, chromosomal translocations that cause nfκb2 gene rearrangement, leading to constitutive processing of p100, are associated with the development of various lymphomas (5Rayet B. Gelinas C. Oncogene. 1999; 18: 6938-6947Crossref PubMed Scopus (1010) Google Scholar, 7Sun S.-C. Xiao G. Cancer Metastasis Rev. 2003; 22: 405-422Crossref PubMed Scopus (73) Google Scholar, 15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 28Fracchiolla N.S. Lombardi L. Salina M. Migliazza A. Baldini L. Berti E. Cro L. Polli E. Maiolo A.T. Neri A. Oncogene. 1993; 8: 2839-2845PubMed Google Scholar, 29Zhang J. Chang C.C. Lombardi L. Dalla-Favera R. Oncogene. 1994; 9: 1931-1937PubMed Google Scholar, 30Thakur S. Lin H.C. Tseng W.T. Kumar S. Bravo R. Foss F. Gelinas C. Rabson A.B. Oncogene. 1994; 9: 2335-2344PubMed Google Scholar). Interestingly, deregulated p100 processing also is found to be associated with T-cell transformation by the human T-cell leukemia virus type 1 and many other human cancers (7Sun S.-C. Xiao G. Cancer Metastasis Rev. 2003; 22: 405-422Crossref PubMed Scopus (73) Google Scholar, 17Xiao G. Civijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.-C. The EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar, 31Lanoix J. Lacoste J. Pepin N. Rice N. Hiscott J. Oncogene. 1994; 9: 841-852PubMed Google Scholar, 32Cogswell P.C. Guttridge D.C. Funkhouser W.K. Baldwin Jr., A.S. Oncogene. 2000; 19: 1123-1131Crossref PubMed Scopus (385) Google Scholar). Because proper processing of p100 plays an essential role in the development and maturation of lymphoid organs, whereas deregulated p100 processing contributes to human malignancies, it is very important to define the detailed mechanisms of p100 processing. In this study, we have demonstrated that NIK not only activates its downstream kinase IKKα but also recruits IKKα into the p100 complex. This “docking” function of NIK requires the C-terminal serines, Ser-866 and Ser-870, of p100. Interestingly, both the N- and C-terminal regions of p100 contain IKKα phosphorylation sites. These IKKα phosphorylation sites together with the IKKα docking sites are essential for NIK-induced ubiquitination and processing of p100. Expression Vectors and Antibodies—Expression vectors encoding p100 and its serine mutants, p100 S866A/S870A and p100 S866A/S870A/S872A (labeled as p100SS/AA and p100SSS/AAA in the figures, respectively), HA-tagged NIK and its mutants, IKKα and its mutants, β-TrCP, and ubiquitin have been described previously (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 17Xiao G. Civijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.-C. The EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar, 33Xiao G. Sun S.C. J. Biol. Chem. 2000; 275: 21081-21085Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Fong A. Sun S.C. J. Biol. Chem. 2002; 277: 22111-22114Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Other p100 serine mutants also were generated by site-directed mutagenesis using pCMV4p100 as template. p100 S99A, p100 S108A, p100 S115A, p100 S123A, and p100 S872A harbor serine to alanine substitution at residues 99, 108, 115, 123, and 872, respectively. p1004 S/A and p1005 S/A mutants were obtained by substituting serines 99, 108, 115, and 123 or 99, 108, 115, 123, and 872 with alanines. GST-p100C and GST-p100C SSS/AAA were generated previously (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 16Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar). GST fusion proteins containing the N-terminal regions of p100 (GST-p100N) and their serine mutants were created by similar strategies. The anti-HA monoclonal antibody conjugated with horseradish peroxidase (anti-HA-HRP, 3F10) was purchased from Roche Applied Science. The antibody recognizing the N terminus of p100 (anti-p100N) was kindly provided by Dr. W. C. Greene (35Beraud C. Sun S.C. Ganchi P. Ballard D.W. Greene W.C. Mol. Cell. Biol. 1994; 14: 1374-1382Crossref PubMed Google Scholar). Cell Culture and Transfection—The kidney carcinoma cell line 293 was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and antibiotics. 293 cells (1 × 105, seeded in a six-well plate) were transfected with DEAE-dextran (36Holbrook N. Gulino A. Ruscetti F. Virology. 1987; 157: 211-219Crossref PubMed Scopus (39) Google Scholar). Immunoblotting (IB) and Coimmunoprecipitation (Co-IP)—293 cells were transfected with the indicated expression vectors using DEAE-dextran and lysed in radioimmunoprecipitation assay buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride) supplemented with a protease inhibitor mixture followed by IB or co-IP assays as described previously (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 17Xiao G. Civijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.-C. The EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). The amounts of cell lysates were ∼7 μg for IB and 250 μg for co-IP assays. In Vitro Kinase Assay—In vitro kinase assays were performed as described previously (16Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar, 17Xiao G. Civijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.-C. The EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). Purified recombinant IKKα and IKKβ proteins (15 ng, gifts from Dr. M. Karin) were incubated at 30 °C for 20 min in a kinase buffer (20 mm Hepes, pH 7.6, 20 mm MgCl2, 20 mm β-glycerophosphate, 1 mm EDTA, and 2 mm dithiothreitol) containing [γ-32P]dATP and labeled substrates. The phosphorylated proteins were fractionated by SDS-PAGE, transferred onto nitrocellulose membranes, and visualized by autoradiography. The membrane was used subsequently for IB to analyze the protein level of substrates. In Vivo Ubiquitin Conjugation Assays—293 cells were transfected with HA-tagged ubiquitin and p100 or its mutants in the presence or absence of NIK. After 36–48 h post-transfection, the cells were lysed in radioimmunoprecipitation assay buffer and immediately subjected to immunoprecipitation (IP) using anti-p100 antibody. The agarose beads were washed three times with radioimmunoprecipitation assay buffer followed by two additional washes with radioimmunoprecipitation assay buffer supplemented with 1 m urea. The eluted ubiquitin-conjugated p100 by SDS loading buffer was analyzed by IB using anti-HA-HRP (16Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar, 17Xiao G. Civijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.-C. The EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). NIK Promotes the Binding of IKKα to p100 —As described earlier (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 17Xiao G. Civijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.-C. The EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar), overexpressed IKKα or its activation by various stimuli that trigger the canonical NF-κB pathway fails to induce productive processing of p100. However, the expression of a very low amount of NIK could strongly induce the processing of p100. These findings suggest that besides activating the catalytic activity of IKKα, NIK may exert additional functions in the induction of p100 processing. In this regard, our previous studies indicated that NIK but not IKKα could interact efficiently with p100, although IKKα could associate physically with NIK (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 33Xiao G. Sun S.C. J. Biol. Chem. 2000; 275: 21081-21085Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Thus, we predicted that NIK might target IKKα into the p100 complex. To test this possibility, we performed co-IP assays. As expected, no significant binding between p100 and IKKα could be detected when these two proteins were coexpressed in 293 cells (Fig. 1A, top panel, lane 2). Interestingly, however, the two proteins became associated stably when NIK also was expressed in cells (lane 3). Because NIK induces the catalytic activity of IKKα (37Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (450) Google Scholar), we examined whether the induction of IKKα/p100 binding by NIK is the result of activation of IKKα. The co-IP studies were performed using dominant-active (SS/EE) and kinase-inactive (SS/AA) forms of IKKα. In the absence of NIK, neither the IKKα SS/EE nor the IKKα SS/AA could interact significantly with p100 (lanes 4 and 6). On the other hand, both of the IKK mutants formed a stable complex with p100 in the presence of NIK (lanes 5 and 7). Thus, the NIK-induced recruitment of IKKα to p100 is independent of the catalytic activity of IKKα. This finding provides an explanation for the low efficiency of IKKα SS/EE in inducing p100 processing (Fig. 1A, bottom panel, lane 4). We then investigated whether the recruitment of IKKα to p100 requires the kinase activity of NIK. When coexpressed with IKKα and p100, both the wild type NIK and its kinase-inactive form (NIK KA) could promote the binding of IKKα to p100 (Fig. 1B, upper panel, lanes 3 and 5), although the NIK KA failed to induce p100 processing (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). Together with the results presented in Fig. 1A, these observations suggest that NIK promotes the IKKα/p100 binding via a physical mechanism rather than via catalytic activation of IKKα. In this regard, prior studies have shown that the alymphoplasia mutation of NIK diminishes its physical interaction with IKKα or p100, although it does not affect its catalytic activity (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 38Luftig M.A. Cahir-McFarland E. Mosialos G. Kieff E. J. Biol. Chem. 2001; 276: 14602-14606Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Interestingly, the NIK alymphoplasia mutant also exhibited reduced activity in promoting the IKKα/p100 association (Fig. 1B, upper panel, lane 4), a defect that was associated with its low efficiency in inducing p100 processing (bottom panel, lane 4). Because NIK KA mutant failed to cooperate with IKKα in inducing p100 processing (bottom panel, lane 5), although it could efficiently recruit IKKα into p100 (upper panel, lane 5), it is very interesting to test whether NIK KA mutant induces p100 processing in the presence of the constitutive form of IKKα. As expected, both NIK wild type and KA could efficiently promote the binding of IKKα SS/EE to p100 (upper panel, lanes 7 and 8). However, coexpression of NIK KA and IKKα SS/EE still failed to trigger p100 processing (bottom panel, lane 8), suggesting that NIK has an additional function besides activating and recruiting IKKα into p100, which is also required for inducible p100 processing. Nevertheless, these results clearly indicate that the ability of NIK to bind to IKKα and p100 is essential for recruiting IKKα to p100 and that this novel function of NIK does not require the catalytic activity of NIK or IKKα. Serines 866 and 870 of p100 Serve as a Docking Site for NIK-induced IKKα Binding—We have previously shown that two C-terminal serines, Ser-866 and Ser-870, of p100 are essential for NIK-induced p100 processing (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). Although these two serines are located in the IKKα phosphorylation site of p100, it remains unclear whether they serve as the direct target of IKKα. We examined whether these two critical residues are involved in the binding of p100 by IKKα. As expected, the wild type p100 formed a stable complex with IKKα in the presence of NIK (Fig. 2A, top panel, lane 2). In contrast, the p100 mutant harboring serine to alanine substitutions at serines 866 and 870 (p100 S866A/S870A) was largely defective in the inducible binding to IKKα (lane 4). The failure of p100 S866A/S870A to recruit IKKα was not attributed to its inability in binding NIK, because a similar level of NIK binding activity was detected with wild type p100 and p100 S866A/S870A (Fig. 2B, top panel, lanes 2 and 3). The expression levels of these two different p100 constructs as well as NIK in the different cells were also comparable (middle and bottom panels). These data strongly imply that serines 866 and 870 may function as a docking site for NIK-induced IKKα binding. Serine 872, but Not Serines 866 and 870, of p100 Serves as the IKKα Phosphorylation Site—Our previous studies have indicated that serines 866, 870, and 872 of p100 are candidates for IKKα phosphorylation sites, because mutation of these serines abolishes the phosphorylation of the C-terminal region of p100 by IKKα (16Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar, 17Xiao G. Civijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.-C. The EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). However, it has remained unclear whether all of the three serines or just some of them are the IKKα targets. To answer this question, GST-p100C mutants harboring serine to alanine substitutions at serines 866 and 870 (S866A/S870A), serine 872 (S872A), or all three serines (SSS/AAA) were examined for phosphorylation by IKKα. As expected, purified recombinant IKKα efficiently phosphorylated wild type GST-p100C but not the GST-p100C SSS/AAA (Fig. 3A, upper panel, lanes 1 and 2). Surprisingly, mutation of serines 866 and 870 did not affect the p100 phosphorylation by IKKα (lane 3). On the other hand, the p100C mutant harboring serine 872 mutation (GST-p100C S872A) completely lost its ability to be phosphorylated by IKKα (lane 4). Thus, serine 872, but not serines 866 and 870, serves as a target of IKKα. Taken together with the data presented in Fig. 2, these findings suggest that serines 866 and 870 are required for the recruitment of IKKα to p100 but are not the actual phosphorylation sites for IKKα. To address the role of serine 872 in the inducible processing of p100, wild type p100 or p100 mutant harboring this serine mutation (p100 S872A) was transfected into 293 cells either in the absence or presence of the p100-processing inducer, NIK. Compared with the wild type p100, the p100 S872A exhibited a dramatic defect in NIK-induced processing (Fig. 3B, upper panel, lanes 2 and 4). Interestingly, the mutation of serine 872 to a phosphomimetic residue (aspartic acid) generated a p100 mutant (p100 S872D) that exhibited a higher inducible processing ability than p100 wild type (lane 6). In contrast, the p100 mutant harboring serine to aspartic acid substitutions at serines 866 and 870 (p100 S866A/S870D) was defective in NIK-induced processing (lane 8), a phenotype similar to that of p100 S866A/S870A (lane 10) (15Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). These results further suggested that serine 872, but not serine 866 or 870, is an IKKα phosphorylation site at p100 C terminus. Thus, the three C-terminal serines of p100 regulate its inducible processing through different mechanisms. Identification of Additional IKKα Phosphorylation Sites within p100 That Regulate p100 Processing—The findings that the C-terminal IKKα phosphorylation site (serine 872) of p100 is only partially required for inducible p100 processing suggested the possibility that p100 contains additional phosphorylation sites for IKKα. To address this possibility, we generated GST fusion proteins containing p100 mutants with progressive C-terminal truncations (Fig. 4A). Indeed, IKKα phosphorylated p100 mutants lacking the C-terminal phosphorylation site (p100 1–753 and 1–680) (see Fig. 4B, upper panel, lanes 1 and 2), thus suggesting the existence of new IKKα targets in the N-terminal region of p100. Further truncations from the C terminus revealed that the N-terminal 132 amino acids were sufficient for IKKα-mediated phosphorylation of the p100 N-terminal region (lane 5); however, a shorter p100 mutant containing the first 84 amino acids, p100-(1–84), was no longer phosphorylated by IKKα (lane 6). Thus, the major N-terminal phosphorylation sites of p100 appear to be located within a 48-amino acid region covering residues 84–132. The N-terminal phosphorylation region of p100 contains four serines, serines 99, 108, 115, and 123. Mutations were introduced to these putative phosphorylation sites either individually or in combination, and the generated GST fusion proteins were subjected to in vitro kinase assays. As shown in Fig. 4C, singular substitution of any of these serines partially affected IKKα-mediated phosphorylation (upper panel, lanes 2, 3, 4, and 5). However, mutation of all of the four serines simultaneously and largely blocked the phosphorylation by IKKα (upper panel, lane 6). These results suggest that the N-terminal region of p100 contains multiple IKKα phosphorylation sites. Consistent with this idea, the N-terminal region of p100 incorporated more radiolabeled phosphates than the C-terminal region of p100 in the in vitro kinase assays (Fig. 4D). Thus, serines 99, 108, 115, and" @default.
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• W2085685524 title "Induction of p100 Processing by NF-κB-inducing Kinase Involves Docking IκB Kinase α (IKKα) to p100 and IKKα-mediated Phosphorylation" @default.
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