Isolation of a Novel Interleukin-1-inducible Nuclear Protein Bearing Ankyrin-repeat Motifs
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.c100075200
ISSN1083-351X
AutoresHirotaka Haruta, Akira Kato, Kazuo Todokoro,
Tópico(s)Inflammasome and immune disorders
ResumoWe isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IκB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor α nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-κB1/p50, NF-κB2/p52, C/EBPβ, and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other IκB family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus. We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IκB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor α nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-κB1/p50, NF-κB2/p52, C/EBPβ, and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other IκB family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus. NF-κB is a transcription factor that is known to play an important role in regulating immune and inflammatory responses (1Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1654) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4572) Google Scholar). There are presently five members of the mammalian NF-κB/Rel family, NF-κB1/p50, NF-κB2/p52, c-Rel, RelA/p65, and RelB (1Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1654) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4572) Google Scholar). The classic form of NF-κB, the heterodimer of the NF-κB1/p50 and RelA/p65, is normally retained in the cytoplasm through interactions with inhibitor protein IκB. The IκB family of proteins includes IκBα, IκBβ, IκBε, Bcl-3, NF-κB1/p105, and NF-κB2/p100, all of which possess 5–7 ankyrin-repeat motifs (1Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1654) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4572) Google Scholar), which form a functional unit able to interact with the Rel homology domain of NF-κB. The cytoplasmic retention of the classic form of NF-κB is primarily carried out by IκBα and IκBβ (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 6Weil R. Laurent-Winter C. Israel A. J. Biol. Chem. 1997; 272: 9942-9949Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 7Phillips R.J. Ghosh S. Mol. Cell. Biol. 1997; 17: 4390-4396Crossref PubMed Google Scholar). Inductive stimuli, such as tumor necrosis factor α (TNFα), 1The abbreviations used are:TNFαtumor necrosis factor αIL-1interleukin-1PMAphorbol 12-myristate 13-acetateDAPI4,6-diamidino-2-phenylindoleLPSlipopolysaccharideRXRretinoid X receptorGSTglutathioneS-transferasePAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salineAP-1activating protein-1INAPIL-1-inducible nuclear ankyrin-repeat proteinFCSfetal calf serumDIGdigoxigeninECLenhanced chemiluminescencebpbase pairCIPcalf intestine alkaline phosphataseHAhemagglutinin interleukin-1 (IL-1), and bacterial endotoxin, lead to the phosphorylation and degradation of IκB, allowing NF-κB to translocate into the nucleus and regulate specific gene expression (1Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1654) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4572) Google Scholar).IκBα is degraded in response to the NF-κB inducers TNFα, IL-1, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and double-stranded RNA. In contrast, IκBβ is degraded only when cells are stimulated with IL-1 or LPS, both of which cause persistent long term activation of NF-κB (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 6Weil R. Laurent-Winter C. Israel A. J. Biol. Chem. 1997; 272: 9942-9949Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 7Phillips R.J. Ghosh S. Mol. Cell. Biol. 1997; 17: 4390-4396Crossref PubMed Google Scholar). Following degradation of the initial pool of IκBβ in response to IL-1 or LPS, newly synthesized IκBβ accumulates as an unphosphorylated protein that forms a stable complex with NF-κB and prevents it from binding to newly synthesized IκBα (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 6Weil R. Laurent-Winter C. Israel A. J. Biol. Chem. 1997; 272: 9942-9949Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 7Phillips R.J. Ghosh S. Mol. Cell. Biol. 1997; 17: 4390-4396Crossref PubMed Google Scholar), resulting in the prolonged activation of NF-κB (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 8Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). This unphosphorylated IκBβ cannot block the nuclear localization signal of NF-κB, thus this NF-κB·IκBβ complex translocates into the nucleus. The function of this complex in the nucleus is yet to be elucidated, and the mechanism by which only IL-1 and LPS can degrade IκBβ remains to be resolved.Unlike the other IκB family members, Bcl-3 is a nuclear protein (9Zhang Q. Didonato J.A. Karin M. McKeithan T.W. Mol. Cell. Biol. 1994; 14: 3915-3926Crossref PubMed Scopus (53) Google Scholar, 10Wulczyn F.G. Naumann M. Scheidereit C. Nature. 1992; 358: 597-599Crossref PubMed Scopus (181) Google Scholar, 11Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar). It does not bind to RelA/p65 but specifically binds to NF-κB1/p50 or NF-κB2/p52 homodimers (10Wulczyn F.G. Naumann M. Scheidereit C. Nature. 1992; 358: 597-599Crossref PubMed Scopus (181) Google Scholar, 12Franzoso G. Bours V. Park S. Tomita-Yamaguchi M. Kelly K. Siebenlist U. Nature. 1992; 359: 339-342Crossref PubMed Scopus (264) Google Scholar, 13Kerr L.D. Duckett C.S. Wamsley P. Zhang Q. Chiao P. Nabel G. McKeithan T.W. Baeuerle P.A. Verma I.M. Genes Dev. 1992; 6: 2352-2363Crossref PubMed Scopus (92) Google Scholar, 14Nolan G.P. Fujita T. Bhatia K. Huppi C. Liou H.C. Scott M.L. Baltimore D. Mol. Cell. Biol. 1993; 13: 3557-3566Crossref PubMed Google Scholar) and takes them into the nucleus where it exhibits transactivating activity (11Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar, 15Fujita T. Nolan G.P. Liou H.C. Scott M.L. Baltimore D. Genes Dev. 1993; 7: 1354-1363Crossref PubMed Scopus (348) Google Scholar). The formation of Bcl-3·(NF-κB1/p50)2·κB complex or Bcl-3·(NF-κB2/p52)2·κB complex is regulated by the phosphorylation status of Bcl-3 (14Nolan G.P. Fujita T. Bhatia K. Huppi C. Liou H.C. Scott M.L. Baltimore D. Mol. Cell. Biol. 1993; 13: 3557-3566Crossref PubMed Google Scholar, 16Bundy D.L. McKeithan T.W. J. Biol. Chem. 1997; 272: 33132-33139Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Bcl-3 also interacts with retinoid X receptor (RXR) or activating protein-1 (AP-1) and functions as their transcription coactivator (17Na S.Y. Choi H.S. Kim J.W. Na D.S. Lee J.W. J. Biol. Chem. 1998; 273: 30933-30938Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Na S.Y. Choi J.E. Kim H.J. Jhun B.H. Lee Y.C. Lee J.W. J. Biol. Chem. 1999; 274: 28491-28496Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, the detailed characters of this unique IκB family member remain mysterious.Here we identified a novel IκB family member, termed IL-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression is specifically induced by IL-1. INAP was found to be weakly homologous to Bcl-3 and localized in the nucleus like Bcl-3. We discuss here the possible function of this novel IκB family member.DISCUSSIONWe isolated a novel IL-1-inducible nuclear factor, INAP, which is related to the IκB family and the Rel family. It is well known that IκB family members bind to the RelA/p65·NF-κB1/p50 complex and prevents the complex from activating and translocating into the nucleus. Therefore, possible interaction of INAP with the RelA/p65·NF-κB1/p50 complex was examined by immunoprecipitation followed by immunoblot analysis. However, we failed to detect the direct and/or indirect binding of INAP to RelA/p65 (data not shown). Furthermore, one of the most important factors induced by IL-1 stimulation is IL6, of which gene expression is regulated by C/EBPβ (NF-IL6), AP-1, and NF-κB (21Sanceau J. Kaisho T. Hirano T. Wietzerbin J. J. Biol. Chem. 1995; 270: 27920-27931Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 22Miyazawa K. Mori A. Yamamoto K. Okudaira H. J. Biol. Chem. 1998; 273: 7620-7627Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Therefore, we also examined the possible interactions of INAP with C/EBPβ and c-Fos/c-Jun in nuclear extracts prepared from IL-1α-stimulated OP9 cells. Once again, we could not detect the interactions of INAP with C/EBPβ or with c-Fos/c-Jun (data not shown). Moreover, the fact that Bcl-3, the protein most closely related to INAP, associates NF-κB1/p50 or NF-κB2/p52 homodimers and modulates their transactivation activities (11Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar, 16Bundy D.L. McKeithan T.W. J. Biol. Chem. 1997; 272: 33132-33139Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) motivated us to examine whether INAP associates with NF-κB1/p50 or NF-κB2/p52 in IL-1-stimulated nucleus. None of these interactions, however, was detected by immunoprecipitation followed by immunoblot analysis (data not shown). It has also been reported that Bcl-3 binds to RXR (17Na S.Y. Choi H.S. Kim J.W. Na D.S. Lee J.W. J. Biol. Chem. 1998; 273: 30933-30938Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) or to AP-1 (18Na S.Y. Choi J.E. Kim H.J. Jhun B.H. Lee Y.C. Lee J.W. J. Biol. Chem. 1999; 274: 28491-28496Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and regulate specific gene expression, and thus we also examined the possible interactions of INAP with RXR or with AP-1 but failed to detect the bindings (data not shown). We concluded that INAP could not bind to any of the binding partners with which Bcl-3 has been reported to interact and that INAP is a very unique protein in the IκB family and is clearly distinct even from the most closely related IκB family member, Bcl-3. To determine the biological function of INAP on IL-1 signalings it is very important to identify the INAP-binding proteins in the IL-1-stimulated nucleus by other means such as yeast two-hybrid screening, pull-down experiments, and far-Western screening.INAP was found to be a novel nuclear factor related to the IκB family, but by IL-1 stimulation INAP was newly produced and accumulated in the nucleus, rather than being degraded as were other IκB family members. From a gene expression point of view, INAP is quite a distinct protein from these family members. There exists no obvious nuclear localization signal in INAP, but it does exist in the nucleus. Furthermore, INAP was not phosphorylated no matter whether it was localized in the cytoplasm or nucleus, indicating that the phosphorylation status of INAP does not affect its subcellular localization.A few potential NF-κB binding sites were found around −340 bp upstream from the initiation codon in human INAP gene promoter regions. However, we demonstrated that INAP gene expression was rapidly induced by IL-1 but not by TNFα nor by PMA, all of which are known to activate the NF-κB signaling pathway. Thus, INAP gene expression is not simply regulated by NF-κB signaling. IL-1- and LPS-specific persistent activation of NF-κB has been reported (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar), but rapid IL-1- (and LPS-) specific INAP gene expression cannot be explained by this mechanism. The mechanism is thus obscure at this moment, and further analyses are required. NF-κB is a transcription factor that is known to play an important role in regulating immune and inflammatory responses (1Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1654) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4572) Google Scholar). There are presently five members of the mammalian NF-κB/Rel family, NF-κB1/p50, NF-κB2/p52, c-Rel, RelA/p65, and RelB (1Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1654) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4572) Google Scholar). The classic form of NF-κB, the heterodimer of the NF-κB1/p50 and RelA/p65, is normally retained in the cytoplasm through interactions with inhibitor protein IκB. The IκB family of proteins includes IκBα, IκBβ, IκBε, Bcl-3, NF-κB1/p105, and NF-κB2/p100, all of which possess 5–7 ankyrin-repeat motifs (1Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1654) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4572) Google Scholar), which form a functional unit able to interact with the Rel homology domain of NF-κB. The cytoplasmic retention of the classic form of NF-κB is primarily carried out by IκBα and IκBβ (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 6Weil R. Laurent-Winter C. Israel A. J. Biol. Chem. 1997; 272: 9942-9949Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 7Phillips R.J. Ghosh S. Mol. Cell. Biol. 1997; 17: 4390-4396Crossref PubMed Google Scholar). Inductive stimuli, such as tumor necrosis factor α (TNFα), 1The abbreviations used are:TNFαtumor necrosis factor αIL-1interleukin-1PMAphorbol 12-myristate 13-acetateDAPI4,6-diamidino-2-phenylindoleLPSlipopolysaccharideRXRretinoid X receptorGSTglutathioneS-transferasePAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salineAP-1activating protein-1INAPIL-1-inducible nuclear ankyrin-repeat proteinFCSfetal calf serumDIGdigoxigeninECLenhanced chemiluminescencebpbase pairCIPcalf intestine alkaline phosphataseHAhemagglutinin interleukin-1 (IL-1), and bacterial endotoxin, lead to the phosphorylation and degradation of IκB, allowing NF-κB to translocate into the nucleus and regulate specific gene expression (1Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1654) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4572) Google Scholar). tumor necrosis factor α interleukin-1 phorbol 12-myristate 13-acetate 4,6-diamidino-2-phenylindole lipopolysaccharide retinoid X receptor glutathioneS-transferase polyacrylamide gel electrophoresis phosphate-buffered saline activating protein-1 IL-1-inducible nuclear ankyrin-repeat protein fetal calf serum digoxigenin enhanced chemiluminescence base pair calf intestine alkaline phosphatase hemagglutinin IκBα is degraded in response to the NF-κB inducers TNFα, IL-1, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and double-stranded RNA. In contrast, IκBβ is degraded only when cells are stimulated with IL-1 or LPS, both of which cause persistent long term activation of NF-κB (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 6Weil R. Laurent-Winter C. Israel A. J. Biol. Chem. 1997; 272: 9942-9949Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 7Phillips R.J. Ghosh S. Mol. Cell. Biol. 1997; 17: 4390-4396Crossref PubMed Google Scholar). Following degradation of the initial pool of IκBβ in response to IL-1 or LPS, newly synthesized IκBβ accumulates as an unphosphorylated protein that forms a stable complex with NF-κB and prevents it from binding to newly synthesized IκBα (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 6Weil R. Laurent-Winter C. Israel A. J. Biol. Chem. 1997; 272: 9942-9949Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 7Phillips R.J. Ghosh S. Mol. Cell. Biol. 1997; 17: 4390-4396Crossref PubMed Google Scholar), resulting in the prolonged activation of NF-κB (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 8Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). This unphosphorylated IκBβ cannot block the nuclear localization signal of NF-κB, thus this NF-κB·IκBβ complex translocates into the nucleus. The function of this complex in the nucleus is yet to be elucidated, and the mechanism by which only IL-1 and LPS can degrade IκBβ remains to be resolved. Unlike the other IκB family members, Bcl-3 is a nuclear protein (9Zhang Q. Didonato J.A. Karin M. McKeithan T.W. Mol. Cell. Biol. 1994; 14: 3915-3926Crossref PubMed Scopus (53) Google Scholar, 10Wulczyn F.G. Naumann M. Scheidereit C. Nature. 1992; 358: 597-599Crossref PubMed Scopus (181) Google Scholar, 11Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar). It does not bind to RelA/p65 but specifically binds to NF-κB1/p50 or NF-κB2/p52 homodimers (10Wulczyn F.G. Naumann M. Scheidereit C. Nature. 1992; 358: 597-599Crossref PubMed Scopus (181) Google Scholar, 12Franzoso G. Bours V. Park S. Tomita-Yamaguchi M. Kelly K. Siebenlist U. Nature. 1992; 359: 339-342Crossref PubMed Scopus (264) Google Scholar, 13Kerr L.D. Duckett C.S. Wamsley P. Zhang Q. Chiao P. Nabel G. McKeithan T.W. Baeuerle P.A. Verma I.M. Genes Dev. 1992; 6: 2352-2363Crossref PubMed Scopus (92) Google Scholar, 14Nolan G.P. Fujita T. Bhatia K. Huppi C. Liou H.C. Scott M.L. Baltimore D. Mol. Cell. Biol. 1993; 13: 3557-3566Crossref PubMed Google Scholar) and takes them into the nucleus where it exhibits transactivating activity (11Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar, 15Fujita T. Nolan G.P. Liou H.C. Scott M.L. Baltimore D. Genes Dev. 1993; 7: 1354-1363Crossref PubMed Scopus (348) Google Scholar). The formation of Bcl-3·(NF-κB1/p50)2·κB complex or Bcl-3·(NF-κB2/p52)2·κB complex is regulated by the phosphorylation status of Bcl-3 (14Nolan G.P. Fujita T. Bhatia K. Huppi C. Liou H.C. Scott M.L. Baltimore D. Mol. Cell. Biol. 1993; 13: 3557-3566Crossref PubMed Google Scholar, 16Bundy D.L. McKeithan T.W. J. Biol. Chem. 1997; 272: 33132-33139Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Bcl-3 also interacts with retinoid X receptor (RXR) or activating protein-1 (AP-1) and functions as their transcription coactivator (17Na S.Y. Choi H.S. Kim J.W. Na D.S. Lee J.W. J. Biol. Chem. 1998; 273: 30933-30938Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Na S.Y. Choi J.E. Kim H.J. Jhun B.H. Lee Y.C. Lee J.W. J. Biol. Chem. 1999; 274: 28491-28496Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, the detailed characters of this unique IκB family member remain mysterious. Here we identified a novel IκB family member, termed IL-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression is specifically induced by IL-1. INAP was found to be weakly homologous to Bcl-3 and localized in the nucleus like Bcl-3. We discuss here the possible function of this novel IκB family member. DISCUSSIONWe isolated a novel IL-1-inducible nuclear factor, INAP, which is related to the IκB family and the Rel family. It is well known that IκB family members bind to the RelA/p65·NF-κB1/p50 complex and prevents the complex from activating and translocating into the nucleus. Therefore, possible interaction of INAP with the RelA/p65·NF-κB1/p50 complex was examined by immunoprecipitation followed by immunoblot analysis. However, we failed to detect the direct and/or indirect binding of INAP to RelA/p65 (data not shown). Furthermore, one of the most important factors induced by IL-1 stimulation is IL6, of which gene expression is regulated by C/EBPβ (NF-IL6), AP-1, and NF-κB (21Sanceau J. Kaisho T. Hirano T. Wietzerbin J. J. Biol. Chem. 1995; 270: 27920-27931Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 22Miyazawa K. Mori A. Yamamoto K. Okudaira H. J. Biol. Chem. 1998; 273: 7620-7627Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Therefore, we also examined the possible interactions of INAP with C/EBPβ and c-Fos/c-Jun in nuclear extracts prepared from IL-1α-stimulated OP9 cells. Once again, we could not detect the interactions of INAP with C/EBPβ or with c-Fos/c-Jun (data not shown). Moreover, the fact that Bcl-3, the protein most closely related to INAP, associates NF-κB1/p50 or NF-κB2/p52 homodimers and modulates their transactivation activities (11Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar, 16Bundy D.L. McKeithan T.W. J. Biol. Chem. 1997; 272: 33132-33139Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) motivated us to examine whether INAP associates with NF-κB1/p50 or NF-κB2/p52 in IL-1-stimulated nucleus. None of these interactions, however, was detected by immunoprecipitation followed by immunoblot analysis (data not shown). It has also been reported that Bcl-3 binds to RXR (17Na S.Y. Choi H.S. Kim J.W. Na D.S. Lee J.W. J. Biol. Chem. 1998; 273: 30933-30938Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) or to AP-1 (18Na S.Y. Choi J.E. Kim H.J. Jhun B.H. Lee Y.C. Lee J.W. J. Biol. Chem. 1999; 274: 28491-28496Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and regulate specific gene expression, and thus we also examined the possible interactions of INAP with RXR or with AP-1 but failed to detect the bindings (data not shown). We concluded that INAP could not bind to any of the binding partners with which Bcl-3 has been reported to interact and that INAP is a very unique protein in the IκB family and is clearly distinct even from the most closely related IκB family member, Bcl-3. To determine the biological function of INAP on IL-1 signalings it is very important to identify the INAP-binding proteins in the IL-1-stimulated nucleus by other means such as yeast two-hybrid screening, pull-down experiments, and far-Western screening.INAP was found to be a novel nuclear factor related to the IκB family, but by IL-1 stimulation INAP was newly produced and accumulated in the nucleus, rather than being degraded as were other IκB family members. From a gene expression point of view, INAP is quite a distinct protein from these family members. There exists no obvious nuclear localization signal in INAP, but it does exist in the nucleus. Furthermore, INAP was not phosphorylated no matter whether it was localized in the cytoplasm or nucleus, indicating that the phosphorylation status of INAP does not affect its subcellular localization.A few potential NF-κB binding sites were found around −340 bp upstream from the initiation codon in human INAP gene promoter regions. However, we demonstrated that INAP gene expression was rapidly induced by IL-1 but not by TNFα nor by PMA, all of which are known to activate the NF-κB signaling pathway. Thus, INAP gene expression is not simply regulated by NF-κB signaling. IL-1- and LPS-specific persistent activation of NF-κB has been reported (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar), but rapid IL-1- (and LPS-) specific INAP gene expression cannot be explained by this mechanism. The mechanism is thus obscure at this moment, and further analyses are required. We isolated a novel IL-1-inducible nuclear factor, INAP, which is related to the IκB family and the Rel family. It is well known that IκB family members bind to the RelA/p65·NF-κB1/p50 complex and prevents the complex from activating and translocating into the nucleus. Therefore, possible interaction of INAP with the RelA/p65·NF-κB1/p50 complex was examined by immunoprecipitation followed by immunoblot analysis. However, we failed to detect the direct and/or indirect binding of INAP to RelA/p65 (data not shown). Furthermore, one of the most important factors induced by IL-1 stimulation is IL6, of which gene expression is regulated by C/EBPβ (NF-IL6), AP-1, and NF-κB (21Sanceau J. Kaisho T. Hirano T. Wietzerbin J. J. Biol. Chem. 1995; 270: 27920-27931Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 22Miyazawa K. Mori A. Yamamoto K. Okudaira H. J. Biol. Chem. 1998; 273: 7620-7627Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Therefore, we also examined the possible interactions of INAP with C/EBPβ and c-Fos/c-Jun in nuclear extracts prepared from IL-1α-stimulated OP9 cells. Once again, we could not detect the interactions of INAP with C/EBPβ or with c-Fos/c-Jun (data not shown). Moreover, the fact that Bcl-3, the protein most closely related to INAP, associates NF-κB1/p50 or NF-κB2/p52 homodimers and modulates their transactivation activities (11Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar, 16Bundy D.L. McKeithan T.W. J. Biol. Chem. 1997; 272: 33132-33139Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) motivated us to examine whether INAP associates with NF-κB1/p50 or NF-κB2/p52 in IL-1-stimulated nucleus. None of these interactions, however, was detected by immunoprecipitation followed by immunoblot analysis (data not shown). It has also been reported that Bcl-3 binds to RXR (17Na S.Y. Choi H.S. Kim J.W. Na D.S. Lee J.W. J. Biol. Chem. 1998; 273: 30933-30938Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) or to AP-1 (18Na S.Y. Choi J.E. Kim H.J. Jhun B.H. Lee Y.C. Lee J.W. J. Biol. Chem. 1999; 274: 28491-28496Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and regulate specific gene expression, and thus we also examined the possible interactions of INAP with RXR or with AP-1 but failed to detect the bindings (data not shown). We concluded that INAP could not bind to any of the binding partners with which Bcl-3 has been reported to interact and that INAP is a very unique protein in the IκB family and is clearly distinct even from the most closely related IκB family member, Bcl-3. To determine the biological function of INAP on IL-1 signalings it is very important to identify the INAP-binding proteins in the IL-1-stimulated nucleus by other means such as yeast two-hybrid screening, pull-down experiments, and far-Western screening. INAP was found to be a novel nuclear factor related to the IκB family, but by IL-1 stimulation INAP was newly produced and accumulated in the nucleus, rather than being degraded as were other IκB family members. From a gene expression point of view, INAP is quite a distinct protein from these family members. There exists no obvious nuclear localization signal in INAP, but it does exist in the nucleus. Furthermore, INAP was not phosphorylated no matter whether it was localized in the cytoplasm or nucleus, indicating that the phosphorylation status of INAP does not affect its subcellular localization. A few potential NF-κB binding sites were found around −340 bp upstream from the initiation codon in human INAP gene promoter regions. However, we demonstrated that INAP gene expression was rapidly induced by IL-1 but not by TNFα nor by PMA, all of which are known to activate the NF-κB signaling pathway. Thus, INAP gene expression is not simply regulated by NF-κB signaling. IL-1- and LPS-specific persistent activation of NF-κB has been reported (4Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar, 5Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar), but rapid IL-1- (and LPS-) specific INAP gene expression cannot be explained by this mechanism. The mechanism is thus obscure at this moment, and further analyses are required.
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