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• W2005133554 abstract "Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-κB activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-κB), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKKβ-, TNF-, and IL1-induced NF-κB activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-κB activation. Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-κB activation pathways. Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-κB activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-κB), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKKβ-, TNF-, and IL1-induced NF-κB activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-κB activation. Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-κB activation pathways. tumor necrosis factor receptor 1 Janus N-terminal kinase tumor necrosis factor receptor associated death domain protein receptor-interacting protein tumor necrosis factor receptor associated factor Fas associated death domain protein inhibitory κB kinase nuclear factor kappa B interferon response factor 1 zinc finger protein inhibiting NF-κB rapid amplification of cDNA end ring-like domain proline-rich domain interleukin-1 hemagglutinin interferon cytomegalovirus 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside β-galactosidase Tumor necrosis factor receptor 1 (TNF-R1)1 is a prototypical member of the TNF receptor family (1Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar). TNF stimulation of TNF-R1 simultaneously induces three divergent effects: apoptosis, activation of the transcription factor NF-κB, and the serine/threonine protein kinase JNK (1Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar). TNF-R1 contains a death domain, which interacts with the cytoplasmic death domain-containing protein TRADD in a TNF-dependent process (2Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 3Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1747) Google Scholar, 4Shu H.B. Takeuchi M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13973-13978Crossref PubMed Scopus (366) Google Scholar, 5Tartaglia L.A. Ayres T.M. Wong G.H.W. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1169) Google Scholar). Once TRADD is recruited to TNF-R1, it functions as an adapter protein to recruit several structurally and functionally divergent proteins, including FADD, RIP, TRAF2, and cellular inhibitor of apoptosis protein (cIAP) (2Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 4Shu H.B. Takeuchi M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13973-13978Crossref PubMed Scopus (366) Google Scholar). The interaction of TRADD with FADD leads to apoptosis through activation of a caspase cascade, which is initiated by the interaction of FADD with caspase-8 (2Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 6Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2111) Google Scholar, 7Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2161) Google Scholar, 8Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 9Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Ni A. Shevchenko J. Scaffid C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2741) Google Scholar, 10Yeh W.C. Pompa J.L. McCurrach M.E. Shu H.B. Elia A.J. Ng A. Shahinian M. Wajegam A. El-Mithchell K. Deiry W.S. Lowe S.W. Goeddel D.V. Mak T.W. Science. 1998; 279: 1954-1958Crossref PubMed Scopus (803) Google Scholar). The interaction of TRADD with TRAF2 and RIP activates a downstream IκB kinase complex called IKK, which contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ/NEMO (11DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar, 12Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4084) Google Scholar, 13Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 14Mercurio F. Zhu H. Murray B.W. Shevchenko A. Li B.L. Bennett J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 15Regnier 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 (1072) Google Scholar, 16Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-330Crossref PubMed Scopus (853) Google Scholar, 17Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 18Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1589) Google Scholar). The activated IKK phosphorylates IκBs, leading to their degradation and subsequent activation of NF-κB (11DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar, 12Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4084) Google Scholar, 13Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 14Mercurio F. Zhu H. Murray B.W. Shevchenko A. Li B.L. Bennett J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 15Regnier 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 (1072) Google Scholar, 16Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-330Crossref PubMed Scopus (853) Google Scholar, 17Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 18Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1589) Google Scholar). RIP is a unique signal transducer in the TNF-R1-mediated NF-κB activation pathway. RIP was first identified as a Fas-interacting protein by the yeast two-hybrid system (20Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (865) Google Scholar). It was later demonstrated that RIP is a component of the TNF-R1 signaling complex (4Shu H.B. Takeuchi M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13973-13978Crossref PubMed Scopus (366) Google Scholar, 21Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar). Gene knock-out experiments suggest that RIP is required for TNF-R1-mediated NF-κB activation but is not required for Fas- and TNF-R1-mediated apoptosis (22Kelliher M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 23Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (469) Google Scholar). RIP is a serine/threonine kinase that contains three domains, including an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (4Shu H.B. Takeuchi M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13973-13978Crossref PubMed Scopus (366) Google Scholar, 20Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (865) Google Scholar). RIP interacts with TRADD through their respective death domains. The intermediate domain of RIP interacts with the RING finger domain of TRAF2, and this interaction is required for RIP-mediated NF-κB activation (21Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar). Recently, it has been suggested that RIP directly interacts with IKKγ and therefore recruits IKK to the TNF-R1 complex (24Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). However, studies with RIP- and TRAF2-deficient cells indicate that TRAF2, but not RIP, is required for recruitment of the IKK complex to TNF-R1, whereas RIP is required for activating IKK (25Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z.G. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 26Devin A. Lin Y. Li S. Yamaoka Z. Karin M. Liu Z.G. Cell. Biol. 2001; 21: 3986-3994Google Scholar). Although RIP is a serine/threonine kinase, its kinase activity is not required for RIP-mediated NF-κB activation (21Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar, 22Kelliher M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 23Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (469) Google Scholar, 25Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z.G. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). It has been proposed that RIP may activate IKK through a putative IKK kinase (25Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z.G. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar), which is probably MEKK3 (27Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (353) Google Scholar). However, the precise mechanisms responsible for RIP-mediated IKK activation are not known. In addition, it is not known whether or how TNF-R1-mediated NF-κB activation pathway is regulated at the level of RIP. To better understand how RIP signals, we performed yeast two-hybrid screening for additional RIP-interacting proteins. This search identified a novel RING-like zinc finger domain-containing protein designated as ZIN (zinc finger proteininhibiting NF-κB). Our results suggest that ZIN is an inhibitor of RIP-mediated NF-κB activation pathways. The recombinant human TNF, IL1, and IFN-γ (R&D Systems Inc., Minneapolis, MN), the monoclonal antibodies against the FLAG (Sigma), Myc (Santa Cruz Biotechnology, Santa Cruz, CA), and the HA epitopes (Covance, Berkely, CA) were purchased from the indicated resources. The human embryonic kidney 293, the B lymphoma RPMI8226, and the T lymphoma Jurkat cells were purchased from ATCC (Manassas, VA). The rabbit polyclonal antiserum against human ZIN was raised against a 21-mer peptide having the following amino acid sequence: QKEAEEEQKRKNGENTFKRIG. The NF-κB (Dr. Gary Johnson, University of Colorado Health Sciences Center) and IRF-1 (Dr. Uli Schindler, Tularik Inc.) luciferase reporter constructs were provided by the indicated investigators. Mammalian expression vectors for HA- or FLAG-tagged RIP, ZIN, and its deletion mutants were constructed by PCR amplification of the corresponding cDNA fragments and subsequently cloning into a CMV promoter-based vector containing a 5′-HA or FLAG tag. To construct a RIP bait vector, a cDNA fragment encoding full-length RIP was inserted in-frame into the Gal4 DNA-binding domain vector pGBT (CLONTECH, Palo Alto, CA). The human B cell cDNA library (ATCC, Manassas, VA) was screened as described (2Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 3Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1747) Google Scholar,28Hu W.H. Johnson H. Shu H.B. J. Biol. Chem. 2000; 275: 10838-10844Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). 5′ RACE was performed using a mixture of several yeast two-hybrid cDNA libraries as template. The 5′ primer corresponds to the sequence of the GAL4 activation domain: ACCGTCGACTGAAGATACCCCACCAAACC. The 3′ primer corresponds to the coding sequence of ZIN: AAGCGGCCGCCATCAGAAGCGATGC. Human multiple tissue mRNA blots were purchased from CLONTECH. The cDNA probe was an ∼1.0-kb fragment that encodes for amino acids 9–363. The hybridization was performed with the radiolabeled ZIN cDNA probe in the Rapid Hybridization buffer (CLONTECH) under high stringency condition. 293 cells (∼2 × 105) were seeded on 6-well (35-mm) dishes and were transfected the following day by the standard calcium phosphate precipitation (29Sambrook J. Fritch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Within the same experiment, each transfection was performed in triplicate, and where necessary, enough of an amount of empty control plasmid was added to ensure that each transfection continued to receive the same amount of total DNA. To normalize for transfection efficiency, 0.3 μg of RSV-β-gal plasmid was added to each transfection. Luciferase reporter assays were performed using a luciferase assay kit (BD PharMingen) and following the manufacturer’s protocols. β-galactosidase activity was measured using the Galacto-Light chemiluminescent kit (TROPIX, Bedford, MA). Luciferase activities were normalized on the basis of β-galactosidase expression levels. Transfected 293 cells from each 100-mm dish were lysed in 1 ml of lysis buffer (20 mm Tris (pH 7.5), 150 mm NaCl, 1% Triton, 1 mm EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride). For each immunoprecipitation, 0.4-ml aliquots of lysates were incubated with 0.5 μg of the indicated monoclonal antibody or control mouse IgG and 25 μl of a 1:1 slurry of GammaBind G Plus-Sepharose (Amersham Biosciences) for at least 1 h. The Sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mm NaCl. The precipitates were fractionated on SDS-PAGE, and subsequent Western blot analyses were performed as described (2Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 3Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1747) Google Scholar, 28Hu W.H. Johnson H. Shu H.B. J. Biol. Chem. 2000; 275: 10838-10844Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). β-Galactosidase co-transfection assays for determination of cell death were performed as described previously (2Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 3Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1747) Google Scholar, 10Yeh W.C. Pompa J.L. McCurrach M.E. Shu H.B. Elia A.J. Ng A. Shahinian M. Wajegam A. El-Mithchell K. Deiry W.S. Lowe S.W. Goeddel D.V. Mak T.W. Science. 1998; 279: 1954-1958Crossref PubMed Scopus (803) Google Scholar, 28Hu W.H. Johnson H. Shu H.B. J. Biol. Chem. 2000; 275: 10838-10844Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 30Shu H.B. Halpins D.R. Goeddel D.V. Immunity. 1997; 6: 751-763Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Briefly, 293 cells (∼2 × 105) were seeded on 6-well (35-mm) dishes and were transfected the following day with 0.1 μg of pCMV-β-galactosidase plasmid and the indicated testing plasmids. Within the same experiment, each transfection was performed in triplicate, and where necessary, enough of an amount of empty control plasmid was added to ensure that each transfection kept receiving the same amount of total DNA. Approximately 24 h after transfection, the cells were stained with X-gal as described previously (30Shu H.B. Halpins D.R. Goeddel D.V. Immunity. 1997; 6: 751-763Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). The numbers of survived blue cells from five representative viewing fields was determined microscopically. Data shown are averages and standard deviations of one representative experiment in which each transfection has been performed in triplicate. 293 cells cultured on glass coverslips were sequentially plunged into methanol and acetone at −20 °C, each for 10 min. Cells were rehydrated in phosphate-buffered saline and stained with primary antibodies for 1 h at room temperature. Cells were then rinsed with phosphate-buffered saline and stained with either a CyTM3-conjugated Affinipure donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) or Alexa FluorTM 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for 45 min at room temperature. The cells were rinsed with phosphate-buffered saline and mounted in Gel/MountTM(Biomeda Corp., Foster City, CA). Cells were observed with a Leica DMR/XA immunofluorescence microscope using ×100 plan objective. To identify potential RIP-interacting proteins, we used the yeast two-hybrid system to screen a human B cell cDNA library with full-length RIP as bait. We screened a total of 5 × 106 independent library clones and obtained 26 β-galactosidase-positive clones. The inserts of 9 of the 26 clones are not in-frame with the GAL4 activation domain in the library vector. Among the other 17 clones, two encode for FADD, a death domain-containing protein that has been reported to interact with RIP (21Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar), and one encodes part of a novel RING-like zinc finger domain-containing protein, which we designated as ZIN. We further studied ZIN because some of the known RIP-interacting proteins, such as TRAF2 and A20, also contain RING or zinc finger domains. Since the ZIN clone obtained from the yeast two-hybrid screening is not full-length, we obtained its full-length cDNA by a combination of GenBankTM data base searches for ZIN-encoding expressed sequence tag clones and 5′ RACE. These efforts identified a ZIN cDNA of ∼2.1 kb that is capable of encoding a 488-amino acid protein (Fig. 1A). The 5′ of the putative start codon (ATG) has an in-frame stop codon, and the 3′ of the cDNA has a poly(A) tail, suggesting that we obtained a cDNA fragment encoding full-length ZIN (data not shown). Blast searches of the GenBankTM data bases indicate that ZIN has no significant homolog to known proteins except that the C-terminal part of ZIN is almost identical to an uncharacterized, hypothetical protein called TRIAD3 (GenBankTM accession number (NP_061884). Structural analysis suggests that ZIN contains four RING-like zinc finger domains (RLDs) at the middle (amino acids 137–352) and a proline-rich domain (PRD) at the C terminus (amino acids 396–482) (Fig. 1A). The N terminus of ZIN has no detectable similarity with any other proteins. The structural properties suggest that ZIN is probably a zinc-binding protein. Northern blot analysis suggests that RIN is ubiquitously expressed in all examined tissues as two transcripts of ∼3.0 and ∼6.0 kB, respectively (Fig. 1B). ZIN is expressed at relatively higher levels in peripheral blood leukocytes and testis (Fig.1B). To determine whether ZIN is expressed in mammalian cells at protein level, we raised a rabbit polyclonal antiserum against a peptide corresponding to amino acids 370–390 of ZIN. Western blot analysis suggests that ZIN is expressed as an ∼56-kDa protein in all examined human cell lines, including B lymphoma PRMI8226, T lymphoma Jurkat, and embryonic kidney 293 cells (Fig. 2). The size of the endogenous ZIN protein is similar to that of overexpressed ZIN, confirming that the identified ZIN cDNA encodes a full-length protein (Fig. 2). In 293 cells, the ZIN antiserum also recognized a second higher molecular weight band, which may represent a post-translationally modified or alternatively spliced form of ZIN or a different protein in 293 cells. To determine whether full-length ZIN interacts with RIP in mammalian cells, we transfected 293 cells with expression plasmids for FLAG-tagged ZIN and HA-tagged RIP and performed co-immunoprecipitation experiments. These experiments suggest that ZIN interacts with RIP in 293 cells (Fig.3). To determine which domains of ZIN are required for interaction with RIP, we constructed three deletion mutants of ZIN. These include ZIN-(1–364) that contains the N-terminal domain and the RLDs, ZIN-(127–488) that contains the RLDs and the C-terminal PRD, and ZIN-(365–488) that contains only the C-terminal PRD (Fig.3A). Transient transfection and co-immunoprecipitation experiments suggest that the two RLD-containing mutants, ZIN-(1–364) and ZIN-(127–488), but not the RLD-lacking mutant ZIN-(365–488), interact with RIP (Fig. 3B). These data suggest that the RLDs of ZIN are required for interaction with RIP. It has been shown that RIP is absolutely required for TNF-R1-induced NF-κB activation (4Shu H.B. Takeuchi M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13973-13978Crossref PubMed Scopus (366) Google Scholar, 21Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar, 22Kelliher M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 23Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (469) Google Scholar, 24Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 25Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z.G. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 26Devin A. Lin Y. Li S. Yamaoka Z. Karin M. Liu Z.G. Cell. Biol. 2001; 21: 3986-3994Google Scholar). To determine whether ZIN has a similar function, we performed NF-κB luciferase reporter gene assays. These experiments indicated that overexpression of ZIN could not activate NF-κB in 293 cells (Fig. 4,A and C). Instead, overexpression of ZIN inhibited RIP-induced NF-κB activation in a dose-dependent manner (Fig. 4A). To exclude the possibility that ZIN affects RIP expression but not RIP signaling, we examined RIP levels in the same lysates by Western blot. As shown in Fig. 4A, RIP levels were not significantly changed with the increased expression of ZIN. These data suggest that ZIN inhibits RIP-mediated NF-κB activation. The two RLD-containing and RIP-interacting ZIN mutants, ZIN-(1–364) and ZIN-(127–488) (Fig. 3), also inhibited RIP-mediated NF-κB activation in reporter gene assays (data not shown). In contrast, ZIN-(365–488), which does not contain the RLDs and does not interact with RIP (Fig. 3), did not inhibit RIP-mediated NF-κB activation (Fig. 4B). In fact, ZIN-(365–488) could weakly activate NF-κB and potentiate RIP-mediated NF-κB activation (Fig.4B). These data suggest that the RLDs of ZIN are required for inhibition of RIP-mediated NF-κB activation. Previous studies indicate that RIP activates NF-κB through IKK (11DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar, 12Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4084) Google Scholar, 13Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 14Mercurio F. Zhu H. Murray B.W. Shevchenko A. Li B.L. Bennett J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 15Regnier 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 (1072) Google Scholar, 16Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-330Crossref PubMed Scopus (853) Google Scholar, 17Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 18Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1589) Google Scholar). We examined whether ZIN could inhibit IKKβ-mediated NF-κB activation. As shown in Fig. 4C, ZIN also inhibited IKKβ-mediated NF-κB activation, whereas ZIN-(365–488) weakly potentiated IKKβ-mediated NF-κB activation (Fig. 4D). In these experiments, neither ZIN nor ZIN-(365–488) affected expression levels of IKKβ. These data, although unexpected because IKKβ is a downstream protein of RIP, suggest that ZIN can inhibit IKKβ-mediated NF-κB activation. Since ZIN can inhibit RIP- and IKKβ-mediated NF-κB activation, we determined whether ZIN inhibits TNF- and IL1-induced NF-κB activation. As shown in Fig. 5,A and B, ZIN, but not ZIN-(365–488), inhibited TNF- and IL1-induced NF-κB activation in a dose-dependent manner. In contrast, ZIN did not inhibit IFN-γ-induced IRF-1 activation (Fig. 5C), suggesting that ZIN specifically inhibits NF-κB activation by TNF and IL1. Previously, it has been suggested that overexpression of RIP potently induces apoptosis (20Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (865) Google Scholar, 21Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar). Since ZIN is a RIP-interacting protein, we examined whether ZIN is involved in RIP-induced apoptosis. As shown in Fig.6A, overexpression of ZIN did not induce apoptosis, but potentiated RIP-induced apoptosis in a dose-dependent manner. In 293 cells, TNF alone does not induce apoptosis. However, overexpression of ZIN could consistently sensitize ∼30% of transfected 293 cells to TNF-induced apoptosis (Fig. 6B). IKKβ(K/A), an IKKβ kinase-inactive mutant that can inhibit TNF-induced NF-κB activation (17Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 28Hu W.H. Johnson H. Shu H.B. J. Biol. Chem. 2000; 275: 10838-10844Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), could also sensitize 293 cells to TNF-induced apoptosis (Fig. 6B). One of the possible explanations for inhibition of RIP-mediated NF-κB activation by ZIN is that ZIN may dissociate TRAF2 from RIP. TRAF2 contains one RING finger domain and four zinc finger domains at its N terminus (43Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (932) Google Scholar). It has been shown that the RING finger domain of TRAF2 interacts with the intermediate domain of RIP and that this interaction is important for TRAF2- and RIP-mediated NF-κB activation (21Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar). Since the RLDs of ZIN are also responsible for interacting with RIP, we investigated the possibility that ZIN may compete with TRAF2 for binding to RIP. To do this, we transfected 293 cells with constant amounts of expression plasmids for TRAF2 and RIP and increased amounts of expression plasmid for ZIN. Co-immunoprecipitation experiments indicated that ZIN could not compete with TRAF2 for interaction with RIP (data not shown). ZIN has a putative nuclear localization signal sequence (amino acids 47–52). To determine the cellular localization of ZIN, we performed immunofluorescent microscopy. These experiments suggest that ZIN is mainly localized in the cytoplasm (Fig. 7). To determine whether RIP colocalizes with ZIN, we transfected 293 cells with an expression plasmid for HA-tagged RIP and performed double immunofluorescent staining. These experiments suggest that overexpressed RIP overlaps with endogenous ZIN (Fig. 7). In addition, we noticed that overexpression of RIP caused substantial aggregation of ZIN (Fig. 7), pointing to the possibility that overexpression of RIP leads to the formation of complexes that contain RIP, ZIN, and other molecules. During the past several years, tremendous progress has been achieved on the molecular mechanisms of TNF-R1 signaling. TNF stimulation of TNF-R1 leads to recruitment of the adapter protein TRADD to the TNF-R1 signaling complex (2Hsu H. Shu H.B. Pan M.G. Goeddel D.V. 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Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 15Regnier 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 (1072) Google Scholar, 16Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-330Crossref PubMed Scopus (853) Google Scholar, 17Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 18Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1589) Google Scholar). These models have now become paradigms of how all TNF receptor family members signal. One of the major unsolved questions on TNF-R1 signaling is how TRAF2 and RIP activate downstream IKK. One group proposed a direct interaction between RIP and the IKKγ subunit of the IKK complex (24Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). However, studies with RIP- and TRAF2-deficient cells indicate that TRAF2, but not RIP, is required for recruitment of the IKK complex to TNF-R1, whereas RIP is required for activating IKK, probably through MEKK3 (25Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z.G. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 26Devin A. Lin Y. Li S. Yamaoka Z. Karin M. Liu Z.G. Cell. Biol. 2001; 21: 3986-3994Google Scholar, 27Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (353) Google Scholar). Currently, the precise mechanisms responsible for RIP-mediated IKK activation are not known. We have used the yeast two-hybrid system to identify additional RIP-interacting proteins. This search identified ZIN as a novel RIP-interacting protein. ZIN contains four RLDs at the middle and a proline-rich domain at its C terminus. Overexpression of ZIN inhibits RIP-mediated NF-κB activation, and the RLDs of ZIN are required for this inhibitory activity. Unexpectedly, overexpression of ZIN also inhibited IKKβ-mediated NF-κB activation. In co-immunoprecipitation experiments, however, we failed to detect an interaction between IKKβ and ZIN. The simplest explanation for this observation is that ZIN also targets a downstream signaling component of IKKβ. ZIN can inhibit TNF-induced NF-κB activation in 293 cells. Since only TNF-R1, but not TNF-R2, is expressed in 293 cells, these data suggest that ZIN inhibits TNF-R1-induced NF-κB activation. This is consistent with the notion that RIP is required for TNF-R1-induced NF-κB activation. Surprisingly, ZIN also inhibits IL1-induced NF-κB activation. Inhibition of TNF- and IL1-induced NF-κB activation is not due to a general inhibitory effect of transcription by ZIN because ZIN does not inhibit IFN-γ-induced IRF-1 activation. Our findings suggest that ZIN has multiple targets in TNF- and IL1-induced NF-κB activation pathways. Currently, we do not know which protein(s) in the IL-1 signaling pathway are targeted by ZIN. Interestingly, ZIN-(365–488), a mutant that does not interact with RIP, can weakly activate NF-κB and potentiate RIP-, IKKβ-, TNF-, and IL1-induced NF-κB activation. It is possible that ZIN-(365–488) can at least partially neutralize the inhibitory effect of full-length ZIN. The structural and functional properties of ZIN are very similar to a previously characterized protein A20 (24Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 36Dixit V.M. Green S. Sarma V. Prochownik E.V. J. Biol. Chem. 1990; 265: 2973-2978Abstract Full Text PDF PubMed Google Scholar, 37Heyninck K. Beyaert R. FEBS Lett. 1999; 442: 147-150Crossref PubMed Scopus (150) Google Scholar, 38Heyninck K. Valck D.D. Vanden Berghe W. van Criekinge W. Contreras R.R. Fiers W. Haegeman G Beyaert R. J. Cell Biol. 1999; 145: 1471-1482Crossref PubMed Scopus (253) Google Scholar, 39Jäättelä M. Mouritzen H. Elling F. Bastholm L. J. Immunol. 1996; 156: 1166-1173PubMed Google Scholar, 40Klinkenberg M. Huffel S.V. Heyninck K. Beyaert R. FEBS Lett. 2001; 498: 93-97Crossref PubMed Scopus (65) Google Scholar, 41Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1194) Google Scholar, 42Song H.Y. Rothe M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6721-6725Crossref PubMed Scopus (374) Google Scholar). Although the sequence of ZIN has no significant homology to A20, both contain putative zinc finger structures. A20 can interact with multiple molecules, including TRAF1, -2, and -6, IKKγ/NEMO, and ABIN (24Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 36Dixit V.M. Green S. Sarma V. Prochownik E.V. J. Biol. Chem. 1990; 265: 2973-2978Abstract Full Text PDF PubMed Google Scholar, 37Heyninck K. Beyaert R. FEBS Lett. 1999; 442: 147-150Crossref PubMed Scopus (150) Google Scholar, 38Heyninck K. Valck D.D. Vanden Berghe W. van Criekinge W. Contreras R.R. Fiers W. Haegeman G Beyaert R. J. Cell Biol. 1999; 145: 1471-1482Crossref PubMed Scopus (253) Google Scholar, 39Jäättelä M. Mouritzen H. Elling F. Bastholm L. J. Immunol. 1996; 156: 1166-1173PubMed Google Scholar, 40Klinkenberg M. Huffel S.V. Heyninck K. Beyaert R. FEBS Lett. 2001; 498: 93-97Crossref PubMed Scopus (65) Google Scholar, 41Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1194) Google Scholar, 42Song H.Y. Rothe M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6721-6725Crossref PubMed Scopus (374) Google Scholar). Overexpression of A20 inhibits TNF- and IL-1-induced NF-κB activation (36Dixit V.M. Green S. Sarma V. Prochownik E.V. J. Biol. Chem. 1990; 265: 2973-2978Abstract Full Text PDF PubMed Google Scholar, 37Heyninck K. Beyaert R. FEBS Lett. 1999; 442: 147-150Crossref PubMed Scopus (150) Google Scholar, 38Heyninck K. Valck D.D. Vanden Berghe W. van Criekinge W. Contreras R.R. Fiers W. Haegeman G Beyaert R. J. Cell Biol. 1999; 145: 1471-1482Crossref PubMed Scopus (253) Google Scholar, 39Jäättelä M. Mouritzen H. Elling F. Bastholm L. J. Immunol. 1996; 156: 1166-1173PubMed Google Scholar, 40Klinkenberg M. Huffel S.V. Heyninck K. Beyaert R. FEBS Lett. 2001; 498: 93-97Crossref PubMed Scopus (65) Google Scholar, 41Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1194) Google Scholar, 42Song H.Y. Rothe M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6721-6725Crossref PubMed Scopus (374) Google Scholar). Gene knock-out studies have demonstrated a critical role for A20 in inhibition of TNF-induced NF-κB activation and inflammation (41Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1194) Google Scholar). Interestingly, it has been shown that the zinc finger domains of A20 are also required for its inhibition of TNF- and IL-1-induced NF-κB activation (40Klinkenberg M. Huffel S.V. Heyninck K. Beyaert R. FEBS Lett. 2001; 498: 93-97Crossref PubMed Scopus (65) Google Scholar). Since the RLDs of ZIN are responsible for interacting with RIP, it is possible that ZIN may compete with TRAF2 for binding to RIP and therefore inhibit RIP-mediated NF-κB activation. However, co-immunoprecipitation experiments indicate that this is not the case, suggesting that other mechanisms are involved in ZIN-mediated inhibition of RIP-induced NF-κB activation. Overexpression of ZIN potentiates RIP- and TNF-induced apoptosis in 293 cells. Previously, it has been shown that NF-κB activation can prevent cells from apoptosis induced by TNF and other stimuli (13Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar,44Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2935) Google Scholar, 45Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2449) Google Scholar, 46Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2580) Google Scholar). The simplest explanation for ZIN′s potentiation of RIP- and TNF-induced apoptosis is that ZIN inhibits RIP-induced NF-κB activation and thus sensitizes cells to apoptosis. Sequence analysis suggests that a bipartite nuclear localization signal sequence exists at amino acids 36–53 of ZIN. This raises the possibility that ZIN is a nuclear protein. However, our immunofluorescent staining experiments suggest that ZIN is mainly localized to the cytoplasm. Moreover, these experiments indicate that overexpressed RIP colocalizes with ZIN and causes the aggregation of ZIN. These data provide additional evidences that ZIN is functionally associated with RIP. In conclusion, we have identified a novel RING-like zinc finger protein that is capable of inhibiting TNF- and IL1-induced NF-κB activation. The identification of ZIN, like A20, may shed new light on the negative regulation of TNF- and IL1-induced NF-κB activation pathways. However, the data provided in this study were mostly from protein overexpression; a physiological role for ZIN needs to be defined by experiments dealing with endogenous protein and/or gene knock-out studies." @default.
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• W2005133554 title "A Novel Zinc Finger Protein Interacts with Receptor-interacting Protein (RIP) and Inhibits Tumor Necrosis Factor (TNF)- and IL1-induced NF-κB Activation" @default.
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