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• W2106369180 abstract "The NF-κB signaling pathway plays a crucial role in the immune, inflammatory, and apoptotic responses. Recently, we identified the NF-κB Essential Modulator (NEMO) as an essential component of this pathway. NEMO is a structural and regulatory subunit of the high molecular kinase complex (IKK) responsible for the phosphorylation of NF-κB inhibitors. Data base searching led to the isolation of a cDNA encoding a protein we called NRP (NEMO-related protein), which shows a strong homology to NEMO. Here we show that NRP is present in a novel high molecular weight complex, that contains none of the known members of the IKK complex. Consistently, we could not observe any effect of NRP on NF-κB signaling. Nonetheless, we could demonstrate that treatment with phorbol esters induces NRP phosphorylation and decreases its half-life. This phosphorylation event could only be inhibited by K-252a and stauroporin. We also show that de novo expression of NRP can be induced by interferon and tumor necrosis factor α and that these two stimuli have a synergistic effect on NRP expression. In addition, we observed that endogenous NRP is associated with the Golgi apparatus. Analogous to NEMO, we find that NRP is associated in a complex with two kinases, suggesting that NRP could play a similar role in another signaling pathway. The NF-κB signaling pathway plays a crucial role in the immune, inflammatory, and apoptotic responses. Recently, we identified the NF-κB Essential Modulator (NEMO) as an essential component of this pathway. NEMO is a structural and regulatory subunit of the high molecular kinase complex (IKK) responsible for the phosphorylation of NF-κB inhibitors. Data base searching led to the isolation of a cDNA encoding a protein we called NRP (NEMO-related protein), which shows a strong homology to NEMO. Here we show that NRP is present in a novel high molecular weight complex, that contains none of the known members of the IKK complex. Consistently, we could not observe any effect of NRP on NF-κB signaling. Nonetheless, we could demonstrate that treatment with phorbol esters induces NRP phosphorylation and decreases its half-life. This phosphorylation event could only be inhibited by K-252a and stauroporin. We also show that de novo expression of NRP can be induced by interferon and tumor necrosis factor α and that these two stimuli have a synergistic effect on NRP expression. In addition, we observed that endogenous NRP is associated with the Golgi apparatus. Analogous to NEMO, we find that NRP is associated in a complex with two kinases, suggesting that NRP could play a similar role in another signaling pathway. interleukin-1 lipopolysaccharide tumor necrosis factor α phorbol 12-myristate 13-acetate NF-κBEssential Modulator NEMO-related protein dithiothreitol phosphate-buffered saline glutathioneS-transferase protein kinase C The transcription factor NF-κB plays a pivotal role in many cellular processes such as immune responses, inflammation, and apoptosis (1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5515) Google Scholar, 2Karin M. Oncogene. 1999; 18: 6867-6874Crossref PubMed Scopus (983) Google Scholar). NF-κB is composed of homo- and heterodimers of various members of the NF-κB/Rel family (3Siebenlist U. Fransozo G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-430Crossref PubMed Scopus (2003) Google Scholar, 4Miyamoto S. Schmitt M. Verma I.M. Adv. Cancer Res. 1995; 66: 255-292Crossref PubMed Google Scholar) and is retained in an inactive form in the cytoplasm by an inhibitory protein belonging to the IκB family, mainly represented by IκBα, IκBβ, and IκBε (5Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampson-Johannes A. Mondal K. Ralph P. Baldwin A.S. Cell. 1991; 65: 1281-1289Abstract Full Text PDF PubMed Scopus (581) Google Scholar, 6Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (691) Google Scholar, 7Whiteside S.T. Epinat J.C. Rice N.R. Israël A. EMBO J. 1997; 16: 1413-1426Crossref PubMed Scopus (337) Google Scholar). In response to diverse stimuli, including IL-1,1 LPS, TNFα, or PMA, as well as several viral proteins, active NF-κB translocates to the nucleus as a result of the complete proteolytic degradation of the IκB proteins. This mechanism has been best studied for the inhibitor IκBα and demonstrated to involve phosphorylation on two specific serine residues (8Didonato J. Mercurio F. Rosette C. Wuli J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar, 9Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X.X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar, 10Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1307) Google Scholar, 11Rodriguez M.S. Michalopoulos I. Arenzana-Seisdedos F. Hay R.T. Mol. Cell. Biol. 1995; 15: 2413-2419Crossref PubMed Google Scholar, 12Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (927) Google Scholar, 13Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N.R. Israël A. Mol. Cell. Biol. 1995; 15: 5339-5345Crossref PubMed Google Scholar) followed by polyubiquitination and degradation by the 26 S proteasome (14Chen Z.J. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1159) Google Scholar). More recently a specific serine protein kinase activity responsible for IκBα phosphorylation has been identified as a large cytoplasmic complex (600–800 kDa) containing two catalytic subunits (IKK1/α and IKK2/β) (15Didonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1890) Google Scholar, 16Mercurio F. Zhu H.Y. Murray B.W. Shevchenko A. Bennett B.L. Li J.W. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1831) Google Scholar, 17Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z.D. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1060) 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 (1562) Google Scholar). IKKα and IKKβ are related molecules of 85 and 87 kDa, respectively, and share 50% sequence similarity. Both proteins contain NH2-terminal kinase domains, leucine zipper, and helix-loop-helix motifs (16Mercurio F. Zhu H.Y. Murray B.W. Shevchenko A. Bennett B.L. Li J.W. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1831) 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 (1562) Google Scholar). In vitro phosphorylation studies have shown that both kinases can phosphorylate IκBα on serines 32 and 36, but IKKβ is more active in this regard. Recently, we have cloned, by complementation of an NF-κB unresponsive cell line, a third component of the IKK complex (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar), that we called NEMO (NF-κB Essential Modulator). NEMO is a 48-kDa glutamine-rich protein, which lacks a catalytic subunit, but contains two coiled-coil motifs, a leucine zipper, and a COOH-terminal zinc finger (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar). NEMO is a regulatory and structural subunit of the complex which seems to interact directly with IKKβ, but not with IKKα (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar). Studies with NH2-terminal deletion mutants of NEMO show that the first 235 residues contain the site of interaction with IKKβ (21Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar). The human homolog of NEMO, IKKγ/IKKAP, has been cloned after purification of the IKK complex (21Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 22Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (836) Google Scholar). Data base searching led to the isolation of a cDNA encoding aNEMO-related protein (NRP), which shares 53% sequence similarity with NEMO. NEMO and NRP were also identified in a yeast two-hybrid screen using an adenovirus protein (Ad E3-14.7K) as a bait. Interestingly, these proteins, named FIP-3 and FIP-2, respectively, could block the anti-apoptotic activity of the E3-14.7K protein after TNFα stimulation (23Li Y. Kang J. Friedman J. Tarassishin L. Ye J. Kovalenko A. Wallach D. Horwitz M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1042-1047Crossref PubMed Scopus (155) Google Scholar, 24Li Y. Kang J. Horwitz M.S. Mol. Cell. Biol. 1998; 18: 1601-1610Crossref PubMed Scopus (175) Google Scholar). In this study, we have characterized NRP more thoroughly. In order to determine whether NRP is involved in NF-κB signaling, we investigated whether it is associated with the IKK complex, or whether it could complement a NEMO-deficient cell line. We could not observe any role of NRP in NF-κB signaling, but we found that the COOH-terminal zinc finger of NRP can functionally replace that of NEMO. We could demonstrate that NRP is present in a high molecular weight complex, smaller than the IKK complex, and is associated with two kinase activities. These results demonstrate that NRP could fulfill a similar regulatory function to that of NEMO in a non-NF-κB-dependent pathway. Moreover in an effort to characterize the signaling pathways that might target NRP, we showed that PMA stimulation induces its phosphorylation. This phosphorylation was inhibited by K-252a and stauroporin. Finally, we also demonstrated that NRP expression is synergistically induced by interferon and TNFα. 70Z/3 is a murine pre-B cell line and its variant 1.3E2 is a NEMO-deficient cell line (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar). E29.1 is a CD4-negative, CD8-negative mouse T-cell hybridoma (kindly provided by P. Truffa Bachi). Jurkat is a human leukemia T cell line. These cells were maintained in RPMI medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. In the case of 70Z/3 and 1.3E2, 50 μmβ-mercaptoethanol were added in the medium. HeLa cells and 293T cells were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 7% CO2. The antisera used were the following: anti-IKK2 (Santa Cruz, ref. H470) is a polyclonal rabbit antiserum. Anti-CD28.2 is a murine monoclonal antibody directed against human CD28 (Immunotech). T cells were activated by stimulation with an anti-TCR murine monoclonal antibody (Vit-3) (kindly provided by W. Knapp). Anti-NEMO rabbit polyclonal antibody (serum 44106) was raised against a TrpE fusion protein encompassing amino acids 30–329 of murine NEMO (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar). Anti-NRP (serum 46096) is a polyclonal rabbit antiserum generated against a TrpE fusion protein encompassing amino acids 84–164 of human NRP. NRP open reading frames were amplified by the polymerase chain reaction. Expression vectors for transfection into 1.3E2 cells were obtained by subcloning cDNAs encoding NEMO, NRP, or their derivatives into the plasmid pcDNA-3 (Invitrogen). NEMO Δ ZF represents amino acids 1–385 of NEMO. For the cloning of NEMO Δ ZF-NRP ZF, NRP plasmid was digested with BstBI/EcoRI and this fragment was ligated into the XbaI site of the NEMO plasmid. Details of all the constructions used in this article are available upon request. Cells (5 × 106) were incubated for the indicated time at 37 °C in 1 ml of regular growth medium containing 100 IU/ml TNFα (Pharmingen), 20 ng/ml IL-1, 10 ng/ml interferon α/β, 10 ng/ml interferon γ, 15 μg/ml LPS (Sigma), 50 ng/ml PMA (Sigma), 1 μm calcium ionophore (Sigma), or 50 μg/ml cycloheximide (Sigma). Treatment of the cells with kinase inhibitors or activators was performed at 37 °C for 20 min and was followed by PMA stimulation for 30 additional minutes. The following kinase inhibitors and activators were purchased from Calbiochem and were used at the indicated doses: K-252a (2 μm), KN-93 (10 μm), W7 (50 μm), KN-62 (200 μm), calmidazolium (5 μm), GF 109203X (GFX, 20, 4 nm), GÖ6976 (3 μm), Rottlerin (10 μm), K-252b (250 μm), H7 (1, 5 μm), calphostin C (1 μm), stauroporin (1 μm), genistein (100 μm), thapisgargin (10 μm), PD098059 (50 μm), SB203580 (10 μm), Bt2cAMP (50 μm). Pertussis toxin (CyA), a gift from D. Ladant, Institut Pasteur, was used at 10 μm. 10 × 106 Jurkat cells were activated at 37 °C with 6 μg of the anti-TCR Vit-3 and/or 20 μg of the anti-CD28 antibody at 37 °C. Cells were lysed by adding an equal volume of 1 × CHRIS buffer (50 mm Tris, pH 8.0, 0.5% Nonidet P-40, 200 mm NaCl, and 0.1 mm EDTA), supplemented with 20 μg/ml each of the protease inhibitors leupeptin, aprotinin, and phenylmethylsulfonyl fluoride, as well as the phosphatase inhibitors sodium fluoride (100 mm) and sodium orthovanadate (2 mm). This analysis was performed using the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB). Cells were lysed as described above. Specific polypeptides were then recovered by immunoprecipitation from equivalent amounts of cellular proteins (1 mg). Immune complexes were collected with Staphylococcus aureus protein A (Pansorbin, Calbiochem). After washing the immunoprecipitates 3 times in lysis buffer, the proteins were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Subsequent immunoblots were obtained using the protocol outlined below. Immunoblots were performed according to a previously described protocol (25Weil R. Laurent-Winter C. Israël A. J. Biol. Chem. 1997; 272: 9942-9949Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). We used antiserum, biotinylated protein A (Interchim Pierce), and streptavidin-horseradish peroxidase (Amersham Pharmacia Biotech) at 1/1000 dilution. Proteins transferred to Immobilon membranes (Millipore) were revealed with the SuperSignal chemiluminescent substrate (Interchim Pierce) and immunoreactive products were detected by autoradiography. 1.3E2 cells were transfected by the DEAE dextran method. The total DNA content in each transfection was adjusted to 10 μg. The Ig-(κB)3-LUC reporter has been described previously (26LeBail O. Schmidt Ullrich R. Israël A. EMBO J. 1993; 12: 5043-5049Crossref PubMed Scopus (290) Google Scholar). In the control, the cells were transfected with the appropriate empty parental expression vector pcDNA3. 24 h after transfection, LPS was added 5 h before harvest. Then, the cells were lysed on ice for 10 min in 300 μl of lysis buffer (25 mm Tris phosphate, pH 7.8, 8 mm MgCl2, 1 mmDTT, 1% Triton, 15% glycerol). Cell debris was removed by centrifugation at 13,000 r.p.m. at 4 °C for 5 min. The whole cell extract was used to measure luciferase activity in the lysis buffer containing 1 mmd-luciferin (Roche Molecular Biochemicals) and 20 mm ATP. Cells were lysed with CHRIS buffer as described above. 1 mg of proteins from the lysates were incubated with 5 μl of the NEMO or NRP antibody. The immunoprecipitations were performed as described above, except that the immunoprecipitates were washed 3 times with 1 × CHRIS followed by 3 washes with a kinase buffer containing: 10 mm Hepes, pH 7.5, 10 mm MgCl2, 100 μm Na3VO4, 20 mmβ-glycerophosphate, 2 mm DTT, 50 mm NaCl. The kinase reactions were conducted as described (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar) for 30 min at 30 °C in the kinase buffer, in the presence of 12.5 μCi of [γ-32P]ATP and GST-IκBα 1–72 wild type (referred as IκBα wt) or GST-IκBα 1–72 A32A36 (IκBα 2A) as substrates. RNA was extracted in TRIzol (Life Technologies), according to the manufacturer's instructions. 10 μg of total RNA were separated on 1% denaturing formaldehyde-agarose gel, transferred onto nylon membrane (Amersham Pharmacia Biotech), and hybridized with a32P-radiolabeled probe corresponding to full-length humanNRP cDNA. Hybridization was carried out at 60 °C in 7% SDS, 0.5 m sodium phosphate, pH 7.0. The membrane was washed successively in 0.1% SDS, 2 × SSC at 25 °C for 5 min and in 0.1% SDS, 0.1 × SSC at 60 °C for 30 min. Cells were lysed in 100 μl of 1 × CHRIS supplemented with 10 μg/ml of the protease inhibitors leupeptin, aprotinin, phenylmethylsulfonyl fluoride. 100 μg of total cell extracts were diluted 4 times with 50 mm Tris-HCl, 5 mm DTT, 2 mm MnCl2, 100 μg/ml bovine serum albumin, pH 8.0, solution containing 500 units of λ-phosphatase (Calbiochem) and incubated at 30 °C for 30 min. The reaction was inhibited with the phosphatase inhibitors sodium fluoride (50 mm), β-glycerophosphate (50 mm), and sodium orthovanadate (1 mm). HeLa cells were trypsinized, pelleted, and resuspended in complete medium at 5 × 106 cells/1 ml with a total amount of 20 μg of plasmid, and the cells were electroporated with the Easyject system (Eurogentec, Seraing, Belgium) in 4-mm cuvettes. Electroporation parameters were: 240 V, 1350 mivrofarads, 156 ohm, pulse time 211 ms. Transfected HeLa cells were seeded in 5 ml of minimal essential medium in 6-well plates and grown on glass coverslips. After 24 h the medium was changed. After an additional 24 h the cells were washed in PBS two times, fixed in 3% paraformaldehyde in PBS for 15 min at 37 °C, rinsed twice in PBS and permeabilized in phosphate-buffered saline (PBS) containing 0.5% (v/v) Triton X-100 in PBS for 10 min. After two washes with PBS, unspecific hybridization was blocked with 3% (v/v) bovine serum albumin in PBS for 15 min and incubated with primary antibody (purified NRP antibody) at 1/100 for 1 h at 37 °C. After washes, coverslips were incubated with Cy3-conjugated secondary antibody (Sigma) diluted 1:200. After being washed in PBS, coverslips were mounted on slides with Mowiol, and cells were examined with a Leica DMRXA-HC microscope. Fifty million 293T cells were washed in PBS and resuspended in 500 μl of 50 mm Tris, pH 7.5, and 1 mm EGTA. Cells were lysed by 30 passages through a 26-gauge needle. After centrifugation for 10 min at 1,500 rpm, the supernatant was recovered and complemented with 1 mm DTT, 0.025% Brij 35, and a mixture of proteases and phosphatases inhibitors. S100 were prepared by centrifuging the cytoplasmic extracts for 30 min at 52,000 rpm in a TLA 100.2 rotor (Beckman). After adding 10% glycerol, the S100 extracts were quickly frozen in dry ice and stored in liquid nitrogen. Gel filtration chromatography was carried out on a Superose 6 column (Amersham Pharmacia Biotech) precalibrated with aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa). Five-hundred μl fractions were recovered and directly analyzed by Western blotting with anti-NEMO and anti-NRP antisera. Cells were lysed and proteins were immunoprecipitated as described above. Immunoprecipitates were applied to a 10% polyacrylamide-SDS gel which had been polymerized in the presence of 0.5 mg/ml myelin basic protein (bovine brain, Sigma). Following electrophoresis, the gel was subjected to several rounds of denaturation and renaturation. For the denaturation procedure, the gel was washed twice for 30 min with a 50 mm Hepes, pH 7.6, solution containing 20% isopropyl alcohol, and twice for 30 min with buffer A (50 mm Hepes, pH 7.6, 5 mmβ-mercaptoethanol), and finally twice with 6 m urea in buffer A. Then the gel was renatured progressively at 4 °C by washing the gel once in a 3 m urea solution in buffer A containing 0.025% Tween 20 for 15 min, once in a 1.5 murea solution in buffer A containing 0.025% Tween 20 for 15 min, and once in a 0.75 m urea solution in buffer A containing 0.025% Tween 20 for another 15 min. For the kinase reaction, the gel was preincubated in kinase buffer (20 mm Hepes, pH 7.6, 20 mm MgCl2) at 30 °C for 30 min and finally incubated at 30 °C for 2 h in the kinase buffer containing 2 mm DTT, 20 μm ATP, and 100 μCi of [γ-32P]ATP. The gel was then washed 4 times for 15 min with trichloroacetic acid/PPi (5% trichloroacetic acid, 1% NaPPi), dried, and exposed to x-ray film. Our previous results have shown that NEMO-deficient cell lines are refractory to all tested NF-κB-activating stimuli, suggesting that IKK is the unique complex involved in NF-κB signaling (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar, 27Courtois G. Whiteside S.T. Sibley C.H. Israël A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar). However, the possibility exists that kinase complexes different from the IKK complex might exhibit a tissue-restricted expression, or might respond to uncharacterized NF-κB activating stimuli. In an effort to identify components of such complexes, we looked for NEMO homologues by data bank searching. BLAST search allowed us to identify a cDNA encoding a 67-kDa protein we called NRP. NRP was also isolated under the name of FIP-2 in a yeast two-hybrid screen using the adenovirus E3-14.7K protein as a bait (24Li Y. Kang J. Horwitz M.S. Mol. Cell. Biol. 1998; 18: 1601-1610Crossref PubMed Scopus (175) Google Scholar). Alignment of the amino acid sequence encoded by NRP with that of NEMO shows 53% amino acid similarity (Fig.1). NRP consists of 572 amino acid residues with a predicted molecular mass of 67 kDa. Alignment of the 2 sequences indicates that NRP contains an “insert” of 167 additional amino acids positioned after residue 134 of NEMO (Fig. 1). Secondary structure prediction of the 2 proteins shows similar structural elements. Both proteins have coiled coil domains, a carboxyl-terminal zinc finger, and a putative leucine zipper, which is located at the beginning of the insert in NRP and in the COOH-terminal part of NEMO (Fig. 1). We have recently reported that NEMO could complement the 70Z/3-derived mutant cell line 1.3E2, that exhibits a defect in NF-κB activation (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar). As NRP and NEMO are related, we asked whether NRP could also complement the 1.3E2 cell line. Transient transfection of a vector expressing NEMO in 1.3E2 resulted in a 30–40-fold increase in NF-κB activity following LPS stimulation, whereas transfection of the parental expression vector had no effect (Fig.2 A). In contrast to NEMO, transfection of NRP did not result in NF-κB activation under the same conditions, suggesting that NRP could not replace NEMO in NF-κB signaling. We recently showed that the zinc finger region of NEMO is essential for its activity, and that its deletion strongly reduces its ability to complement the 1.3E2 cell line (Fig.2 A). 2G. Courtois and S. Yamaoka, manuscript in preparation. As the zinc finger domains of NRP and NEMO are very similar (Fig. 1), we tested the possibility that the zinc finger domain of NRP could functionally replace that of NEMO. A hybrid molecule consisting of the NRP zinc finger fused to the rest of the NEMO molecule (NEMO ΔZF-NRP ZF) could activate NF-κB to the same extent as wild-type NEMO, suggesting a possible functional similarity between the two molecules. The significant homology between NEMO and NRP led us to investigate whether NRP was associated with the IKK complex. To examine this possibility, E29.1 T cell hybridoma lysates were subjected to immunoprecipitation with normal rabbit serum as a control (Fig. 2 B, lane 1), NRP antibody (lane 2), or NEMO antibody (lane 3). As described previously (20Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar), immunoprecipitation of NEMO followed by immunoblotting for IKKβ demonstrated the association between these proteins (lane 3); however, we did not find IKKβ in NRP immunoprecipitates (lane 2) and we could not detect an association between NEMO and NRP by immunoprecipitating one of these two proteins followed by immunodetection of the other (lanes 2 and 3). Taken together, these results show that NRP is not associated with NEMO, IKKβ, IKKα (data not shown) and is probably not a component of the IKK complex. Since NEMO is an essential component of the 600–800-kDa kinase complex that phosphorylates IκB, we investigated whether NRP is also present in a high molecular mass complex. S100 extracts were prepared from 293T cells and fractionated on a Superose 6 gel filtration column. The fractions were analyzed by Western blotting using antibodies directed against NRP and NEMO. As shown in Fig.2 C, NRP is present in a high molecular mass complex ranging from 400 to 700 kDa (lanes 7–10), whereas the larger IKK complex containing NEMO migrates, as expected, at 600 to 800 kDa (lanes 9–11). These results suggest that these two proteins are essentially present in different complexes. The results presented above demonstrate that NRP is not associated with known IκB kinases but, they do not address whether NRP is associated with other putative IκB kinases. In order to elucidate this point, NEMO and NRP were immunoprecipitated from resting or stimulated cells. The immune complexes were assayed for phosphorylation of a glutathioneS-transferase (GST) fusion protein containing the amino-terminal part of IκBα in the wild type context (IκBα) or mutated on its two phosphorylation sites (IκBα 2A). In Fig. 2 D, top panel, Jurkat cells were stimulated for 10 min with PMA and ionomycin (lanes 3 and 4), anti-CD28 (lanes 5 and 6) or both (lanes 7 and 8). As expected, NEMO was found to be associated with an inducible kinase activity as highlighted by IκBα phosphorylation (compare lanes 3, 5, 7 to lane 1). This activity was directed against the phosphorylation sites of IκBα since IκBα 2A was not phosphorylated (lanes 2, 4, 6, and 8). This phosphorylation was maximum when the cells were co-stimulated with PMA, ionomycin, and anti-CD28 (compare lanes 7 to lanes 3 and 5). In contrast, we could not observe any kinase activity associated with NRP (lanes 9–16). In the bottom panels, we have evaluated the effect of PMA and TNFα treatment on NEMO and NRP kinase activity in HeLa and E29.1 cells. We could observe an inducible kinase activity associated with NEMO after TNFα stimulation (compare lanes 5 and 1) and to a lesser extent after PMA stimulation (compare lanes 3 and 1); however, we could not observe any kinase activity associated with NRP. These data indicate that NRP, in contrast to NEMO, is not associated with an IκBα kinase activity. In order to investigate the effect of different NF-κB stimuli on NRP expression, 70Z/3 preB cells were stimulated for the indicated time with PMA. As depicted in Fig. 3, top panel, precipitation of NRP followed by immunoblotting with the same antibody demonstrated an increase in the appearance of a slower migrating form of the molecule after 15 min of PMA stimulation (lanes 2–5). This upper band progressively returned to basal level after 1 h of stimulation (lanes 4 and 5). We then tested whether other stimuli could also increase the amount of the upper band. 70Z/3 cells were subjected to IL-1 (middle panel) or LPS stimulation (bottom panel), two other stimuli able to activate NF-κB in this cell line; we could not detect any effect on NRP expression following these treatments. To evaluate whether similar events could occur in T cells, the murine hybridoma E29.1 was subjected to PMA treatment (Fig. 3 B, top panel). We could observe a strong increase in the appearance of the slow migrating band after 5 min of stimulation (compare lanes 2and 1). However, TNFα treatment of E29.1 cells did not result in any change in NRP expression (bottom panel), although prolonged treatment with this cytokine strongly increased the level of the NRP mRNA (see below). We then investigated whether this slowly migrating band represents a hyperphosphorylated form of NRP. E29.1 cells were left untreated or were stimulated for 30 min with PMA and then lysed with Nonidet P-40 containing buffer (Fig.4, lanes 1 and 2). Following this treatment, cell extracts were treated with λ-phosphatase (lanes 3 and 4) or with λ-phosphatase plus phosphatase inhibitors (lanes 5 and 6). PMA treatment induced the appearance of the upper band (lane 2), which disappeared following phosphatase treatment; this disappearance could be blocked by phosphatas" @default.
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• W2106369180 date "2000-07-01" @default.
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• W2106369180 title "Phorbol Esters and Cytokines Regulate the Expression of theNEMO-related Protein, a Molecule Involved in a NF-κB-independent Pathway" @default.
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• W2106369180 cites W1991781635 @default.
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• W2106369180 cites W2029607802 @default.
• W2106369180 cites W2033098442 @default.
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• W2106369180 cites W2101257770 @default.
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• W2106369180 doi "https://doi.org/10.1074/jbc.m001500200" @default.
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