NF-κB-inducing Kinase Phosphorylates and Blocks the Degradation of Down Syndrome Candidate Region 1
2007; Elsevier BV; Volume: 283; Issue: 6 Linguagem: Inglês
10.1074/jbc.m706707200
ISSN1083-351X
AutoresEun Jung Lee, Su Ryeon Seo, Ji Won Um, Joongkyu Park, Yohan Oh, Kwang Chul Chung,
Tópico(s)Down syndrome and intellectual disability research
ResumoDown syndrome, the most frequent genetic disorder, is characterized by an extra copy of all or part of chromosome 21. Down syndrome candidate region 1 (DSCR1) gene, which is located on chromosome 21, is highly expressed in the brain of Down syndrome patients. Although its cellular function remains unknown, DSCR1 expression is linked to inflammation, angiogenesis, and cardiac development. To explore the functional role of DSCR1 and the regulation of its expression, we searched for novel DSCR1-interacting proteins using a yeast two-hybrid assay. Using a human fetal brain library, we found that DSCR1 interacts with NF-κB-inducing kinase (NIK). Furthermore, we demonstrate that NIK specifically interacts with and phosphorylates the C-terminal region of DSCR1 in immortalized hippocampal cells as well as in primary cortical neurons. This NIK-mediated phosphorylation of DSCR1 increases its protein stability and blocks its proteasomal degradation, the effects of which lead to an increase in soluble and insoluble DSCR1 levels. We show that an increase in insoluble DSCR1 levels results in the formation of cytosolic aggregates. Interestingly, we found that whereas the formation of these inclusions does not significantly alter the viability of neuronal cells, the overexpression of DSCR1 without the formation of aggregates is cytotoxic. Down syndrome, the most frequent genetic disorder, is characterized by an extra copy of all or part of chromosome 21. Down syndrome candidate region 1 (DSCR1) gene, which is located on chromosome 21, is highly expressed in the brain of Down syndrome patients. Although its cellular function remains unknown, DSCR1 expression is linked to inflammation, angiogenesis, and cardiac development. To explore the functional role of DSCR1 and the regulation of its expression, we searched for novel DSCR1-interacting proteins using a yeast two-hybrid assay. Using a human fetal brain library, we found that DSCR1 interacts with NF-κB-inducing kinase (NIK). Furthermore, we demonstrate that NIK specifically interacts with and phosphorylates the C-terminal region of DSCR1 in immortalized hippocampal cells as well as in primary cortical neurons. This NIK-mediated phosphorylation of DSCR1 increases its protein stability and blocks its proteasomal degradation, the effects of which lead to an increase in soluble and insoluble DSCR1 levels. We show that an increase in insoluble DSCR1 levels results in the formation of cytosolic aggregates. Interestingly, we found that whereas the formation of these inclusions does not significantly alter the viability of neuronal cells, the overexpression of DSCR1 without the formation of aggregates is cytotoxic. Down syndrome (DS), 2The abbreviations used are: DS, Down syndrome; DSCR1, Down syndrome candidate region 1; HEK293, human embryonic kidney 293; IKKα, IκB kinase α; MTT, 3,(4,5-dimethyldiazol-2-yl)2,5-diphenyl-tetrazolium bromide; NF-ATs, The nuclear factors of activated T cells; NIK, NF-κB-inducing kinase; PBS, phosphate-buffered saline; HA, hemagglutinin; GFP, green fluorescent protein; GST, glutathione S-transferase; TUNEL, terminal dUTP nick-end labeling. 2The abbreviations used are: DS, Down syndrome; DSCR1, Down syndrome candidate region 1; HEK293, human embryonic kidney 293; IKKα, IκB kinase α; MTT, 3,(4,5-dimethyldiazol-2-yl)2,5-diphenyl-tetrazolium bromide; NF-ATs, The nuclear factors of activated T cells; NIK, NF-κB-inducing kinase; PBS, phosphate-buffered saline; HA, hemagglutinin; GFP, green fluorescent protein; GST, glutathione S-transferase; TUNEL, terminal dUTP nick-end labeling. the most common genetic disorder, occurs in one of every 700–800 births. Patients with DS display many typical phenotypes, such as immune deficiency, characteristic facial features, mental retardation, congenital heart disease, and early onset Alzheimer disease-like symptoms (1Epstein C.J. Prog. Clin. Biol. Res. 1995; 393: 241-246PubMed Google Scholar, 2Toyoda A. Noguchi H. Taylor T.D. Ito T. Pletcher M.T. Sakaki Y. Reeves R.H. Hattori M. Genome Res. 2002; 12: 1323-1332Crossref PubMed Scopus (45) Google Scholar). DS is associated with having three copies of chromosome 21, also known as trisomy 21 (3Antonarakis S.E. Lyle R. Dermitzakis E.T. Reymond A. Deutsch S. Nat. Rev. Genet. 2004; 5: 725-738Crossref PubMed Scopus (488) Google Scholar, 4Roizen N.J. Patterson D. Lancet. 2003; 361: 1281-1289Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar), and the overexpression of a number of genes located on this chromosome is thought either directly or indirectly to be responsible for these clinical features. Down syndrome candidate region 1 (DSCR1, also called as Adapt78, MCIP1, calcipressin 1, or RCAN1) gene is located near the Down syndrome critical region of chromosome 21 (5Fuentes J.J. Pritchard M.A. Estivill X. Genomics. 1997; 44: 358-361Crossref PubMed Scopus (162) Google Scholar, 6Pfister S.C. Machado-Santelli G.M. Han S.W. Henrique-Silva F. BMC Cell Biol. 2002; 3: 24Crossref PubMed Scopus (22) Google Scholar). It is highly expressed in the brain, heart, and skeletal muscles of DS fetuses (7Fuentes J.J. Pritchard M.A. Planas A.M. Bosch A. Ferrer I. Estivill X. Hum. Mol. Genet. 1995; 4: 1935-1944Crossref PubMed Scopus (223) Google Scholar), and it is known to interact physically and functionally with Ca2+/calmodulin-dependent protein phosphatase 2B (also known as calcineurin A; see Ref. 8Fuentes J.J. Genesca L. Kingsbury T.J. Cunningham K.W. Perez-Riba M. Estivill X. de la Luna S. Hum. Mol. Genet. 2000; 9: 1681-1690Crossref PubMed Google Scholar). DSCR1 has been proposed to be a feedback inhibitor of calcineurin based on two controversial findings. First, the overexpression of DSCR1 suppresses calcineurin signaling, and second, calcineurin activity in the hearts of DSCR1 knock-out mice is greatly diminished (9Rothermel B. Vega R.B. Yang J. Wu H. Bassel-Duby R. Williams R.S. J. Biol. Chem. 2000; 275: 8719-8725Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 10Vega R.B. Rothermel B.A. Weinheimer C.J. Kovacs A. Naseem R.H. Bassel-Duby R. Williams R.S. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 669-674Crossref PubMed Scopus (192) Google Scholar). Moreover, the proper expression of DSCR1 appears to be linked to inflammation, angiogenesis, and cardiac development (11Harris C.D. Ermak G. Davies K.J. Cell. Mol. Life Sci. 2005; 62: 2477-2486Crossref PubMed Scopus (94) Google Scholar, 12Hesser B.A. Liang X.H. Camenisch G. Yang S. Lewin D.A. Scheller R. Ferrara N. Gerber H.P. Blood. 2004; 104: 149-158Crossref PubMed Scopus (133) Google Scholar, 13Ryeom S. Greenwald R.J. Sharpe A.H. Mckeon F. Nat. Immunol. 2003; 4: 874-881Crossref PubMed Scopus (107) Google Scholar, 14Yao Y.G. Duh E.J. Biochem. Biophys. Res. Commun. 2004; 321: 648-656Crossref PubMed Scopus (63) Google Scholar). NF-κB-inducing kinase (NIK), a Ser/Thr kinase, is a member of mitogen-activated protein kinase (MAPK) kinase kinase family. It preferentially phosphorylates and therefore activates IκB kinase α (IKKα) (15Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (446) Google Scholar, 16Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1067) Google Scholar). NIK and IKKα are known to induce the processing of p100 and the generation of p52, thereby activating the alternative NF-κB pathway (17Dejardin E. Biochem. Pharmacol. 2006; 72: 1161-1179Crossref PubMed Scopus (303) Google Scholar). Although the physiological role of NIK in NF-κB signaling is unclear, gene knock-out studies have suggested that NIK regulates the transcriptional activity of NF-κB in a receptor-restricted manner, such as by both tumor necrosis factor-α and interleukin-1β (18Yin L. Wu L. Wesche H. Arthur C.D. White J.M. Goeddel D.V. Schreiber R.D. Science. 2001; 291: 2162-2165Crossref PubMed Scopus (347) Google Scholar). NIK-/- cells are also unable to induce NF-κB-dependent gene transcription in response to treatment with lymphotoxin-β despite IκBα degradation (18Yin L. Wu L. Wesche H. Arthur C.D. White J.M. Goeddel D.V. Schreiber R.D. Science. 2001; 291: 2162-2165Crossref PubMed Scopus (347) Google Scholar). NIK has both functional nuclear import and export signals that result in its continuous shuttling between the cytoplasm and the nucleus, which suggests that NIK, like IKKα, has an intranuclear function (19Birbach A. Bailey S.T. Ghosh S. Schmid J.A. J. Cell Sci. 2004; 117: 3615-3624Crossref PubMed Scopus (69) Google Scholar). The nuclear factors of activated T cells (NF-ATs) are a family of transcription factors that transduce calcium signals in the immune, cardiac, muscular, and nervous systems (20Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Like their distant relatives the Rel family, which includes NF-κB, NF-ATs are located in the cytoplasm of resting cells and are activated by their induced nuclear import (21Zhu J. McKeon F. Cell. Mol. Life Sci. 2000; 57: 411-420Crossref PubMed Scopus (45) Google Scholar). Calcium signaling activates calcineurin and induces the movement of NF-AT proteins into the nucleus, where they cooperate with other proteins to form complexes on DNA. Some NF-ATs are characterized by a highly conserved DNA-binding domain as well as a calcineurin-binding domain (22Luo C. Shaw K.T.Y. Raghavan A. Aramburu J. Garcia-Cozar F. Perrino B.A. Hogan P.G. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8907-8912Crossref PubMed Scopus (156) Google Scholar). The binding of calcineurin to the latter domain controls the nuclear transport of these NF-ATs (22Luo C. Shaw K.T.Y. Raghavan A. Aramburu J. Garcia-Cozar F. Perrino B.A. Hogan P.G. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8907-8912Crossref PubMed Scopus (156) Google Scholar). Although many reports have suggested a physiological link between the NF-κB and NF-AT pathways, the functional linkage between these two signaling pathways has yet to be determined. In the study presented here, we investigated the cellular function of DSCR1 by searching for novel binding partner(s). We found that NIK selectively binds to and phosphorylates the C-terminal region of DSCR1. Furthermore, we demonstrate that this phosphorylation of DSCR1 enhances its stability, which in turn leads to an increase in the levels of soluble and insoluble DSCR1. MaterialsThe following materials were used. Synthetic dropout medium (SD/T, SD/L, and SD/HLT) and yeast extract peptone dextrose medium containing adenine were purchased from MP Biomedicals (Solon, OH). The human fetal brain cDNA library was purchased from Clontech. Peroxidase-conjugated anti-rabbit and anti-mouse IgGs were purchased from Zymed Laboratories Inc.. All cell culture reagents, including Dulbecco's modified Eagle's medium and fetal bovine serum, and TRIzol reagent were purchased from Invitrogen. Protein A-Sepharose was purchased from Amersham Biosciences, and enhanced chemiluminescence reagents and [γ-32P]ATP were purchased from PerkinElmer Life Sciences. Polyclonal and monoclonal anti-HA, anti-GFP, and anti-NIK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-FLAG antibody and cycloheximide were purchased from Sigma. Clasto-lactacystin β-lactone and MG132 were purchased from Calbiochem. A mammalian expression vector for HA-tagged human wild type DSCR1 was kindly provided by S. de la Luna (Genomics Regulation Center, Barcelona, Spain), and plasmids encoding wild type and deletions of GFP-tagged DSCR1 and HA-tagged wild type calcineurin A were kindly provided by B. A. Rothermel (University of Texas Southwestern Medical Center, Dallas). GST-tagged bacterial expression vectors encoding amino acids 1–197 (full length), 1–90, and 90–197 of DSCR1 were constructed by subcloning PCR products from HA-tagged DSCR1 into pGEX-4T1 (Amersham Biosciences) and confirmed by DNA sequencing. Mammalian expression vectors for Myc-tagged wild type NIK and its kinase-deficient mutants, such as Myc-NIK-KD (in which the catalytic domain of NIK from amino acids 100–250 is deleted) and Myc-NIK-KKAA (in which lysine residues at position 429 and 430 are mutated to alanine), were gifts from T. H. Lee (Yonsei University, Seoul, Korea). Rabbit polyclonal antibody of DSCR1 was produced commercially (Lab Frontier, Seoul, Korea) by injecting purified GST-DSCR1. The serum from these rabbits was then affinity-purified using standard methods. Yeast Two-hybrid AssayScreening was performed using a 13-week human fetal brain Matchmaker cDNA library subcloned into the activation domain of pACT2 vector, and yeast strain carrying the pHybTrp/Zeo-plasmid that encoded for wild type DSCR1 was used as bait. The yeast strain L40, which contains the reporter genes lacZ and HIS3 under the control of LexA promoter, was sequentially transformed with bait vector then the cDNA library vectors, and the cells were plated on a synthetic medium containing 5 mm 3-amino-1,2,4-triazole and lacking histidine, leucine, and tryptophan. After 10–14 days at 30 °C, the transformants were grown on synthetic medium containing 50 μg/ml of 5-bromo-4-chloro-3-indolyl-d-galactoside, but lacking histidine, leucine, and tryptophan. After 2–3 days at 30 °C, the resulting yeast colonies displaying a blue color were selected as positive clones. The plasmids from these positive clones were extracted in lysis buffer containing 2% Triton X-100, 1% SDS, 100 mm NaCl, 10 mm Tris, pH 8.0, and 1.0 mm EDTA and then transformed into Escherichia coli DH5α by electroporation. The inserts of the plasmids from the positive library clones were analyzed using an automatic DNA sequencer (ALF Express, Amersham Biosciences). Cell CultureHuman embryonic kidney 293 (HEK293) cells and rat embryonic hippocampal (H19-7) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. These cells were transfected with the Lipofectamine plus reagent (Invitrogen) using the supplier's recommended instructions. To prepare lysates, the cells were rinsed twice with ice-cold phosphate-buffered saline (PBS), solubilized in lysis buffer (Tris, pH 7.9, containing 1.0% Nonidet P-40, 150 mm NaCl, 1 mm EGTA, 1 mm EDTA, 10% glycerol, 1 mm Na3VO4, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 10 mm NaF, and 0.2 mm phenylmethylsulfonyl fluoride), and then scraped. Supernatant was collected after centrifugation for 10 min at 14,000 × g at 4 °C. Protein concentrations were determined using the Bio-Rad detergent-compatible protein assay kit according to the manufacturer's instructions. Immunoprecipitation and Western Blot AnalysisOne microgram of antibody was incubated with 2 mg of cell extracts in lysis buffer overnight at 4 °C. Fifty microliters of a 1:1 suspension of protein A-Sepharose beads were added, and the mixture was incubated for 2 h at 4 °C with gentle rotation. The beads were pelleted and washed extensively with cell lysis buffer. Bound proteins were dissociated by boiling in SDS-PAGE sample buffer, and whole protein samples were separated on an SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Millipore, Japan). The membranes were blocked in TBST buffer (20 mm Tris, pH 7.6, 137 mm NaCl, 0.05% Tween 20, and 3% nonfat dried milk) for 3 h and then incubated overnight at 4 °C in 3% nonfat dried milk containing the appropriate antibodies. The membranes were washed several times in TBST and then incubated with IgG-coupled horseradish peroxidase secondary antibody (Zymed Laboratories Inc.). After 60 min, the blots were washed several times with TBST, and resulting immunocomplexes were visualized using enhanced chemiluminescence according to the manufacturer's instructions. Reverse Transcription-PCRTotal cellular mRNA was extracted using the TRIzol reagent. cDNA was synthesized by reverse transcription using random primers. Two micrograms of cDNA were used per PCR. The primers used were as follows: rat dscr1 forward, 5′-C CGGAATTCATGCATTTTAGGGACTTTA-3′, and rat dscr1 reverse, 5′-CCGCTCAAGGCTGAGGTGGATGGG-3′; human dscr1 forward, 5′-GAGGAGGTGGACCTGCAGGACCTG-3′, and human dscr1 reverse, 5′-TCAGCTGAGGTGGATCGGCGTGTAC-3′. Preparation of Nonidet P-40-soluble/insoluble FractionsCells were solubilized with 1.0% Nonidet P-40, and the resulting cellular suspension was fractionated by centrifugation at 15,000 × g for 30 min. The supernatants (i.e. the Nonidet P-40-soluble fraction) were directly immunoprecipitated. The resulting pellet (i.e. the Nonidet P-40-insoluble fraction) was washed three times with ice-cold lysis buffer and then solubilized in 4% SDS sample buffer (0.5 m Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% mercaptoethanol) for 1 h at 60 °C. ImmunocytochemistryCells were seeded overnight at 70% confluence onto coverslips in 6-well dishes. The following day, the cells were transfected. Twenty four hours later, the cells were washed with PBS, fixed with neutrally buffered 4% (w/v) paraformaldehyde, and permeabilized with 1% PBS solution containing 0.1% Triton X-100 for 1 h. The cells were then incubated for 24 h at 4 °C with the indicated primary antibody diluted in PBS containing 1% bovine serum albumin. After washing three times with PBS, the cells were incubated with secondary antibody for 2 h at room temperature. The cells were then analyzed using either fluorescence (IX71, Olympus) or confocal microscopy (LSM 510 META, Carl Zeiss). Preparation of Cytosolic and Nuclear FractionsCells were washed with ice-cold PBS, resuspended in hypotonic buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl) supplemented with protease inhibitors (dithiothreitol, aprotinin, and leupeptin), and then incubated on ice for 30 min. The cells were then lysed using a disposable syringe, and the lysates were subjected to centrifugation at 3,000 rpm for 15 min at 4 °C. The resulting supernatant was used as the cytosolic fraction. The resulting pellet, which was used as the nuclear fraction, was washed with hypotonic buffer and lysed with 1.0% Nonidet P-40 lysis buffer. Supernatants from each fraction were collected after centrifugation at 14,000 × g for 10 min at 4 °C. In Vitro Kinase AssayConfluent cells were harvested using lysis buffer. The soluble lysates were incubated with the indicated antibodies for 2 h at 4 °C. Following the addition of protein A-Sepharose beads, the mixture was incubated for 2 h at 4 °C and then rinsed with lysis and kinase buffers. Immunocomplex kinase assays were performed by incubating the cell lysates with the substrate in the reaction buffer (0.2 mm sodium orthovanadate, 2 mm dithiothreitol, 10 mm MgCl2, 5 mCi of [γ-32P]ATP, 100 μm ATP, and 20 mm HEPES, pH 7.4) for 2 h at 30 °C. The reactions were terminated, and the mixtures were subject to SDS-PAGE. Phosphorylated substrates were visualized by autoradiography. Assessment of Cell Survival (MTT Extraction Assay)3,(4,5-Dimethyldiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT, 62.5 μl of a 5 mg/ml stock solution) was added to the 250 μl of medium in each well of a 24-well plate. After a 2-h incubation at 37 °C, 250 μl of extraction buffer (20% SDS and 50% N,N-dimethylformamide, pH 7.4) was added. After an overnight incubation at 37 °C, the absorbance at 570 nm was measured using a VERSA MAX enzyme-linked immunosorbent assay reader (Molecular Devices, Sunnyvale, CA) with an extraction buffer negative control. TUNEL AssayCells were seeded overnight at 70% confluence onto coverslips in 6-well dishes. The following day, the cells were transfected with the indicated plasmids, incubated for 24 h, and then treated with zinc for 1 h. Cell apoptosis was analyzed using an in situ cell death detection kit (TMR red, Roche Applied Science) that is based on the terminal TUNEL technology. Identification of Novel DSCR1-interacting Proteins Using a Yeast Two-hybrid AssayTo identify novel DSCR1-interacting partners, a yeast two-hybrid assay was performed using a human fetal brain cDNA library with full-length DSCR1 as the bait. We found several previously identified DSCR1-interacting proteins, such as calcineurin A (8Fuentes J.J. Genesca L. Kingsbury T.J. Cunningham K.W. Perez-Riba M. Estivill X. de la Luna S. Hum. Mol. Genet. 2000; 9: 1681-1690Crossref PubMed Google Scholar) and Raf-1 (23Cho Y.J. Abe M. Kim S.Y. Sato Y. Arch. Biochem. Biophys. 2005; 439: 121-128Crossref PubMed Scopus (25) Google Scholar), which indicates that the assay is a reliable method for identifying DSCR1-interacting proteins. In addition to the known DSCR1-interacting proteins, our assay identified NIK as a protein that interacts with DSCR1 (data not shown). Based on the known physiological substrate of NIK and the fact that DSCR1 function is regulated by its protein stability, which in turn is affected by its phosphorylation status, the functional connection between DSCR1 and NIK was further characterized. DSCR1 Interacts with NIK in Neuronal CellsUsing an immunoprecipitation binding assay, we examined the potential interaction of NIK and DSCR1 in mammalian neuronal cells. H19-7 cells were transiently transfected with HA-tagged human wild type DSCR1 alone, or in the presence of Myc-tagged NIK. Extracts from the transfected cells were then subjected to immunoprecipitation using either anti-HA or anti-Myc antibodies and then immunoblot analysis using anti-Myc or anti-HA antibodies (Fig. 1A). This analysis revealed that DSCR1 associated with NIK (Fig. 1A). The use of preimmune IgG and empty protein A beads as negative controls (Fig. 1A) demonstrates that transiently transfected DSCR1 selectively binds to NIK in H19-7 cells. Next we examined whether IKKα, the only known substrate of NIK, also associates with DSCR1. Cells were transfected with HA-tagged IKKα and/or GFP-tagged DSCR1 and then subjected to immunoprecipitation. The results demonstrate that IKKα does not interact with DSCR1 (Fig. 1B). HA-tagged calcineurin A was used as a positive control in the GFP-tagged DSCR1 binding assay in H19-7 cells to demonstrate the reliability of the co-immunoprecipitation assay (Fig. 1C). To determine whether DSCR1 and NIK interact in the absence of exogenous DNA addition, we examined the specific binding of endogenous DSCR1 and NIK in primary neuronal cells and transformed H19-7 cell line using co-immunoprecipitation assays. As shown in Fig. 2, A and B, we found that endogenous NIK binds to endogenous DSCR1 in both H19-7 cells and primary cortical neurons. Immunoprecipitation using nonspecific IgG was used as a negative control. This interaction was also observed in rat whole brain lysates (Fig. 2C), suggesting that the specific interaction between DSCR1 and NIK is not an artifact of DNA transfection and transformation but actually occurs in the mammalian central nervous system. Wild Type NIK Binds to the C-terminal Domain of DSCR1To determine the protein domain(s) responsible for the interaction between DSCR1 and NIK, we used several deletion constructs encoding DSCR1 fragments fused to GFP in similar co-immunoprecipitation assays. As shown in Fig. 3A, we found that NIK co-immunoprecipitated with the full-length DSCR1 protein as well as with the DSCR1 peptide fragments containing amino acids 30–197 and 90–197. Interestingly, NIK did not co-immunoprecipitate with a DSCR1 peptide fragment containing the N-terminal 90 amino acids (Fig. 3), suggesting that the DSCR1 domain critical for interacting with NIK is included within amino acid residues 90–197. Furthermore, when we examined whether wild type DSCR1 binds to the kinase-deficient NIK mutant, DSCR1 binds to wild type NIK but not to its kinase-dead mutant, such as NIK-KD or NIK-KK/AA (Fig. 3B). NIK Phosphorylates DSCR1 in H19-7 CellsNext, we examined whether DSCR1 is a substrate of NIK. We have previously shown that zinc induces the phosphorylation and activation of IKK in H19-7 cells (24Min Y.K. Park J.H. Chong S.A. Kim Y.S. Ahn Y.S. Seo J.T. Bae Y.S. Chung K.C. J. Neurosci. Res. 2003; 71: 689-700Crossref PubMed Scopus (24) Google Scholar). NIK is known to function as an upstream activator of the NF-κB signaling pathway that ultimately results in the activation of IKKα (15Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (446) Google Scholar). Therefore, we first examined whether NIK is also phosphorylated and activated by zinc. H19-7 cells were either transfected with Myc-tagged NIK plasmid or mock-transfected and then stimulated with zinc plus the zinc ionophore, pyrithione. Lysates from the resulting transfected cells were subjected to immunoblotting using anti-phospho-NIK antibodies. As shown in Fig. 4A, treatment with zinc plus pyrithione induced the phosphorylation of endogenous NIK. Furthermore, the cells transfected with wild type NIK had much higher levels of phosphorylated NIK than the mock-transfected cells (Fig. 4A). To examine the effects of zinc treatment on NIK-associated DSCR1 phosphorylation, H19-7 cells were transfected with Myc-tagged NIK and then either untreated or treated with zinc plus pyrithione. Lysates from the resulting transfected cells were immunoprecipitated using anti-NIK antibodies, and these immunocomplexes were then used in in vitro kinase assays with recombinant GST-DSCR1 as the substrate. The results, shown in Fig. 4B, demonstrate that whereas DSCR1 was phosphorylated by anti-NIK immunocomplexes from untreated cells, the phosphorylation was significantly higher with anti-NIK immunocomplexes from cells treated with zinc plus pyrithione. Furthermore, this increase in DSCR1 phosphorylation was not observed in cells transfected with a kinase-inactive dominant-negative NIK mutant (Fig. 4B). These results demonstrate that wild type NIK specifically phosphorylates DSCR1, and the phosphorylation of DSCR1 is significantly induced by zinc treatment. NIK Phosphorylates C-terminal Domain of DSCR1To determine which domain(s) of DSCR1 are specifically phosphorylated by NIK, three bacterially expressed DSCR1 fragments fused with GST were purified and used as a substrate of NIK in in vitro kinase assay. As shown in Fig. 5, full-length DSCR1 and DSCR1 peptide fragment containing amino acid residues 90–197 could be phosphorylated by NIK following zinc plus pyrithione treatment. However, DSCR1 peptide fragment containing amino acid residues 1–90 could not be phosphorylated by NIK (Fig. 5). These data suggest that NIK specifically phosphorylates DSCR1 on a domain within amino acid residues 90–197. The NIK-mediated Phosphorylation of DSCR1 Leads to an Increased Half-life and Reduced Proteasomal DegradationNext, we examined the physiological consequence(s) of NIK-mediated DSCR1 phosphorylation. First, we examined whether this phosphorylation affects the half-life of DSCR1. Cells were transfected with HA-tagged DSCR1 alone or together with either wild type NIK or kinase-deficient mutants. The transfected cells were then incubated with cycloheximide for the indicated times. The levels of DSCR1 in these cells were measured by Western blot analysis using an anti-HA antibody. The cells transfected with DSCR1 and wild type NIK had ∼2-fold more DSCR1 than the cells transfected with DSCR1 alone (Fig. 6, A and B). Interestingly, this increase in DSCR1 protein levels was not observed in the cells transfected with DSCR1 and the kinase-dead NIK mutants (Fig. 6, A and B). These data suggest that the half-life of DSCR1 is increased by its NIK-mediated phosphorylation. Next, we examined whether NIK alters the levels of 1% Nonidet P-40-soluble and -insoluble DSCR1. H19-7 cells were transfected with HA-tagged DSCR1 and/or Myc-tagged NIK. Lysates from the resulting transfected cells were blotted using anti-HA antibodies. As shown in Fig. 7, the levels of Nonidet P-40-soluble and -insoluble DSCR1 were increased in the cells co-transfected with wild type NIK. However, the levels of DSCR1 were not markedly changed in cells co-transfected with kinase-deficient NIK mutant, as compared with control cells transfected with DSCR1 alone (Fig. 7, top, left panel). These results indicate that the NIK-mediated phosphorylation of DSCR1 increases its stability in two different ways. We have previously shown that the endogenous levels of DSCR1 are modulated by protein ubiquitination and proteasomal degradation in H19-7 cells (25Lee E.J. Lee J.Y. Seo S.R. Chung K.C. Mol. Cell. Neurosci. 2007; 35: 585-595Crossref PubMed Scopus (20) Google Scholar). Based on the results described above, we examined whether NIK affects the proteasome-mediated degradation of DSCR1. We mock-transfected or transiently transfected cells with DSCR1 plus NIK and then treated the cells with the proteasomal inhibitor MG132. Lysates from these cells were then immunoblotted using anti-HA antibodies. As expected, MG132 treatment resulted in an increase in the levels of soluble DSCR1 and no apparent reduction in the level of insoluble DSCR1 in the absence of NIK (Fig. 7, top right panel). This finding supports the hypothesis that DSCR1 is constitutively degraded by proteasome. The increase in soluble DSCR1 levels was not altered by the presence of NIK or its kinase activity. Interestingly, MG132 treatment of cells transfected with wild type NIK resulted in the accumulation of insoluble DSCR1 relative to the mock-transfected control cells (Fig. 7A, middle panel). This result indicates that, in addition to the inhibitory effect on the proteasomal targeting, NIK also affects the stability of DSCR1 by altering its solubility. Similar results were observed when another proteasomal inhibitor, clasto-lactacystin β-lactone, was used (Fig. 7B). Next we tested whether NIK also affects the half-life of DSCR1 in 1% Nonidet P-40-insoluble fraction. As shown in Fig. 7C, the cells transfected with DSCR1 and wild type NIK had also greatly increased the stability of DSCR1 in Nonidet P-40-insoluble pellet fraction than the cells trans
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