NF-κB-inducing Kinase Phosphorylates and Blocks the Degradation of Down Syndrome Candidate Region 1

Abstract

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, 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 were from and and were purchased from and were purchased from expression for human DSCR1 was by S. de la Luna and and of DSCR1 and calcineurin were by B. A. Rothermel of expression and of DSCR1 were by from DSCR1 into and by expression for NIK and its such as which the domain of NIK from is and which and are to were from T. H. of DSCR1 was by The from these was using was using a human fetal brain cDNA library into the domain of and yeast the that for DSCR1 was used as The yeast which the genes and the of was with the cDNA library and the cells were on a medium containing and and the were on medium containing of and the yeast a were as The from these were in containing and and into by The of the from the library were using an Amersham Cell embryonic kidney cells and embryonic hippocampal cells were in Dulbecco's modified Eagle's medium with fetal bovine serum, and cells were with the reagent using the To the cells were with phosphate-buffered in containing 1 1 1 1 1 and and was for Protein were using the protein to the and of was with of cell in of a of protein A-Sepharose were and the was for with The were and with cell proteins were by in and protein were on an and to a The were in and for and in containing the The were in and with Laboratories the were with and were using enhanced chemiluminescence to the cellular was using the TRIzol cDNA was by transcription using of cDNA were used The used were as and human and human of were with and the cellular was by for The the were directly The the was three with and in for 1 were in The following the cells were the cells were with PBS, with and with containing for 1 The cells were for with the primary in containing bovine three with PBS, the cells were with for The cells were using either or of and were with PBS, in with and and on for The cells were using a and the were to for The was used as the cytosolic The which was used as the nuclear was with and with from were for In cells were using The soluble were with the for the of protein A-Sepharose the was for and with and kinase kinase were by the cell with the in the of and for The were and the were to were by of Cell of a was to the of medium in well of a a of and was an the was using a with an were in The following the cells were with the for and with for 1 Cell was using an in cell that is based on the terminal of DSCR1-interacting Using a novel DSCR1-interacting a yeast two-hybrid was using a human fetal brain cDNA library with DSCR1 as the We found DSCR1-interacting such as calcineurin 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 and M. Y. Biochem. Biophys. 2005; PubMed Scopus Google Scholar), which that the is a for DSCR1-interacting In to the known DSCR1-interacting NIK as a protein that interacts with DSCR1 not on the known physiological of NIK and the that DSCR1 function is by its protein stability, which in turn is by its phosphorylation the functional between DSCR1 and NIK was DSCR1 with NIK in an binding we the of NIK and DSCR1 in neuronal cells were with human DSCR1 or in the of from the cells were to using either or and using or This that DSCR1 associated with NIK The of and protein as controls that DSCR1 selectively binds to NIK in we IKKα, the known of NIK, also with DSCR1. were with IKKα DSCR1 and to The results demonstrate that IKKα does not interact with DSCR1 calcineurin was used as a in the DSCR1 binding in cells to demonstrate the of the To DSCR1 and NIK interact in the of we the binding of DSCR1 and NIK in primary neuronal cells and cell using in and we found that NIK binds to DSCR1 in both cells and primary cortical neurons. using was used as a This was also in brain that the between DSCR1 and NIK is not an of and occurs in the nervous NIK to the C-terminal of the protein responsible for the between DSCR1 and NIK, we used DSCR1 to in in we found that NIK with the DSCR1 protein as well as with the DSCR1 containing and Interestingly, NIK not with a DSCR1 containing the that the DSCR1 domain critical for with NIK is Furthermore, we DSCR1 binds to the NIK DSCR1 binds to NIK not to its such as or NIK DSCR1 in we DSCR1 is a of We have that induces the phosphorylation and of in cells J. Res. 2003; PubMed Scopus Google Scholar). NIK is known to function as an of the NF-κB signaling pathway that results in the 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). we NIK is also and activated by cells were either with NIK or and with the from the cells were to using in treatment with induced the phosphorylation of Furthermore, the cells with NIK levels of NIK the cells To the effects of treatment on DSCR1 cells were with NIK and either or with from the cells were using and these were used in in kinase with as the The in demonstrate that whereas DSCR1 was by from cells, the phosphorylation was significantly with from cells with Furthermore, this increase in DSCR1 phosphorylation was not in cells with a NIK results demonstrate that NIK specifically phosphorylates DSCR1, and the phosphorylation of DSCR1 is significantly induced by NIK C-terminal of which of DSCR1 are specifically by NIK, three expressed DSCR1 with were and used as a of NIK in in kinase assay. in DSCR1 and DSCR1 containing be by NIK following DSCR1 containing not be by NIK that NIK specifically phosphorylates DSCR1 on a domain The NIK-mediated of DSCR1 to an and we the physiological of NIK-mediated DSCR1 First, we this phosphorylation the of DSCR1. were with DSCR1 or with either NIK or The cells were with for the The levels of DSCR1 in these cells were by using an The cells with DSCR1 and NIK DSCR1 the cells with DSCR1 and Interestingly, this increase in DSCR1 protein levels was not in the cells with DSCR1 and the NIK and that the of DSCR1 is by its NIK-mediated we NIK the levels of and DSCR1. cells were with DSCR1 from the cells were using in the levels of and DSCR1 were in the cells with the levels of DSCR1 were not in cells with NIK as with cells with DSCR1 results that the NIK-mediated phosphorylation of DSCR1 increases its stability in two We have that the levels of DSCR1 are by protein and proteasomal degradation in cells E.J. Mol. Cell. PubMed Scopus Google Scholar). on the results we NIK the degradation of DSCR1. We or cells with DSCR1 NIK and the cells with the proteasomal inhibitor from these cells were using treatment in an increase in the levels of soluble DSCR1 and in the of insoluble DSCR1 in the of NIK This the that DSCR1 is by The increase in soluble DSCR1 levels was not by the of NIK or its kinase Interestingly, treatment of cells with NIK in the of insoluble DSCR1 to the cells This result in to the on the proteasomal NIK also the stability of DSCR1 by its results were proteasomal was used we NIK also the of DSCR1 in in the cells with DSCR1 and NIK also greatly the stability of DSCR1 in the cells with DSCR1 Furthermore, this increase was not in the cells with DSCR1 and the NIK which is the to that in DSCR1 that the stability of and DSCR1 is by NIK-mediated NIK the of DSCR1 we the NIK-mediated increase in the levels of insoluble DSCR1 leads to the formation of DSCR1 aggregates. cells were either with GFP, DSCR1, or DSCR1 and was using a in cells with an of in the as well as in the In the cells with and cytosolic DSCR1 aggregates In of the cells using an that the DSCR1 aggregates a of protein which is also found in Furthermore, using the DSCR1 aggregates also To cytosolic DSCR1 the cells were into cytosolic and nuclear and the of DSCR1 in was determined. The levels of DSCR1 in cytosolic of cells with NIK was enhanced to that in the cells with DSCR1 or DSCR1 a NIK In DSCR1 was not in the nuclear of cells The of the were by with such as nuclear and cytosolic IκB protein these that the NIK-mediated phosphorylation of DSCR1 its degradation, which results in its cytosolic The of DSCR1 by NIK-mediated on Cell we NIK-mediated phosphorylation of DSCR1 that leads to its cytosolic cell cells were with DSCR1 or NIK cell viability was by an in the overexpression of DSCR1 induced cell that this cell is a result of In cells were with DSCR1, and either NIK or in to cells with DSCR1 that DSCR1 phosphorylation and the induced by its overexpression are not that the NIK-mediated increase in soluble and insoluble DSCR1 levels are not the of neuronal cell associated with DSCR1 DSCR1 is known to the signaling of thereby cell and 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, T. M. Noguchi N. T. X. H. T. T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). NF-AT has been to cooperate with NF-κB to the expression of many of cells between NF-AT and NF-κB signaling pathways and their feedback DSCR1 and IκB T. M. Noguchi N. T. X. H. T. T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). many reports have calcineurin in the NF-κB signaling calcineurin was to NF-κB activity by the and degradation of IκB B. G. N. J. PubMed Scopus Google Scholar, B. J. Immunol. 1995; Google Scholar), and in T protein kinase and calcineurin have been to IκB kinase and NF-κB A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, G. M. J. Cell Biol. 2003; PubMed Scopus Google that a with calcineurin in and in and is a for calcineurin In calcineurin a role and NF-κB activity A. A. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). DSCR1 was to the expression of genes in the response and to transcriptional by its IκBα Y. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we that NIK, which in the alternative NF-κB signaling physically interacts with DSCR1 in neuronal We also that NIK the C-terminal region of DSCR1, which leads to an increase in protein stability by the degradation of DSCR1. results that DSCR1 or a role in the NF-κB The phosphorylation of DSCR1 or DSCR1 was to the regulation of the treatment of human and cells with and results in the of DSCR1 the the region to which results in the of calcineurin S. D.A. Davies Ermak G. S. N. Biol. 2003; PubMed Scopus Google Scholar). kinase has been to the DSCR1 in which on to calcineurin signaling Z. D.A. S.T. Kingsbury T.J. C.J. A. Cunningham K.W. 2004; PubMed Scopus Google Scholar). Furthermore, and are to DSCR1 S. J.D. B. X. Yang J. Y. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). DSCR1 is from calcineurin and binds with thereby its on calcineurin activity S. J.D. B. X. Yang J. Y. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we found that the overexpression of DSCR1 in cells results in and its phosphorylation results in the formation of cytosolic aggregates. The of a role in the of many M.A. Nat. Rev. Mol. Cell Biol. 2005; Scopus Google Scholar, M.A. Nat. 2004; PubMed Scopus Google Scholar). DS characteristic of the Alzheimer disease, such as the of and J.D. Res. PubMed Scopus Google and the of neuronal cells B. J. Sci. Full Text PDF PubMed Scopus Google Scholar, Nat. 1996; PubMed Scopus Google Scholar, M. N. J. PubMed Scopus Google Scholar). It has been suggested that the overexpression of genes located on chromosome 21 leads to the development of these Alzheimer Interestingly, DSCR1 has been to be in the of Alzheimer J.M. J. 2005; PubMed Scopus Google Scholar, G. Davies K.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the overexpression of DSCR1 the phosphatase activity of which the of protein of and in the two to the development of Alzheimer K.J. C.D. Ermak G. 2001; PubMed Scopus Google Scholar, T. H. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar, Res. 2000; PubMed Google Scholar). that expression of DSCR1 a role in The presented in this study whereas the expression of DSCR1 induces in neuronal cells, the NIK-mediated phosphorylation of DSCR1 is which leads to the formation of aggregates. demonstrate that DSCR1 formation does not the associated with its This the that the formation of an DSCR1 is not and not a role in the of the associated with Down it has been that are not the of the that lead to the formation these are the M.A. Nat. 2004; PubMed Scopus Google Scholar, 2006; PubMed Scopus Google Scholar). It is that the formation of is a of a that is to the cell from such This is by studies that and not are the in Alzheimer disease, disease, and The of in Alzheimer is in the that of of the in whereas and have I. R. 2002; PubMed Scopus Google Scholar). results are by study that that two proteins were in which were the and not the M. E. F. F. L. J. N. G. M. 2002; PubMed Scopus Google Scholar). be and it be to these DSCR1 aggregates protein and the or in and in with In study that NIK interacts with and phosphorylates DSCR1. Although the overexpression of DSCR1 the viability of cells, the NIK-mediated phosphorylation of DSCR1 leads to the formation of its which not to have an on cell studies are to and DSCR1 regulates the NIK activity and the alternative NF-κB signaling The results of such studies with a of the functional role of DSCR1 in NIK-mediated of the NF-κB signaling We are to S. de la B. A. and T. H. for