Genetic Evidence for the Essential Role of β-Transducin Repeat-containing Protein in the Inducible Processing of NF-κB2/p100

Abstract

Processing of the nfκb2gene product p100 to generate p52 is an important step in NF-κB regulation. This step is regulated by a nonclassical NF-κB signaling pathway involving the NF-κB-inducing kinase (NIK). NIK induces p100 processing by triggering phosphorylation of specific C-terminal serines of p100. However, the downstream molecular events leading to p100 processing remain unclear. Here we show that NIK induced the physical recruitment of β-transducin repeat-containing protein (β-TrCP), a component of the SCF ubiquitin ligase complex, to p100. This event required the phosphorylation sites as well as the death domain of p100. Using the RNA interference technique, we demonstrated that β-TrCP is essential for NIK-induced p100 ubiquitination and processing. Interestingly the constitutive processing of p100 mutants was independent of β-TrCP. These results suggest that β-TrCP is an essential component of NIK-induced p100 processing. Processing of the nfκb2gene product p100 to generate p52 is an important step in NF-κB regulation. This step is regulated by a nonclassical NF-κB signaling pathway involving the NF-κB-inducing kinase (NIK). NIK induces p100 processing by triggering phosphorylation of specific C-terminal serines of p100. However, the downstream molecular events leading to p100 processing remain unclear. Here we show that NIK induced the physical recruitment of β-transducin repeat-containing protein (β-TrCP), a component of the SCF ubiquitin ligase complex, to p100. This event required the phosphorylation sites as well as the death domain of p100. Using the RNA interference technique, we demonstrated that β-TrCP is essential for NIK-induced p100 ubiquitination and processing. Interestingly the constitutive processing of p100 mutants was independent of β-TrCP. These results suggest that β-TrCP is an essential component of NIK-induced p100 processing. IκB kinase β-transducin repeat-containing protein death domain processing-inhibitory domain NF-κB-inducing kinase RNA interference small interfering RNA green fluorescent protein immunoblotting coimmunoprecipitation immunoprecipitation RNAi-resistant β-TrCP hemagglutinin reverse transcription radioimmune precipitation buffer Skp1-Cullin-1/Cdc53-F box protein The NF-κB family of transcription factors participates in the regulation of diverse biological processes, including innate and adaptive immune responses, lymphoid organ development and maturation, inflammation, and cell growth and survival (1Silverman N. Maniatis T. Genes Dev. 2001; 15: 2321-2342Crossref PubMed Scopus (777) Google Scholar, 2Gilmore T.D. Koedood M. Piffat K.A. White D.W. Oncogene. 1996; 13: 1367-1378PubMed Google Scholar, 3Barkett M. Gilmore T.D. Oncogene. 1999; 69: 6910-6924Crossref Scopus (1082) Google Scholar, 4Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2470) Google Scholar). Deregulated function of NF-κB contributes to the development of various cell malignancies (5Rayet B. Gelinas C. Oncogene. 1999; 18: 6938-6947Crossref PubMed Scopus (1010) Google Scholar, 6Baldwin A.S. J. Clin. Investig. 2001; 107: 241-246Crossref PubMed Scopus (1199) Google Scholar). Mammalian cells have five NF-κB proteins, RelA, RelB, c-Rel, p50, and p52, which function as various homo- and heterodimers (7Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar). In most cell types, the NF-κB factors are sequestered in the cytoplasm through physical interaction with specific ankyrin repeat-containing inhibitors, including IκBα and homologues (8Baldwin A.S., Jr. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar). The latent forms of NF-κB can be activated by a large variety of chemical, environmental, and microbial agents, which act by inducing the phosphorylation and subsequent degradation of IκBα (9Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1317) Google Scholar, 10Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M., Qi, X. Lee W.Y. Ballard D.A. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). This canonical NF-κB signaling pathway depends on a multisubunit IκB kinase (IKK),1 which is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ (also named NEMO, IKKαP1, or FIP-3) (11Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). When activated by upstream signals, IKK directly catalyzes the phosphorylation of IκBα at two specific serines, which in turn triggers its ubiquitination. Recent biochemical studies suggest that the β-transducin repeat-containing protein (β-TrCP, also named E3RSIκB) is involved in the ubiquitination of phosphorylated IκBα, although genetic evidence is lacking (11Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). Another level of NF-κB regulation is via processing of the NF-κB1 and NF-κB2 precursor proteins p105 and p100, a proteasome-mediated event required for generating p50 and p52, respectively (7Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar, 12Fan C.M. Maniatis T. Nature. 1991; 354: 395-398Crossref PubMed Scopus (239) Google Scholar). Both p105 and p100 contain ankyrin repeats at their C-terminal portions and function as IκB-like NF-κB inhibitors by forming cytoplasmic complexes with mature NF-κB subunits (13Rice N.R. MacKichan M.L. Israel A. Cell. 1992; 71: 243-253Abstract Full Text PDF PubMed Scopus (343) Google Scholar, 14Mercurio F. DiDonato J.A. Rosette C. Karin M. Genes Dev. 1993; 7: 705-718Crossref PubMed Scopus (252) Google Scholar). Thus, the processing of p105 and p100 also serves to liberate the sequestered NF-κB members. It seems clear that the processing of p105 is largely a constitutive event (11Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar), while the processing of p100 is tightly controlled by both negative and positive regulatory mechanisms (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). The C-terminal region of p100 contains a death domain (DD) that functions as a processing-inhibitory domain (PID). Mutant forms of p100 lacking the PID undergo constitutive processing. Interestingly, in some lymphoma cells, the nfκb2 gene is involved in chromosomal translocations that produce C-terminal truncation mutants of p100 lacking the PID, and at least some of these p100 mutants have been shown to undergo constitutive processing (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). These findings raise the possibility that deregulated processing of p100 may contribute to the development of lymphoid malignancies. The processing of wild type p100 can be induced by a noncanonical NF-κB signaling pathway involving the NF-κB-inducing kinase (NIK) (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). NIK induces p100 phosphorylation at specific C-terminal serines, which serves as a trigger for its inducible processing. Consistently anik gene mutation in the alymphoplasia mice is associated with the absence of p100 processing, resulting in severe deficiencies in lymphoid organ development (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). More recent studies suggest that NIK induces p100 phosphorylation through a downstream kinase, IKKα (16Senftleben U. Cao Y. Xiao G. Kraehn G. Greten F. Chen Y., Hu, Y. Fong A. Sun S.-C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar). Interestingly IKKα is also involved in p100 processing induced by the retroviral oncoprotein Tax (17Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). In sharp contrast, IKKβ, which is essential for the canonical NF-κB signaling, is completely dispensable for the inducible processing of p100 (16Senftleben U. Cao Y. Xiao G. Kraehn G. Greten F. Chen Y., Hu, Y. Fong A. Sun S.-C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar, 17Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). These findings reveal a novel signaling pathway specifically regulating the processing of p100, which is essential for the development and maturation of lymphoid organs. While it is clear that phosphorylation triggers the processing of p100, the downstream molecular events involved in this signaling process remain poorly defined. In this study, we have investigated the mechanism of p100 ubiquitination and the role of this posttranslational modification in regulating p100 processing. Using the small interfering RNA (siRNA)-mediated gene suppression technique, we have demonstrated that β-TrCP is an essential component involved in NIK-induced ubiquitination of p100. In NIK-expressing cells, β-TrCP forms a stable complex with p100 but not with a p100 mutant lacking its phosphorylation site. We further demonstrate that β-TrCP-mediated p100 ubiquitination is essential for inducible, but not constitutive, processing of p100. Expression vectors encoding NIK and derivatives, p100 and derivatives, and HA-ubiquitin have been described (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 17Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). To construct the HA-tagged β-TrCP, the β-TrCP cDNA from Jurkat T cells was amplified by RT-PCR and inserted into the pcDNA-HA vector (18Harhaj E.W. Sun S.-C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The β-TrCPiR is a mutant harboring sense mutations in the siRNA targeting site that prevent the binding and degradation by the specific siRNA. The anti-HA monoclonal antibody (horseradish peroxidase-conjugated, 3F10) was purchased from Roche Molecular Biochemicals. The antibody recognizing the N terminus of p100 (anti-p100) was kindly provided by Dr. W. C. Greene (19Sun S.-C. Ganchi P.A. Beraud C. Ballard D.W. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1346-1350Crossref PubMed Scopus (162) Google Scholar). siRNA for human β-TrCP and green fluorescent protein (GFP) were synthesized by Dharmacon Research, Inc. (Lafayette, CO). The sequences of β-TrCP siRNA are: GUG GAA UUU GUG GAA CAU CTT (sense) and GAU GUU CCA CAA AUU CCA CTT (antisense). The sequences of GFP siRNA are: GCU ACC UGU UCC AUG GCC ATT (sense) and UGG CCA UGG AAC AGG UAG CTT. Transfection of 293 cells was performed using LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. Briefly, the cells were seeded into six-well plates 12–16 h prior to transfection. About 0.6 nmol of the β-TrCP siRNA was mixed with 800 ng of carrier DNA (pcDNA) and transfected into the 293 cells in 2 ml of culturing medium. 24 h later, the same transfection was performed to achieve high efficiency gene suppression. 24 h following the second siRNA transfection, DNA expression vectors encoding p100 and the other indicated proteins were transfected into the cells. Protein extracts and RNA were prepared at 24–30 h after the DNA transfection. The efficiency of β-TrCP gene suppression was monitored by RT-PCR. 293 cells were transfected using DEAE-dextran (20Holbrook N. Gulino A. Ruscetti F. Virology. 1987; 157: 211-219Crossref PubMed Scopus (39) Google Scholar) and lysed in RIPA buffer supplemented with protease inhibitors (17Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). The cell lysates (about 7 μg) were subjected to SDS-PAGE and IB as described previously (21Uhlik M. Good L. Xiao G. Harhaj E.W. Zandi E. Karin M. Sun S.-C. J. Biol. Chem. 1998; 273: 21132-21136Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). CoIP assays (using 250 μg of cell lysates) were performed as described previously (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). 293 cells were transfected in six-well plates with HA-ubiquitin and p100 together with the indicated expression vectors. About 30 h posttransfection, the cells were lysed in RIPA buffer and immediately subjected to IP using anti-p100. The agarose beads were washed three times with RIPA buffer, and the attached proteins were eluted in SDS loading buffer. The eluted ubiquitin-conjugated p100 was analyzed by IB using horseradish peroxidase-conjugated anti-HA. As described earlier (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar), the inducible processing of p100 is associated with its ubiquitination, although it remains unclear whether this is an essential step in p100 processing. Additionally the ubiquitin ligase regulating p100 ubiquitination remains to be identified. We investigated the role of βTrCP in the processing of p100 using siRNA-mediated gene suppression. As expected from various other studies (22Caplen N.J. Trends Biotechnol. 2002; 20: 49-51Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 23Harborth J. Elbashir S.M. Bechert K. Tuschl T. Weber K. J. Cell Sci. 2001; 114: 4557-4565Crossref PubMed Google Scholar, 24Luo X. Tang Z. Rizo J. Yu H. Mol. Cell. 2002; 9: 59-71Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), the β-TrCP siRNA efficiently depleted the mRNA of βTrCP but not that of a control gene, glyceraldehyde-3-phosphate dehydrogenase, as detected by the sensitive RT-PCR analysis (Fig. 1 A). More importantly the β-TrCP suppression led to almost complete blockade of NIK-induced p100 processing. This effect was specific since the processing of p100 was not affected in cells transfected with the control GFP siRNA (Fig. 1 B, lanes 5–8). Furthermore the β-TrCP RNAi was also able to inhibit NIK-induced processing of endogenous p100 (Fig. 1 C). These results provide genetic evidence for an essential role of β-TrCP in the inducible processing of p100. With the RNAi genetic approach, we were also able to determine whether β-TrCP is essential for proteolysis of IκBα. For these studies, we expressed HA-tagged IκBα with a constitutively activated form of IKKβ (IKKβSS/EE). IKKβSS/EE efficiently induced degradation of IκBα (Fig. 1 D, lane 2); however, the inducible degradation of IκBα was completely blocked in cells transfected with the β-TrCP siRNA (lane 4). We next examined whether β-TrCP is required for the ubiquitination of p100. When expressed in 293 cells, NIK efficiently induced polyubiquitination of p100 as evidenced by the formation of ubiquitin-conjugated heterogeneous p100 adducts (Fig.2 A, lane 2). Remarkably the p100 polyubiquitination was largely abrogated when β-TrCP gene expression was suppressed by RNAi (lane 4). Moreover the RNAi-mediated inhibition of p100 ubiquitination was associated with the marked diminishment of NIK-induced p100 processing (Fig. 2 B, lane 4). To further confirm the inhibitory effect of β-TrCP siRNA was specifically caused by the loss of β-TrCP, functional rescue was performed by transfecting the siRNA-treated cells with a β-TrCP mutant harboring sense mutations in the siRNA targeting site (RNAi-resistant β-TrCP or βTrCPiR). As shown in Fig. 2, both the ubiquitination and the processing of p100 were efficiently restored in cells expressing the RNAi-resistant form of β-TrCP (Fig. 2, A andB, lane 6). Thus, β-TrCP is an essential component for regulating the ubiquitination of p100. We then examined whether β-TrCP physically interacts with p100 by coIP assays. When expressed in 293 cells, β-TrCP only weakly interacted with p100 in the absence of the processing-inducing kinase NIK (Fig.3 A, top panel,lane 1). However, in the presence of NIK, β-TrCP formed a stable complex with p100 and was readily coprecipitated by the p100 antibody (lane 2). In contrast, a catalytically inactive NIK mutant (NIK(K429A/K430A)) failed to induce the binding of β-TrCP to p100. This functional difference was not due to variation in protein expression since similar amounts of NIK and NIK mutant were detected by immunoblotting in the cell lysates (Fig. 3 A,middle panel). The levels of β-TrCP (middle panel) and p100 (bottom panel) were also comparable in the different cell transfectants. Furthermore the NIK-induced p100/β-TrCP interaction was tightly associated with the induction of p100 processing (bottom panel). Finally the molecular interaction between β-TrCP and p100 appeared to be strong since β-TrCP also formed a stable complex with endogenous p100 in the presence of NIK (Fig. 3 B, lane 3). Since NIK, but not NIK(K429A/K430A), is unable to induce p100 phosphorylation (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar), we reasoned that the phosphorylation site of p100 might be required for β-TrCP recognition. This idea was tested by performing the coIP assays using a p100 mutant (p100SS/AA, see Ref. 15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar) harboring serine to alanine mutations at its phosphorylation site. As expected, the p100SS/AA did not respond to NIK for processing (Fig.3 C, bottom panel, lane 5). More importantly this p100 mutant also failed to interact with β-TrCP (top panel, lane 5) or become polyubiquitinated (data not shown and Ref. 15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar) in NIK-expressing cells. These findings strongly suggest the direct involvement of β-TrCP in catalyzing the polyubiquitination of p100. We have previously shown that a DD located in the C-terminal region of p100 functions to suppress the constitutive processing of p100. Mutant forms of p100 harboring a DD deletion (p100ΔDD) or C-terminal truncations undergo constitutive processing (Ref. 15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar and Fig. 4 B). It has remained unclear how the constitutive processing of p100 is mediated. One possibility we considered was that the constitutively processed p100 mutants bind to β-TrCP independently of NIK. This idea was tested by coIP assays using β-TrCP and or a p100 C-terminal truncation mutant lacking the DD and C-terminal sequences To was able to bind β-TrCP (Fig. 4 4 and when NIK was expressed in the cells and Consistently NIK did not the constitutive processing of the p100 mutants B, lanes 4 and but induced the processing of the wild type NIK (lane 2). This to determine whether β-TrCP is required for p100 constitutive processing. In sharp to the results with the inducible processing of p100, the constitutive processing of was not affected by RNAi-mediated β-TrCP gene suppression (Fig. 4 C, lanes and 6). results were with the (data not Thus, the constitutive processing of p100 to be independent of β-TrCP. Processing of the nfκb2 gene p100, to generate p52 is an important step of NF-κB which is specifically involved in the development and maturation of lymphoid organs. evidence that the mechanism of p100 processing from that regulating degradation of the NF-κB IκBα. studies using the alymphoplasia mice demonstrate an essential role for the NIK kinase in regulating p100 processing in lymphoid (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). the other NIK is not required for degradation of IκBα or the of the form of NF-κB K. F. K. K. M. K. T. T. Nat. 1999; PubMed Scopus Google Scholar, L. H. White Science. 2001; PubMed Scopus Google Scholar). studies reveal that IKKβ, which is for the canonical NF-κB signaling is dispensable for NIK-induced p100 processing, while IKKα is an essential component of the pathway (16Senftleben U. Cao Y. Xiao G. Kraehn G. Greten F. Chen Y., Hu, Y. Fong A. Sun S.-C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar). We have the studies by the mechanism of p100 polyubiquitination and its role in p100 processing. Using the siRNA-mediated gene suppression technique, we have provided genetic evidence for the involvement of β-TrCP in p100 polyubiquitination (Fig.2 A). Furthermore inhibition of p100 ubiquitination in cells results in blockade of the inducible processing of p100 1 and 2 B). These findings suggest that polyubiquitination of p100 is through the ubiquitin ligase and serves as an essential step in the inducible p100 processing. studies using a β-TrCP mutant suggest that β-TrCP also participates in ubiquitination of other proteins, including IκBα, human and (11Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). With the siRNA technique, we can determine whether β-TrCP is essential for the ubiquitination and proteolysis of of the RNAi studies have the essential role of β-TrCP in degradation of IκBα (Fig. 1 and p105 (data not A site for β-TrCP phosphorylated serines and has been in the various β-TrCP (Ref. M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google see Fig. 5). The β-TrCP binding site in p100 contains although not of the in the other proteins (Fig. 5). of the two serines to this site in p100 completely its to bind β-TrCP (Fig.3 C). Interestingly, in to the C-terminal phosphorylation the DD of p100 is required for its interaction with β-TrCP (Fig. 4 A). with this the p100 mutant lacking the DD is in ubiquitination (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). We have previously shown that p100 mutants lacking the C-terminal DD undergo constitutive processing (15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). Interestingly the processing of these mutants is independent of β-TrCP. Consistently these mutants not physically interact with β-TrCP. This two how the constitutive processing of p100 is the region of p100 may interact with to be ubiquitin which p100 mutants for constitutive ubiquitination and the constitutive processing of p100 may not ubiquitination. In of the second we have been unable to polyubiquitination of the constitutively processed forms of p100 using the in ubiquitination (Ref. 15Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar and not these results the possibility for involvement of ubiquitination in the constitutive processing of p100 it that strong polyubiquitination is specifically required for the inducible processing of p100. We Dr. Greene for and of the Sun for of the