In Vivo Identification of Inducible Phosphoacceptors in the IKKγ/NEMO Subunit of Human IκB Kinase
2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês
10.1074/jbc.m301705200
ISSN1083-351X
AutoresRobert S. Carter, Kevin N. Pennington, Bradley J. Ungurait, Dean W. Ballard,
Tópico(s)Cell death mechanisms and regulation
ResumoTranscription factor NF-κB plays a pivotal regulatory role in the genetic programs for cell cycle progression and inflammation. Nuclear translocation of NF-κB is controlled by an inducible protein kinase called IKK, which earmarks cytoplasmic inhibitors of NF-κB for proteolytic destruction. IKK contains two structurally related catalytic subunits termed IKKα and IKKβ as well as a noncatalytic subunit called IKKγ/NEMO. Mutations in the X-linked gene encoding IKKγ can interfere with NF-κB signaling and lead to immunodeficiency disease. Although its precise mechanism of action remains unknown, IKKγ is phosphorylated in concert with the induction of NF-κB by the viral oncoprotein Tax and the proinflammatory cytokine tumor necrosis factor α (TNF). We now demonstrate that TNF-induced phosphorylation of IKKγ is blocked in cells deficient for IKKβ but not IKKα. Phosphopeptide-mapping experiments with metabolically radiolabeled cells indicate that IKKβ phosphorylates human IKKγ at Ser-31, Ser-43, and Ser-376 following the enforced expression of either the Tax oncoprotein or the type 1 TNF receptor. Inducible phosphorylation of IKKγ is attenuated following the deletion of its COOH-terminal zinc finger domain (amino acids 397–419), a frequent target for mutations that occur in IKKγ-associated immunodeficiencies. As such, IKKβ-mediated phosphorylation of IKKγ at these specific serine targets may facilitate proper regulation of NF-κB signaling in the immune system. Transcription factor NF-κB plays a pivotal regulatory role in the genetic programs for cell cycle progression and inflammation. Nuclear translocation of NF-κB is controlled by an inducible protein kinase called IKK, which earmarks cytoplasmic inhibitors of NF-κB for proteolytic destruction. IKK contains two structurally related catalytic subunits termed IKKα and IKKβ as well as a noncatalytic subunit called IKKγ/NEMO. Mutations in the X-linked gene encoding IKKγ can interfere with NF-κB signaling and lead to immunodeficiency disease. Although its precise mechanism of action remains unknown, IKKγ is phosphorylated in concert with the induction of NF-κB by the viral oncoprotein Tax and the proinflammatory cytokine tumor necrosis factor α (TNF). We now demonstrate that TNF-induced phosphorylation of IKKγ is blocked in cells deficient for IKKβ but not IKKα. Phosphopeptide-mapping experiments with metabolically radiolabeled cells indicate that IKKβ phosphorylates human IKKγ at Ser-31, Ser-43, and Ser-376 following the enforced expression of either the Tax oncoprotein or the type 1 TNF receptor. Inducible phosphorylation of IKKγ is attenuated following the deletion of its COOH-terminal zinc finger domain (amino acids 397–419), a frequent target for mutations that occur in IKKγ-associated immunodeficiencies. As such, IKKβ-mediated phosphorylation of IKKγ at these specific serine targets may facilitate proper regulation of NF-κB signaling in the immune system. Transcription factor NF-κB and other dimeric members of the Rel polypeptide family regulate the expression of multiple genes involved in inflammation, immunity, mitosis, and cell survival (1Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4597) Google Scholar, 2Sha W.C. J. Exp. Med. 1998; 187: 143-146Crossref PubMed Scopus (183) Google Scholar, 3Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3439) Google Scholar). Biologic inducers of NF-κB include the proinflammatory cytokines tumor necrosis factor α (TNF) 1The abbreviations used are: TNF, tumor necrosis factor α; HTLV1, human T-cell leukemia virus type 1; IKK, IκB kinase; IL-1, interleukin 1; LPS, lipopolysaccharide; TNF-R1, tumor necrosis factor receptor-1; MEF, murine embryonic fibroblasts; WT, wild type; ZF, zinc-finger.1The abbreviations used are: TNF, tumor necrosis factor α; HTLV1, human T-cell leukemia virus type 1; IKK, IκB kinase; IL-1, interleukin 1; LPS, lipopolysaccharide; TNF-R1, tumor necrosis factor receptor-1; MEF, murine embryonic fibroblasts; WT, wild type; ZF, zinc-finger. and interleukin-1 (IL-1), the lipopolysaccharide (LPS) component of Gram-negative bacteria, and the Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV1) (4Hawiger J. Veach R.A. Liu X.Y. Timmons S. Ballard D.W. Blood. 1999; 94: 1711-1716Crossref PubMed Google Scholar, 5Chu Z.L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 6Sun S.C. Ballard D.W. Oncogene. 1999; 18: 6948-6958Crossref PubMed Scopus (167) Google Scholar, 7Karin M. Oncogene. 1999; 18: 6867-6874Crossref PubMed Scopus (1004) Google Scholar). Each of these signal-dependent responses is controlled by labile cytoplasmic inhibitors of NF-κB such as IκBα and a multicomponent IκB kinase called IKK (8Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (304) Google Scholar). The core IKK holoenzyme contains two catalytic subunits termed IKKα and IKKβ. Following cellular stimulation, the IKKβ catalytic subunit phosphorylates IκBα, leading to degradation of the inhibitor and nuclear translocation of NF-κB. Signal-dependent activation of IKKβ is triggered by phosphorylation of two serine residues in its "T loop" regulatory domain (9Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (749) Google Scholar). This modification appears to involve either autophosphorylation or phosphoryl group transfer from an upstream IKKβ kinase to the same acceptor sites following cellular stimulation (9Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (749) Google Scholar). More recent studies of the IκB kinase complex have identified a noncatalytic component called IKKγ (also known as NEMO, IKKAP1, or FIP-3) (10Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar, 11Mercurio 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, 12Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (850) Google Scholar, 13Li 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). IKKγ is required for signal-dependent activation of IKKβ, leading to its assignment as an essential regulatory subunit of the enzyme (10Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar, 12Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (850) Google Scholar, 14Harhaj E.W. Good L. Xiao G. Uhlik M. Cvijic M.E. Rivera-Walsh I. Sun S.C. Oncogene. 2000; 19: 1448-1456Crossref PubMed Scopus (92) Google Scholar). Small deletions or point mutations in the gene encoding IKKγ can cause skin inflammation or humoral immunodeficiencies in humans (15Courtois G. Smahi A. Israel A. Trends Mol. Med. 2001; 7: 427-430Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In terms of structural organization, sequences within the NH2-terminal half of IKKγ mediate its interaction with IKKβ (11Mercurio 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). In contrast, the COOH-terminal half of IKKγ is required for signal-dependent regulation of IκB kinase activity, suggesting that IKKγ links IKKβ to upstream activators (11Mercurio 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, 12Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (850) Google Scholar). Despite all of these findings, the mechanism of IKKγ action remains elusive (16Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3286) Google Scholar). In this regard, we and others (9Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (749) Google Scholar, 17Prajapati S. Gaynor R.B. J. Biol. Chem. 2002; 277: 24331-24339Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 18Tarassishin L. Horwitz M.S. Biochem. Biophys. Res. Commun. 2001; 285: 555-560Crossref PubMed Scopus (16) Google Scholar, 19Carter R.S. Geyer B.C. Xie M. Acevedo-Suarez C.A. Ballard D.W. J. Biol. Chem. 2001; 276: 24445-24448Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) have recently shown that IKKγ is phosphorylated in response to NF-κB agonists such as TNF and the Tax oncoprotein of HTLV1. Considering the key role that phosphorylation plays in the mechanism for IKKβ activation, these findings suggest that IKKγ subunit phosphorylation is important for proper regulation of the NF-κB signaling pathway. To extend these fundamental observations, we conducted new experiments that address the mechanism of IKKγ phosphorylation and the relevant phosphoacceptor sites. In this report, we demonstrate that endogenous IKKβ but not IKKα is required for signal-dependent phosphorylation of IKKγ in vivo. Using a combination of site-directed mutagenesis and phosphopeptide mapping, we have also monitored changes in the phosphorylation status of IKKγ in metabolically radiolabeled cells. Results from these biochemical experiments indicate that human IKKγ is phosphorylated at Ser-31, Ser-43, and Ser-376 in response to cellular stimulation with either TNF or the HTLV1 Tax oncoprotein. Minimal deletion of the zinc finger domain of IKKγ attenuates this inducible response. We conclude that IKKβ mediates phosphorylation of IKKγ at both amino-terminal and carboxyl-terminal sites via a zinc finger-dependent mechanism. The observed in vivo pattern of signal-dependent phosphorylation at distal sites in the protein may reflect post-translational control of IKKγ at two distinct levels. Reagents—Polyclonal anti-IKK and anti-HA antibodies (H-470, FL-419, and Y-11) were purchased from Santa Cruz Biotechnology, Inc. Monoclonal antibodies for IKKγ, the FLAG epitope, and the T7 tag were purchased from BD Biosciences, Sigma, and Novagen, respectively. Polyclonal anti-IKKβ antibodies were provided by Nancy Rice (NCI, National Institutes of Health, Bethesda, MD). Rabbit polyclonal antibodies specific for amino acids 321–353 of Tax were provided by Bryan Cullen (Duke University) (20Smith M.R. Greene W.C. Genes Dev. 1990; 4: 1875-1885Crossref PubMed Scopus (347) Google Scholar). Expression vectors for Tax, TNF-R1, and epitope-tagged subunits of IKK have been described previously (20Smith M.R. Greene W.C. Genes Dev. 1990; 4: 1875-1885Crossref PubMed Scopus (347) Google Scholar, 21Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1850) Google Scholar, 22Ye J. Xie X. Tarassishin L. Horwitz M.S. J. Biol. Chem. 2000; 275: 9882-9889Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 23Tartaglia L.A. Goeddel D.V. J. Biol. Chem. 1992; 267: 4304-4307Abstract Full Text PDF PubMed Google Scholar). Site-directed mutations were generated using the QuikChange kit (Stratagene) as specified by the manufacturer. Metabolic Radiolabeling and Subcellular Fractionation—Murine embryonic fibroblasts (MEFs) derived from mice lacking either IKKα or IKKβ have been described previously (24Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (417) Google Scholar, 25Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (853) Google Scholar). MEFs and human 293T cells (26DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Crossref PubMed Scopus (915) Google Scholar) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, and antibiotics. Human embryonic kidney 293T cells were transfected using the calcium phosphate method (27Cullen B.R. Methods Enzymol. 1987; 152: 684-704Crossref PubMed Scopus (662) Google Scholar). At 36 h post-transfection, 293T cells were labeled for 4 h with [32P]orthophosphate (1 mCi/ml, ICN) in phosphate-free Dulbecco's modified Eagle's medium (Mediatech). Cytoplasmic extracts were prepared from recipient cells by detergent lysis (28Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6420Crossref PubMed Scopus (3916) Google Scholar) in the presence of phosphatase and protease inhibitors (5Chu Z.L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Extracts were subjected to immunoprecipitation with anti-FLAG M2 antibodies coupled to agarose in ELB buffer (250 mm NaCl, 50 mm HEPES, 5 mm EDTA, 0.1% Nonidet P-40) (5Chu Z.L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). MEFs were incubated overnight in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum and then labeled in phosphate-free medium for 4 h with [32P]orthophosphate (1 mCi/ml) prior to agonist treatment. Cytoplasmic extracts were prepared as described above, precleared with polyclonal anti-HA antibodies bound to protein A, and immunoprecipitated with polyclonal anti-IKKγ antibodies. Resultant complexes were washed sequentially with ELB buffer containing 2 m urea and then with 150 mm NaCl, 10 mm sodium phosphate pH 7.2, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40. Immunoprecipitates were fractionated by SDS-PAGE and transferred to polyvinylidine difluoride (PVDF) membranes. Phosphoproteins were identified by autoradiography and analyzed by immunoblotting using an enhanced chemiluminescence system (Pierce) (5Chu Z.L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Resolved phosphoproteins were quantitatively analyzed with a Storm 860 PhosphorImager (Amersham Biosciences). Phosphopeptide Mapping and Phosphoamino Acid Analysis—Phosphoproteins were separated by SDS-PAGE, transferred to PVDF membranes, and visualized by autoradiography. Membrane sections containing radiolabeled IKKγ were excised, incubated with methanol, and then blocked with 50 mm NH4HCO3 containing 0.1% Tween 20 (Bio-Rad) for 30 min at room temperature. Phosphoproteins were digested in situ with N-tosyl-l-lysine chloromethyl ketone (TLCK) treated chymotrypsin (8 μg, Worthington) in 125 μl of NH4HCO3 (37 oC, 12 h) (29Luo K.X. Hurley T.R. Sefton B.M. Methods Enzymol. 1991; 201: 149-152Crossref PubMed Scopus (131) Google Scholar). Released peptides were subjected to two-dimensional phosphopeptide mapping as described by Boyle et al. (30Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar) excluding the performic acid oxidation. Phosphoproteins were first separated by electrophoresis on TLC plates at pH 1.9 (30Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar) using the Hunter thin-layer electrophoresis system (model HTLE-7000, CBS Scientific). Separation in the second dimension was performed by ascending chromatography in n-butanol (37.5%), pyridine (25%), and acetic acid (7.5%) (30Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar). Resolved phosphopeptides were visualized by autoradiography using Biomax MS high speed film (Eastman Kodak Co.). Phosphoaminoacid analysis was performed by hydrolysis of membrane-bound IKKγ with 6 n HCl as described previously (30Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar). IκB Kinase Assays—Cytoplasmic extracts were immunoprecipitated with anti-T7 antibodies in the presence of ELB buffer. IκB kinase activity was measured as described previously (5Chu Z.L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 31DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar) in a reaction mixture containing ATP (10 μm), [γ-32P]ATP (5 μCi), and recombinant glutathione S-transferase protein fused to amino acids 1–54 of IκBα. Radiolabeled products were fractionated by SDS-PAGE, transferred to PVDF membranes, and visualized by autoradiography. IKKβ Is Required for in Vivo Phosphorylation of IKKγ—Two interactive components of IKK termed IKKβ and IKKγ are essential for TNF-induced phosphorylation and degradation of IκBα (7Karin M. Oncogene. 1999; 18: 6867-6874Crossref PubMed Scopus (1004) Google Scholar). Phosphoryl group transfer to IκBα is mediated by IKKβ, which is itself activated via a phosphorylation-dependent mechanism (9Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (749) Google Scholar). However, the functional requirement for IKKγ in this context remains an enigma (16Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3286) Google Scholar). In this regard, we recently found that IKKγ also serves as a phosphorylation substrate in cells expressing the Tax oncoprotein of HTLV1, a potent pathologic inducer of IκB kinase activity (19Carter R.S. Geyer B.C. Xie M. Acevedo-Suarez C.A. Ballard D.W. J. Biol. Chem. 2001; 276: 24445-24448Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Accordingly, signal-dependent phosphorylation of IKKγ may regulate the function of this noncatalytic subunit in the NF-κB signaling pathway. To extend our findings with Tax, we monitored IKKγ subunit phosphorylation in MEFs following treatment with proinflammatory mediators. In initial experiments, wild type MEFs were metabolically radiolabeled with [32P]orthophosphate and then stimulated with either TNF, IL-1, or LPS. Endogenous IKKγ complexes were purified by immunoprecipitation, fractionated by SDS-PAGE, and analyzed by sequential autoradiography and immunoblotting. As shown in Fig. 1A (top panel), TNF induced significant phosphorylation of IKKγ as compared with untreated controls (top panel, lanes 1 and 2). Similar results were obtained with IL-1 and LPS (lanes 3 and 4). Changes in the phosphorylation status of IKKγ could not be attributed to differences in either IKKβ or IKKγ protein content (lower panels). As expected, IKKβ was also phosphorylated and activated under these stimulatory conditions (top panel and data not shown). We conclude that phosphorylation of endogenous IKKγ is induced not only by Tax but also by multiple proinflammatory agonists of NF-κB in murine fibroblasts. In prior studies, we found that IKKβ has the capacity to phosphorylate a recombinant IKKγ substrate in vitro (19Carter R.S. Geyer B.C. Xie M. Acevedo-Suarez C.A. Ballard D.W. J. Biol. Chem. 2001; 276: 24445-24448Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This finding raised the possibility that in vivo phosphorylation of IKKγ is mediated by IKKβ or the structurally related IKKα catalytic subunit. To test this hypothesis, MEFs deficient for either IKKα or IKKβ were cultured in the presence of [32P]orthophosphate and stimulated with TNF under conditions leading to optimal IκB kinase activity (data not shown). We then prepared IKK immunocomplexes and monitored the core subunits for phosphoprotein content. As shown in Fig. 1B (top panel), treatment of wild type MEFs with TNF led to a 13.6-fold increase in IKKγ phosphorylation (lanes 1 and 2). This inducible response was readily detected in MEFs lacking IKKα (lanes 3 and 4), whereas IKKγ phosphorylation was almost completely blocked in MEFs lacking IKKβ (lanes 5 and 6). Coupled with our prior in vitro results (19Carter R.S. Geyer B.C. Xie M. Acevedo-Suarez C.A. Ballard D.W. J. Biol. Chem. 2001; 276: 24445-24448Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), we conclude that IKKβ mediates phosphorylation of IKKγ under physiologic signaling conditions. However, we cannot exclude the possibility that IKKα plays a secondary role in IKKγ phosphorylation. IKKγ Is Phosphorylated on Multiple Serines—A prerequisite for understanding the functional consequences of IKKγ subunit phosphorylation is to identify the relevant IKKβ-responsive acceptor sites. To address this important question, expression vectors for IKKβ, IKKγ, and HTLV1 Tax were transfected into 293T cells. After metabolic radiolabeling with 32Pi, ectopic IKKγ complexes were immunopurified and analyzed by SDS-PAGE. As shown in Fig. 2A, phosphoryl group transfer to IKKγ was significantly increased in the presence of Tax relative to the basal level of IKKγ phosphorylation in Tax-deficient cells. Importantly, IKKβ was chronically phosphorylated and activated by Tax under these experimental conditions, permitting us to capture sufficient quantities of stably modified IKKγ for subsequent phosphoamino acid and phosphopeptide-mapping analyses. As shown in Fig. 2B, these biochemical studies revealed the presence of phosphoserine in IKKγ but no evidence for signal-dependent phosphorylation of either threonine or tyrosine residues. Based on the corresponding cDNA sequences, human IKKγ contains a total of 26 serine residues, 19 of which are conserved in murine IKKγ (10Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar, 11Mercurio 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, 12Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (850) Google Scholar, 13Li 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). Accordingly, IKKγ may contain more than one inducible phosphoacceptor site. To address this possibility, human 293T cells were programmed with expression vectors for IKKγ and IKKβ in the presence or absence of a Tax effector plasmid. Radiolabeled IKKγ was purified from recipient cells and digested with chymotrypsin, and the resultant peptides were resolved by two-dimensional phosphopeptide mapping (30Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar). As shown in Fig. 2C, IKKγ derived from Tax-expressing cells contained four major chymotryptic phosphopeptides, which we designated as PP-1, PP-2, PP-3, and PP-4. Radiolabeling efficiencies for the same set of IKKγ phosphopeptides were significantly reduced in parallel studies with Tax-deficient cells, thus confirming the signal-dependent nature of these post-translational modifications (Fig. 2C). Considering that there are 14 potential sites for chymotryptic cleavage in IKKγ, our biochemical mapping data strongly suggest that Tax induces phosphorylation of IKKγ at multiple serine residues in vivo. Identification of Inducible Phosphoacceptors in IKKγ—To assign specific phosphoacceptors in IKKγ, we next used site-directed mutagenesis to replace individual serine residues with alanine in the full-length protein. In this regard, sequences in the NH2-terminal region of IKKγ (amino acids 1–120) are necessary for its interaction with the IKKβ catalytic subunit (11Mercurio 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, 32May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (615) Google Scholar). To determine whether the IKKβ binding domain of IKKγ is subject to signal-dependent phosphorylation, 293T cells were programmed with expression vectors for Tax, IKKβ, and human IKKγ containing alanine replacements at serine residues 17, 31, 43, 68, and 85. All five of these serines are conserved between mouse and human IKKγ (10Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar, 11Mercurio 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, 12Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (850) Google Scholar, 13Li 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). After labeling recipient cells with 32Pi, IKKγ proteins were isolated, digested with chymotrypsin, and analyzed by two-dimensional phosphopeptide mapping. As shown in Fig. 3 (upper panels), point mutations at Ser-31 and Ser-43 in IKKγ eliminated two of the four major phosphopeptides identified in control-mapping experiments with wild type IKKγ (PP-1 and PP-2, respectively). In sharp contrast, replacement of either Ser-17, Ser-68, or Ser-85 with alanine yielded a phosphopeptide fingerprint that was indistinguishable from the pattern for wild type IKKγ following chymotrypsin digestion (Fig. 3, lower panels). These in vivo results indicate that IKKβ mediates inducible phosphorylation of IKKγ at Ser-31 and Ser-43, whereas other local serine residues in the IKKβ binding domain of IKKγ are spared from modification. In contrast to the NH2-terminal region of IKKγ, sequences in the COOH-terminal half of the protein (amino acids 320–419) appear to couple IKK to upstream signal (11Mercurio 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, 12Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (850) Google Scholar). In the case of human IKKγ, this region contains potential serine phosphoacceptors at positions 341, 346, 350, 364, 376, 377, 383, and 387. Based on their corresponding phosphopeptide fingerprints, mutations affecting the first four members of this set did not significantly change the phosphorylation status of IKKγ in Tax-expressing cells (data not shown). Accordingly, we engineered alanine replacements into IKKγ at the cluster of serines positioned between amino acids 370 and 390 (denoted as S376A, S377A, S383A, and S387A). As shown in Fig. 4 (top panels), point mutations at either Ser-383 or Ser-387 had no significant effect on distribution of the four major phosphopeptides identified in studies with wild type IKKγ. However, in vivo phosphorylation of S376A in Tax-expressing cells yielded a pattern of chymotryptic peptides that was clearly distinct from the wild type IKKγ fingerprint, specifically with respect to PP-3 and PP-4 (Fig. 4, bottom panels). Of particular interest, the mutation of Ser-376 was associated with the appearance of two new phosphopeptides (designated PP-3′ and PP-4′), suggesting the presence of compensatory phosphoacceptors in the same chymotryptic fragments. Consistent with this hypothesis, alanine replacement of Ser-377 in mutant S376A eliminated the appearance of PP-3′ and PP-4′ (mutant S376/377A), whereas the disruption of Ser-377 alone was without affect (mutant S377A). These data indicate that Ser-376 is the primary target for phosphorylation in the COOH-terminal region of IKKγ and the vicinal serine at position 377 serves a compensatory role. Taken together with data shown in Fig. 3, our mapping experiments strongly suggest that Ser-31, Ser-43, and Ser-376 are the major phosphoacceptor sites in human IKKγ. To confirm this interpretation, we next engineered expression vectors for IKKγ containing serial mutations at the identified serine phosphoacceptors and introduced them into 293T cell transfectants along with Tax and IKKβ effector plasmids. IKKβ immunocomplexes were then isolated from 32Pi-labeled recipients and analyzed for phosphoprotein content. As shown in Fig. 5, simultaneous disruption of Ser-31 and Ser-43 in the NH2-terminal region of IKKγ led to a significant reduction in subunit-specific phosphorylation (mutant S31/43A, lane 3). Simultaneous disruption of Ser-376 and compensatory Ser-377 in the COOH-terminal half of IKKγ also attenuated 32Pi incorporation into the protein (mutant S376/377A, lane 4). Consistent with the biochemical phenotype of these double point mutants, the defect in substrate-radiolabeling efficiency was further evident (∼19% wild type control) when all four of the identified serine targets in IKKγ were disrupted in combination by site-directed mutagenesis (lane 5). Observed changes in the phosphorylation status of IKKγ were not attributed to fluctuations in ectopic protein expression, because each of the phosphorylation-defective mutants of IKKγ were comparably expressed in metabolically radiolabeled cells (Fig. 5, bottom panels). Moreover, these results could not be attributed to changes in the overall structural integrity
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