Positive Regulation of IκB Kinase Signaling by Protein Serine/Threonine Phosphatase 2A
2005; Elsevier BV; Volume: 280; Issue: 43 Linguagem: Inglês
10.1074/jbc.m506093200
ISSN1083-351X
AutoresArlene E. Kray, Robert S. Carter, Kevin N. Pennington, Rey J. Gomez, Laura E. Sanders, Joan M. Llanes, Wasif N. Khan, Dean W. Ballard, Brian E. Wadzinski,
Tópico(s)Cell death mechanisms and regulation
ResumoTranscription factor NF-κB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-α (TNF). In the absence of TNF, NF-κB is sequestered in the cytoplasm by inhibitory IκB proteins. Phosphorylation of IκBby the β-catalytic subunit of IKK, a multicomponent IκB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-κB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKβ, which greatly stimulates IκB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IκB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121–179 of the IKKγ regulatory subunit. Third, deletion of the PP2A-binding site in IKKγ attenuates T loop phosphorylation and catalytic activation of IKKβ in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK·PP2A complexes is required for the proper induction of IκB kinase activity in vivo. Transcription factor NF-κB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-α (TNF). In the absence of TNF, NF-κB is sequestered in the cytoplasm by inhibitory IκB proteins. Phosphorylation of IκBby the β-catalytic subunit of IKK, a multicomponent IκB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-κB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKβ, which greatly stimulates IκB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IκB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121–179 of the IKKγ regulatory subunit. Third, deletion of the PP2A-binding site in IKKγ attenuates T loop phosphorylation and catalytic activation of IKKβ in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK·PP2A complexes is required for the proper induction of IκB kinase activity in vivo. Insults to the immune system and various forms of cellular stress activate the transcription factor NF-κB 3The abbreviations used are:NF-κBnuclear factor-kappa BIκBinhibitor of kappa BIKKIκB kinasePP2Aprotein serine/threonine phosphatase 2ATNFtumor necrosis factor αTNF-R1TNF-receptor type 1MEFsmurine embryonic fibroblastsDMEMDulbecco's modified Eagle's mediumPMAphorbol 12-myristate 13-acetateCAPS3-(cyclohexylamino)propanesulfonic acid and other dimeric members of the Rel polypeptide family (reviewed in Refs. 1Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3372) Google Scholar and 2Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3293) Google Scholar). NF-κB regulates the expression of multiple genes involved in the control of cell growth, division, and survival. A variety of stimuli can activate NF-κB-mediated gene transcription, including tumor necrosis factor-α (TNF), interleukin-1, T and B cell mitogens, bacterial products, viral proteins, double-stranded RNA, as well as physical and chemical stress. Most of these agonists converge on a latent, cytoplasmic form of NF-κB that associates with IκBα or other members of the inhibitory IκB family. Following cellular stimulation, IκBα is phosphorylated, ubiquitinated, and degraded by the 26 S proteasome. In turn, NF-κB is free to translocate to the nuclear compartment where it activates transcription units containing the κB-binding site (1Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3372) Google Scholar, 2Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3293) Google Scholar). nuclear factor-kappa B inhibitor of kappa B IκB kinase protein serine/threonine phosphatase 2A tumor necrosis factor α TNF-receptor type 1 murine embryonic fibroblasts Dulbecco's modified Eagle's medium phorbol 12-myristate 13-acetate 3-(cyclohexylamino)propanesulfonic acid Phosphorylation of IκB is catalyzed by a multicomponent protein kinase termed the IKK signalsome (3Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4085) Google Scholar). Within the prototypic complex are two highly homologous IκB kinase subunits, termed IKKα and IKKβ, which form homo- or heterodimers via their leucine zippers (4Mercurio 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 (1853) Google Scholar, 5Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 6Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1589) Google Scholar). In response to various cell stimuli, IKKα and IKKβ are activated by a mechanism involving phosphorylation of their respective T loops at Ser-176/Ser-180 and Ser-177/Ser-181 (7Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (752) Google Scholar). T loop phosphorylation occurs via IKK subunit trans-autophosphorylation and/or the action of upstream kinases (3Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4085) Google Scholar). In addition to phosphorylation of T loop serines, activated IKKα and IKKβ also undergo autophosphorylation at a C-terminal serine cluster, resulting in decreased kinase activity (7Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (752) Google Scholar). Despite extensive study of these kinases, the exact molecular mechanisms involved in the activation and post-inductive repression of IKK are poorly understood. Signal-induced activation of IKKβ is dependent on its interaction with the regulatory subunit IKKγ, which lacks a kinase domain (8Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar, 9Yamaoka 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 (950) Google Scholar, 10Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar). The N-terminal half of IKKγ is required for its assembly into a functional IκB kinase complex containing IKKα and IKKβ. In contrast, the C-terminal half of this regulatory subunit is required for the induction of IκB kinase activity, presumably by serving as an interaction surface for upstream signal transducers. For example, IKKγ serves as a molecular adaptor for the Tax oncoprotein of human T cell leukemia virus, type 1, which stimulates persistent activation of IKK (11Sun S.C. Ballard D.W. Oncogene. 1999; 18: 6948-6958Crossref PubMed Scopus (167) Google Scholar). However, the precise molecular composition of the IKK signalsome remains unclear in terms of the cellular proteins that modulate its transient activity following cellular stimulation with physiologic agonists of NF-κB (12Rothwarf D.M. Karin M. Sci. STKE 1999. 1999; : RE1Google Scholar, 13Karin M. Oncogene. 1999; 18: 6867-6874Crossref PubMed Scopus (1007) Google Scholar). The multilevel regulation of IKK by phosphorylation indicates that protein phosphatases may play an important role in IKK signaling. Consistent with this possibility, prior studies with purified proteins have indicated that protein serine/threonine phosphatase 2A (PP2A) inhibits IκB kinase activity in vitro (14DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar). The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a catalytic C subunit (PP2AC), a structural A subunit, and a variable B subunit (reviewed in Refs. 15Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1542) Google Scholar and 16Zolnierowicz S. Biochem. Pharmacol. 2000; 60: 1225-1235Crossref PubMed Scopus (173) Google Scholar). PP2A associates with many cellular proteins, including cytoskeletal components (17Strack S. Westphal R.S. Colbran R.J. Ebner F.F. Wadzinski B.E. Brain Res. Mol. Brain Res. 1997; 49: 15-28Crossref PubMed Scopus (92) Google Scholar, 18Price N.E. Wadzinski B. Mumby M.C. Brain Res. Mol. Brain Res. 1999; 73: 68-77Crossref PubMed Scopus (31) Google Scholar), receptors (19Fan G.H. Yang W. Sai J. Richmond A. J. Biol. Chem. 2001; 276: 16960-16968Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), monoamine transporters (21Bauman A.L. Apparsundaram S. Ramamoorthy S. Wadzinski B.E. Vaughan R.A. Blakely R.D. J. Neurosci. 2000; 20: 7571-7578Crossref PubMed Google Scholar), metabolic enzymes (22Kabashima T. Kawaguchi T. Wadzinski B.E. Uyeda K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5107-5112Crossref PubMed Scopus (302) Google Scholar), transcription factors (23Yang J. Fan G.H. Wadzinski B.E. Sakurai H. Richmond A. J. Biol. Chem. 2001; 276: 47828-47833Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 24Wadzinski B.E. Wheat W.H. Jaspers S. Peruski Jr., L.F. Lickteig R.L. Johnson G.L. Klemm D.J. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (286) Google Scholar), and several viral proteins (reviewed in Ref. 25Garcia A. Cayla X. Sontag E. Microbes Infect. 2000; 2: 401-407Crossref PubMed Scopus (22) Google Scholar). More recent studies indicate that PP2A forms stable complexes with a growing set of cellular protein kinases, presumably to facilitate substrate recognition and rapid signal-dependent changes in their phosphorylation status (reviewed in Ref. 26Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). Here we provide several in vivo lines of evidence indicating that PP2A binds to IKK and modulates its catalytic activity. Specifically, we show that TNF-induced degradation of IκB is attenuated in cells treated with okadaic acid, an inhibitor of PP2A. PP2A and IKK derived from mammalian cell extracts co-immunoprecipitate and fractionate together on both kinase and phosphatase affinity resins. This phosphatase/kinase interaction is mediated by the IKKγ regulatory subunit. Deletion of the PP2A-binding site in IKKγ (amino acids 121–179) attenuates T loop phosphorylation and catalytic activation of IKKβ induced by enforced cellular expression of either the Tax oncoprotein or the type 1 receptor for TNF. Furthermore, in sharp contrast to wild type IKKγ, IKKγ lacking the PP2A-binding site is unable to restore TNF-induced phosphorylation of IKKβ, degradation of IκBα, and nuclear translocation of NF-κB when the mutant is stably introduced into IKKγ-deficient fibroblasts. Taken together, these data provide strong evidence that the formation of IKK·PP2A complexes is required for TNF-dependent phosphorylation and activation of IKK in vivo. Reagents—Polyclonal IKK subunit antibodies (H744, H470, FL-419, and M280) and the p65 antibody were purchased from Santa Cruz Biotechnology, and the monoclonal IKK subunit (α, β, and γ) antibodies were from Pharmingen. Monoclonal antibodies recognizing the T7 epitope and agarose conjugates of the T7 antibody were obtained from Novagen. The PP2AC monoclonal antibodies were from BD Transduction Laboratories or Upstate Biotechnology, Inc. A phospho-specific antibody recognizing IKKα and IKKβ phosphorylated at Ser-180 and Ser-181, respectively, was purchased from Cell Signaling Technology. Anti-FLAG M2 monoclonal antibodies were purchased from Sigma. Rabbit anti-Tax antibodies were provided by Dr. Bryan Cullen (Duke University). Normal rabbit IgG, normal mouse IgG, and secondary antibodies for alkaline phosphatase colorimetric detection were obtained from The Jackson Laboratory; secondary antibodies for fluorescence detection were obtained from Rockland or Molecular Probes. Horseradish peroxidase-conjugated secondary antibodies were obtained from Pierce. Mammalian expression plasmids for Tax, TNF-R1, and IKK subunits have been described previously (27Carter R.S. Pennington K.N. Ungurait B.J. Arrate P. Ballard D.W. J. Biol. Chem. 2003; 278: 48903-48906Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Ye 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). Expression plasmids for IKKγ used in Fig. 4 were kindly provided by Dr. M. Horwitz (Albert Einstein College of Medicine). pBABE-Puro, pCL-Ampho, and pHSCMV-VSVg were gifts from Dr. Christopher Aiken (Vanderbilt University). PolyFect transfection reagent and plasmid purification kits were purchased from Qiagen. The nuclear and cytoplasmic extraction kit was purchased from Active Motif. Okadaic acid, fostriecin, and microcystin were obtained from Alexis Biochemicals, and microcystin-Sepharose was obtained from Upstate Biotechnology, Inc. ATP-Sepharose was a gift from Dr. Timothy Haystead (Duke University) or purchased from Upstate Biotechnology, Inc. [γ-32P]ATP was obtained from ICN. Western blot Blocking Buffer and protein G-Sepharose were obtained from Zymed Laboratories Inc. Phenyl-Sepharose, MonoQ, and Superdex-200 columns and gel filtration standards were from Amersham Biosciences. Centricon-30 filters were obtained from Millipore. B Lymphocytes, Cell Culture, and Transfections—Primary B lymphocytes were isolated from spleens of C57Bl6 mice and purified by AutoMACS (Miltenyi Biotec) by a negative selection protocol as described previously (29Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar, 30Petro J.B. Khan W.N. J. Biol. Chem. 2001; 276: 1715-1719Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). All mice that were used as the source of splenocytes were treated humanely in accordance with federal and state government guidelines, and their use was approved by the Institutional Animal Care and Use Committee (Vanderbilt University). Jurkat T cells were maintained in Roswell Park Memorial Institute (RPMI) media supplemented with 10% fetal bovine serum, 2 mm glutamine, and antibiotics. Murine embryonic fibroblasts (MEFs) derived from mice lacking IKKγ were a gift from Dr. Joseph DiDonato (Cleveland Clinic) and have been described previously (31Rudolph D. Yeh W.C. Wakeham A. Rudolph B. Nallainathan D. Potter J. Elia A.J. Mak T.W. Genes Dev. 2000; 14: 854-862PubMed Google Scholar). HeLa cells, human embryonic kidney-293T cells (HEK-293T), and MEFs were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mm glutamine, and antibiotics. HEK-293T and HeLa cells were transfected using PolyFect according to the manufacturer's recommended protocol or by the calcium phosphate method as described previously (27Carter R.S. Pennington K.N. Ungurait B.J. Arrate P. Ballard D.W. J. Biol. Chem. 2003; 278: 48903-48906Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Generation of T7-tagged IKKγ Internal Deletion Mutant of IKKγ Lacking Amino Acids 121–179—Amino acids 121–179 were deleted from IKKγ by PCR amplification of the flanking regions, with an engineered overhang, followed by a second round of PCR in which the two flanking regions were allowed to anneal, and the resulting sequence was amplified. PCR amplification of the sequence encoding the T7 tag and first 120 amino acids of IKKγ (with a 3′ overhang) was accomplished by using the sense primer 5′-ATGGGTCGCATGGATCCG-3′, the antisense primer 5′-GCCGCGCCTCCTTCTGCCTC-3′, and T7-tagged full-length IKKγ (T7-IKKγ)/pcDNA3 (28Ye 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) as a template; the second region of IKKγ encoding amino acid residues 180–419 (with a 5′ overhang) was amplified in another PCR by using the sense primer 5′-GAAGGAGGCGCGGCAGCTGG-3′, the antisense primer 5′-TCCGCCTTGTAGATATCCG-3′, and T7-IKKγ/pcDNA3 as a template. The PCR products were allowed to anneal and then amplified by using the outside primers 5′-ATGGGTCGCATGGATCCG-3′ and 5′-TCCGCCTTGTAGATATCCG-3′. The resulting PCR product of IKKγ, lacking the sequence encoding amino acids 121–179, was digested with BamHI and EcoRV and ligated back into BamHI/EcoRV-digested T7-IKKγ/pcDNA3. Proper construction of the plasmid encoding the deletion mutant was verified by automated DNA sequencing (Vanderbilt University DNA Core Facility). Generation of MEF Cell Lines Stably Expressing T7-tagged IKKγ Constructs—Viral stocks were produced by transfecting HEK-293T cells (35-mm dish) with 3 μg of pBABE-Puro encoding the IKK construct, 3 μg of pCL-Ampho, and 1.2 μg of pHSCMV-VSVg (32Yee J.K. Friedmann T. Burns J.C. Methods Cell Biol. 1994; 43: 99-112Crossref PubMed Scopus (396) Google Scholar, 33Naviaux R.K. Costanzi E. Haas M. Verma I.M. J. Virol. 1996; 70: 5701-5705Crossref PubMed Google Scholar). Supernatants containing fully packaged retrovirus were recovered 48 h after transfection and were immediately used for transduction. Transduction was achieved by incubating IKKγ-deficient MEFs with the appropriate viral stock in the presence of Polybrene (0.8 μg/ml). Supernatants were removed 8 h post-infection, and the cells were cultured an additional 48 h in complete DMEM. Transduced cells were selected in DMEM containing 2 μg/ml puromycin. Immunoblotting—Protein samples were separated on SDS-polyacrylamide gels (10%) and electrophoretically transferred to nitrocellulose in 10 mm CAPS, pH 11, containing 10% methanol (1 h at 1 A). Proteins on the membrane were visualized with Ponceau S, followed by washing in TTBS (25 mm Tris-HCl, pH 7.4, 137 mm NaCl, 3 mm KCl, and 0.2% Tween 20). The membrane was blocked in TTBS containing 0.5% bovine serum albumin or in Blocking Buffer (Zymed Laboratories Inc.), followed by incubation with the diluted primary antibody. After washing, the membranes were then incubated with alkaline phosphatase- or fluor-conjugated secondary antibodies; bound antibodies were visualized by colorimetric detection or via the Odyssey Infrared Imaging system (LiCor). Alternatively, immunoblotting was performed using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence detection system from Amersham Biosciences. Fast Performance Liquid Chromatography—Jurkat T cells (100 ml of cell suspension) were stimulated with PMA (50 ng/ml) and ionomycin (1 μm) for 15 min. Cells were pelleted and lysed in 12 ml of Buffer A (20 mm Tris-HCl, pH 7.5, 20 mm β-glycerol phosphate, 5 mm Na4P2O7, 10 mm NaF, 0.5 mm Na3VO4, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 10% glycerol, and 0.25% Triton X-100) containing 0.1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 1 μg/ml pepstatin, 2 μg/ml aprotinin, and 2 mm benzamidine. The clarified cell lysates obtained following centrifugation at 12,000 × g were adjusted to 1 m ammonium sulfate and applied to a phenyl-Sepharose column (20 ml) equilibrated in Buffer A containing 1 m ammonium sulfate and reduced protease inhibitor concentrations (400 ng/ml pepstatin, 200 ng/ml aprotinin, and 2 mm benzamidine). The column was developed (2 ml/min) with a linear gradient (1 to 0 m ammonium sulfate in the same buffer), and 4-ml fractions were collected. The peak fractions of IKK activity (fractions 34–45) were pooled and applied to a MonoQ column (1 ml). The MonoQ column was developed (1 ml/min) using a linear gradient (0 to 0.5 m NaCl in Buffer A), and 1-ml fractions were collected. The peak fractions of IKK, which eluted from the MonoQ column between 370 and 400 mm NaCl (fractions 20–22), were pooled and concentrated to 200 μl using a Centricon-30. The concentrated sample was fractionated (0.5 ml/min) on an analytical Superdex-200 gel filtration column (20 ml) equilibrated in Buffer A containing 150 mm NaCl, and 0.5-ml fractions were collected. Aliquots of each column fraction were subjected to Western analysis or assayed for IKK activity. ATP-Sepharose and Microcystin-Sepharose Affinity Purifications—Jurkat T cell lysates (3.5 mg of protein) in 1 ml of Buffer C (50 mm β-glycerol phosphate, pH 7.4, 1.5 mm EGTA, 0.15 mm Na3VO4, 1 mm DTT, 50 mm MgCl2, and 1% Nonidet P-40) were incubated with 50 μl of a 50% slurry of ATP-Sepharose overnight at 4 °C. ATP-Sepharose-bound proteins were washed five times with Buffer C and once with "wash buffer" (25 mm Tris-HCl, pH 7.4, 1 mm DTT, and 1% Nonidet P-40). After washing, bound proteins were eluted three times with 75 μl of elution buffer containing 25 mm Tris-HCl, pH 7.4, 100 mm ATP, 1 mm DTT, 5 mm EGTA, and 1% Nonidet P-40 (ATP). The eluted material was then incubated with 50 μl of a 50% slurry of microcystin-Sepharose overnight at 4 °C. Microcystin-Sepharose-bound proteins were washed five times with wash buffer and eluted two times with 50 μl of SDS sample buffer (ATP→ MC). Twenty-μl aliquots of the ATP and ATP→ MC samples and a 20-μl aliquot of Jurkat T cell extracts were subjected to SDS-PAGE and analyzed by silver staining or immunoblotting by using IKKα-, IKKβ-, and PP2AC-specific antibodies. Immunoprecipitations—One μg of the PP2AC 1D6 monoclonal antibody (Upstate Biotechnology, Inc.) was preincubated with or without 50 μg of the corresponding PP2AC C-terminal peptide (PHVTRRTPDYFL) for 0.5 h on ice. Jurkat T cell lysates (0.5–1.5 mg of protein) were prepared in Buffer B (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, 0.1% Nonidet P-40, 1 mm Na4P2O7, 1 mm NaF, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 2 μg/ml pepstatin, and 2 mm benzamidine). Cell extracts were incubated with the PP2AC monoclonal antibody (pretreated ± peptide) and 30 μl of a 50% slurry of protein G-Sepharose for 4–16 h at 4 °C. The beads were pelleted and washed four times with "IP wash buffer" (33 mm HEPES, pH 7.5, 167 mm NaCl, 3.5 mm EDTA, and 0.05% Nonidet P-40). Bound proteins were eluted with SDS sample buffer and subjected to immunoblot analysis. Immunoprecipitation of epitope-tagged IKK subunits was accomplished by incubating cellular extracts (50–60 μg of protein) prepared in Buffer B with 10–15 μl of a 50% slurry of anti-T7-agarose or anti-FLAG M2-agarose beads for 2 h at 4 °C. For immunoblot analyses, bound proteins were washed four times with ELB buffer (50 mm HEPES, pH 7.5, 5 mm EDTA, and 250 mm NaCl), eluted with SDS sample buffer, and subjected to SDS-PAGE. For kinase assays, bound proteins were washed sequentially with ELB buffer and kinase buffer (10 mm HEPES, pH 7.4, 5 mm MgCl2, 1 mm MnCl2, 2 mm NaF, and 50 μm Na3VO4) and assayed as described below. For immunoprecipitation studies with primary B lymphocytes, lysates were prepared from 107 cells in RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 mm Na3VO4, 1 mm NaF, 1% Triton X-100, and 0.1% SDS). Extracts were pre-cleared by incubating with 12 μl of either protein G-Sepharose (PP2AC immunoprecipitations) or protein A-Sepharose (IKK immunoprecipitations) for 1 h at 4°C. The pre-cleared cell extracts were incubated with 2 μg of the PP2AC monoclonal antibody (BD Transduction Laboratories) and 12 μl of protein G-Sepharose or with 2 μg of the IKKγ polyclonal antibody (FL-419, Santa Cruz Biotechnology) and 12 μl of protein A-Sepharose for 12–14 h at 4 °C. As controls, precleared extracts were also incubated with equivalent amounts of normal mouse IgG and protein G-Sepharose or normal rabbit IgG and protein A-Sepharose. Bound proteins were washed three times with ELB buffer, eluted with SDS sample buffer, and subjected to immunoblot analysis. In Vitro Kinase Assays—IκB kinase activity was measured in reaction mixtures containing recombinant glutathione S-transferase protein fused to amino acids 1–54 of IκBα(GST-IκBα), 10 μm ATP, and 5 μCi of [γ-32P]ATP, as described previously (14DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar, 34Chu 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). A substrate containing alanine replacements for Ser-32 and Ser-36 of IκBα(GST-IκBα MU) was used to assess kinase specificity. The kinase reactions were incubated at 30 °C for 30 min and then terminated by heat denaturation in the presence of 1% SDS. Radiolabeled phosphoproteins were resolved by SDS-PAGE and visualized by autoradiography. Microcystin-Sepharose Affinity Isolations—Soluble protein extracts (0.5 mg) of HEK-293T or HeLa cells expressing T7-tagged IKKγ proteins were incubated with 30 μl of a 50% slurry of microcystin-Sepharose overnight at 4 °C. The beads were washed four times with Buffer A, and bound proteins were eluted with SDS sample buffer and subjected to immunoblot analysis. Soluble protein extracts of MEF cell lines (0.1 mg) were incubated with 20 μl of a 50% slurry of microcystin-Sepharose in ELB buffer for 2 h at 4 °C. The beads were washed four times with ELB buffer, and bound proteins were eluted with SDS sample buffer and subjected to immunoblot analysis. Effects of PP2A Inhibitors on IκB Degradation—Several studies have implicated a negative regulatory role for PP2A in signal-dependent activation of IKK and NF-κB (7Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (752) Google Scholar, 14DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. 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To investigate this question further, we monitored the fate of IκBα in HeLa cells following acute treatment with okadaic acid, the cytokine TNF, or both. As shown
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