FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023303889> ?p ?o ?g. }

• W2023303889 endingPage "1493" @default.
• W2023303889 startingPage "1487" @default.
• W2023303889 abstract "I-κB kinase (IKK) is a serine/threonine kinase that phosphorylates I-κBα and I-κBβ and targets them for polyubiquitination and proteasome-mediated degradation. IKK consists of two highly related catalytic subunits, α and β, and a regulatory γ subunit, which becomes activated after serine phosphorylation of the activation loops of the catalytic domains. The human T-lymphotropic retrovirus type-I trans-activator, Tax, has been shown to interact directly with IKKγ and activates IKK via a mechanism not fully understood. Here we demonstrate that IKK binds serine/threonine protein phosphatase 2A (PP2A), and via a tripartite protein-protein interaction, Tax, IKKγ, and PP2A form a stable ternary complex.In vitro, PP2A down-regulates active IKK prepared from Tax-producing MT4 cells. In the presence of Tax, however, the ability of PP2A to inactivate IKK is diminished. Despite their interaction with IKKγ, PP2A-interaction-defective Tax mutants failed to activate NF-κB. Our data support the notion that IKKγ-associated PP2A is responsible for the rapid deactivation of IKK, and inhibition of PP2A by Tax in the context of IKK·PP2A·Tax ternary complex leads to constitutive IKK and NF-κB activation. I-κB kinase (IKK) is a serine/threonine kinase that phosphorylates I-κBα and I-κBβ and targets them for polyubiquitination and proteasome-mediated degradation. IKK consists of two highly related catalytic subunits, α and β, and a regulatory γ subunit, which becomes activated after serine phosphorylation of the activation loops of the catalytic domains. The human T-lymphotropic retrovirus type-I trans-activator, Tax, has been shown to interact directly with IKKγ and activates IKK via a mechanism not fully understood. Here we demonstrate that IKK binds serine/threonine protein phosphatase 2A (PP2A), and via a tripartite protein-protein interaction, Tax, IKKγ, and PP2A form a stable ternary complex.In vitro, PP2A down-regulates active IKK prepared from Tax-producing MT4 cells. In the presence of Tax, however, the ability of PP2A to inactivate IKK is diminished. Despite their interaction with IKKγ, PP2A-interaction-defective Tax mutants failed to activate NF-κB. Our data support the notion that IKKγ-associated PP2A is responsible for the rapid deactivation of IKK, and inhibition of PP2A by Tax in the context of IKK·PP2A·Tax ternary complex leads to constitutive IKK and NF-κB activation. human T-lymphotropic virus type I I-κB kinase protein phosphatase 2A glutathione S-transferase long terminal repeat okadaic acid mitogen-activated protein kinase/extracellular signal-regulated kinase kinase cAMP-response element-binding protein p300 CREB-binding protein-associated factor NF-κB/Rel family of transcription factors are controlled by inhibitory I-κB proteins I-κBα and I-κBβ and the I-κB-like domains in NF-κB1 and NF-κB2 that sequester NF-κB/Rel in the cytoplasm as multiprotein complexes (for reviews, see Ref. 1Beg A.A. Baldwin A.S.J. Genes Dev. 1993; 7: 2064-2070Crossref PubMed Scopus (735) Google Scholar, 2Liou H.C. Baltimore D. Curr. Opin. Cell Biol. 1993; 5: 477-487Crossref PubMed Scopus (516) Google Scholar, 3Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2011) Google Scholar). Upon activation by extracellular stimuli such as interleukin-1, tumor necrosis factor-α, bacterial lipopolysaccharide, or by human T-lymphotropic virus type I (HTLV-I)1 Tax, I-κBα and I-κBβ become serine-phosphorylated and polyubiquinated and are rapidly degraded via proteasome-mediated proteolysis, resulting in heightened nuclear levels of NF-κB and increased expression of a plethora of cellular genes under NF-κB regulation, including the genes of many cytokines and their receptors, adhesion molecules, and immune modulators (1Beg A.A. Baldwin A.S.J. Genes Dev. 1993; 7: 2064-2070Crossref PubMed Scopus (735) Google Scholar, 2Liou H.C. Baltimore D. Curr. Opin. Cell Biol. 1993; 5: 477-487Crossref PubMed Scopus (516) Google Scholar, 3Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2011) Google Scholar). Dysregulation and/or hyperactivation of the NF-κB/I-κB regulatory pathway as caused by chromosomal translocation (4Neri A. Chang C.C. Lombardi L. Salina M. Corradini P. Maiolo A.T. Chaganti R.S. Dalla Favera R. Cell. 1991; 67: 1075-1087Abstract Full Text PDF PubMed Scopus (332) Google Scholar), oncogene transduction (5Gilmore T.D. Cancer Surv. 1992; 15: 69-87PubMed Google Scholar), viral infection, or targeted gene disruption (6Beg A.A. Sha W.C. Bronson R.T. Baltimore D. Genes Dev. 1995; 9: 2736-2746Crossref PubMed Scopus (411) Google Scholar, 7Klement J.F. Rice N.R. Car B.D. Abbondanzo S.J. Powers G.D. Bhatt P.H. Chen C.H. Rosen C.A. Stewart C.L. Mol. Cell. Biol. 1996; 16: 2341-2349Crossref PubMed Google Scholar) leads to cancers of the hematopoietic cells or chronic inflammatory diseases. The phosphorylation of I-κBα and I-κBβ is mediated by a kinase called I-κB kinase (IKK) (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar, 9Karin M. Ben Neriah Y.L. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar). The core IKK enzyme consists of two highly homologous catalytic subunits α and β of 85 and 87 kDa in sizes, respectively, and a 48-kDa regulatory subunit, IKK-γ/NEMO (referred to as IKK-γ herein) (9Karin M. Ben Neriah Y.L. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar, 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 (945) Google Scholar). Both IKK-α and IKK-β contain NH2-terminal kinase domains followed by leucine zippers and helix-loop-helix domains that mediate protein-protein interactions important for IKK oligomerization and kinase activity (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar,9Karin M. Ben Neriah Y.L. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar). Likewise, IKK-γ also contains extensive helical regions and leucine zipper domains that engage in protein-protein interaction (9Karin M. Ben Neriah Y.L. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar,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 (945) Google Scholar). In vivo the IKK holoenzyme exists as a large protein complex of at least 700–900 kDa in size (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar, 9Karin M. Ben Neriah Y.L. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar). It is not clear what other protein components are present in the holo-IKK enzyme complex in addition to IKK-α, IKK-β, and IKK-γ. Several members of the I-κB and NF-κB/Rel families of proteins and mitogen-activated protein kinase phosphatase-1 (MKP), MEK kinase, and NF-κB inducing kinase (NIK) have been reported to interact with IKK, although these proteins have not been found to co-elute with the 900-kDa IKK complex chromatographically (9Karin M. Ben Neriah Y.L. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar). The transactivator/oncoprotein of HTLV-I, Tax, has been shown to activate IKK constitutively (11Sun S.C. Elwood J. Beraud C. Greene W.C. Mol. Cell. Biol. 1994; 14: 7377-7384Crossref PubMed Google Scholar, 12Good L. Sun S.C. J. Virol. 1996; 70: 2730-2735Crossref PubMed Google Scholar, 13Sun S.C. Ballard D.W. Oncogene. 1999; 18: 6948-6958Crossref PubMed Scopus (166) Google Scholar, 14Xiao G. Sun S.C. Oncogene. 2000; 19: 5198-5203Crossref PubMed Scopus (52) Google Scholar, 15Chu Z.L., Di Donato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16Chu Z.L. Shin Y.A. Yang J.M., Di Donato J.A. Ballard D.W.L.H. J. Biol. Chem. 1999; 274: 15297-15300Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Tax-mediated IKK activation is due in part to a direct interaction between Tax and IKK-γ (14Xiao G. Sun S.C. Oncogene. 2000; 19: 5198-5203Crossref PubMed Scopus (52) Google Scholar, 16Chu Z.L. Shin Y.A. Yang J.M., Di Donato J.A. Ballard D.W.L.H. J. Biol. Chem. 1999; 274: 15297-15300Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar,18Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T.L.H. J. Biol. Chem. 1999; 274: 17402-17405Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The molecular mechanism via which Tax affects IKK activation after its association with IKK-γ remains incompletely understood, however. We have found recently that Tax can interact directly with the catalytic subunit of the major serine/threonine protein phosphatase 2A (PP2Ac) in vivo and in vitro. 2Y.-L. Kuo, L.-C. Wang, M.-H. Liang, Y. Tang, D.-X. Fu, B.-Y. Liu, R. Harrod, H.-J. Kung, H.-M. Shih, and C.-Z. Gram, submitted for publication.2Y.-L. Kuo, L.-C. Wang, M.-H. Liang, Y. Tang, D.-X. Fu, B.-Y. Liu, R. Harrod, H.-J. Kung, H.-M. Shih, and C.-Z. Gram, submitted for publication. Furthermore, Tax acts as a noncompetitive inhibitor of PP2A in in vitroassays where 32P-labeled glycogen phosphorylasea is used as a PP2A substrate.2 Consistent with the notion that Tax inhibits the activity of PP2A, MEK phosphorylation and to a lesser extent cAMP-response element-binding protein and ATF-1 phosphorylation in Tax-expressing HTLV-I-transformed cells and in Tax-transfected human embryonic kidney 293 cells are greatly elevated.2 Most interestingly, PP2A interaction-defective mutants of Tax fail to stimulate MEK phosphorylation and are unable to activate NF-κB, thus suggesting a link between the inhibition of PP2A by Tax and NF-κB activation.2 PP2A is a major serine/threonine protein phosphatase in all eukaryotic cells (for comprehensive reviews, see Refs. 19Evans D.R. Hemmings B.A. Nature. 1998; 394: 23-24Crossref PubMed Scopus (21) Google Scholar, 20Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 21Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1519) Google Scholar, 22Ronne H. Carlberg M., Hu, G.Z. Nehlin J.O. Mol. Cell. Biol. 1991; 11: 4876-4884Crossref PubMed Scopus (165) Google Scholar, 23Mumby M.C. Walter G. Cell Regul. 1991; 2: 589-598Crossref PubMed Scopus (33) Google Scholar). It is crucial for the negative regulation of multiple cellular processes. The holo-PP2A enzyme is a heterotrimer that consists of a core enzyme formed by the highly conserved A subunit (60 kDa, 2 human isoforms) and C (catalytic) subunit (36 kDa, 2 human isoforms) together with many different regulatory B-subunits that derive from multiple genes (three distinct families: B/B55/PR55 (3 genes), B′/B56/PR61 (>3 genes), and B′′/PR72/PR130 (1 gene)), and their alternatively spliced mRNAs (19Evans D.R. Hemmings B.A. Nature. 1998; 394: 23-24Crossref PubMed Scopus (21) Google Scholar, 20Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 21Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1519) Google Scholar, 23Mumby M.C. Walter G. Cell Regul. 1991; 2: 589-598Crossref PubMed Scopus (33) Google Scholar, 24Kremmer E. Ohst K. Kiefer J. Brewis N. Walter G. Mol. Cell. Biol. 1997; 17: 1692-1701Crossref PubMed Scopus (151) Google Scholar). The crystal structure of the A-subunit shows it to contain 15 tandem helical repeats (HEAT motifs) that assume a shape that resembles a horseshoe (25Groves M.R. Hanlon N. Turowski P. Hemmings B.A. Barford D. Cell. 1999; 96: 99-110Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). It serves as a scaffold to which C- and B-subunits are attached (19Evans D.R. Hemmings B.A. Nature. 1998; 394: 23-24Crossref PubMed Scopus (21) Google Scholar, 20Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 21Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1519) Google Scholar, 23Mumby M.C. Walter G. Cell Regul. 1991; 2: 589-598Crossref PubMed Scopus (33) Google Scholar). The heterodimer formed by A- and C-subunits and the heterotrimer containing all three subunits are termed core enzyme and holoenzyme, respectively (19Evans D.R. Hemmings B.A. Nature. 1998; 394: 23-24Crossref PubMed Scopus (21) Google Scholar, 20Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 21Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1519) Google Scholar, 23Mumby M.C. Walter G. Cell Regul. 1991; 2: 589-598Crossref PubMed Scopus (33) Google Scholar). A role of PP2A in controlling the signal transduction pathway that leads to IKK/NF-κB activation has been reported previously (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar). Okadaic acid, an inhibitor of PP2A, has been shown to activate NF-κB in vivo (26Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (930) Google Scholar, 27Sun S.C. Maggirwar S.B. Harhaj E. J. Biol. Chem. 1995; 270: 18347-18351Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Furthermore, PP2A can inactivate the kinase activity of IKK in vitro (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar). Here we provide evidence to show that via a tripartite interaction, Tax, PP2A, and IKKγ form a ternary complex. In in vitro IKK assays, Tax reduces the ability of PP2A to inactivate IKK. Consistent with the notion that activation of IKK requires that Tax bind to both IKKγ and PP2A, several Tax mutants that are abrogated or attenuated for PP2A binding fail to activate NF-κB even though they continue to bind IKKγ. These results indicate that, in the context of the IKK·PP2A·Tax complex, PP2A activity is inhibited or diminished. In essence, IKK is activated by serine phosphorylation of its activation loop upon extracellular stimulation. In normal cells, phospho-IKK becomes rapidly inactivated by IKKγ-associated PP2A, returning IKK to a resting state. In HTLV-I infected or transformed cells, PP2A inhibition by IKKγ-bound Tax maintains IKK in the phosphorylated and active form, causing constitutive phosphorylation and degradation of I-κB, which in turn leads to nuclear presence of NF-κB/Rel and potent activation of genes under NF-κB/Rel control. The anti-Tax monoclonal antibody 4C5 is of IgG2a subtype and reacts with amino acid residues 333–353 of Tax. 3D.-X. Fu, Y.-L. Kuo, B.-Y. Liu, K.-T. Jeang, and C.-Z. Giam, unpublished results. The anti-PP2A C-subunit monoclonal antibody (the COOH-terminal amino acid residues 295–309 of the catalytic subunit of human PP2A) and anti-IKKγ (amino acids 1–419, representing full-length human IKKγ) monoclonal antibody were from Upstate Biotechnology, Inc. and Santa Cruz Biotechnology, Inc., respectively. The purified PP2A was derived from human red blood cells and consists of both A and C subunits (Upstate Biotechnology). Jurkat and the HTLV-I-transformed T-cell line MT-4 were cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and 100 units/ml penicillin (Invitrogen) in the presence of 10% CO2. The glutathione S-transferase (GST) fusion of wild-type and mutant forms of Tax, GST-Tax, GST-H43Q, GST-K85N, GST-M22, GST-M47, were constructed as reported previously (28Adya N. Giam C.Z. J. Virol. 1995; 69: 1834-1841Crossref PubMed Google Scholar). GST-IκBα-(1–54) and Ser-32 and Ser-36 phosphorylation sites mutant GST-IκBα-(1–54)AA were generous gifts of Drs. DiDonato and Karin (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar). GST-IKKγΔC was constructed by replacing in GST-Tax an NcoI, SmaI fragment that contains the coding sequence for wild-type Tax with an NcoI,SmaI fragment that encodes the NH2-terminal 306 amino acid residues of IKKγ. GST fusion proteins were expressed and prepared by standard protocols and stored frozen in buffer D (20 mm Hepes (pH 7.9), 100 mm KCl, 0.2% (v/v) 2-mercaptoethanol, 1 μm phenylmethylsulfonyl fluoride, and 20% glycerol) at −80 °C. Immunoprecipitations were carried out using extracts prepared from Jurkat and HTLV-I-transformed MT-4 cells. Ten million cells were harvested, washed 3 times, each with 10 ml of phosphate-buffer saline, and lysed by repeated passage through a 27.5-gauge syringe in 1 ml of lysis buffer (100 mm NaCl, 50 mm Tris-HCl, pH 8.0, 1% Nonidet P-40, 2 mmEDTA, 5 mm NaF, 1 mmNa3VO4, 0.2 mm phenylmethylsulfonyl fluoride) containing 50 μg/ml each of the protease inhibitors pepstatin, leupeptin, bestatin, and aprotinin (Roche Molecular Biochemicals). Cell debris was removed by centrifugation at 12,000 rpm in a microcentrifuge for 10 min at 4 °C. Immunoprecipitation was carried out with 200 μg of proteins in 500 μl of cell lysates. Briefly, cell lysates were precleared by incubation with 100 μl of a 50% slurry of protein G-agarose (Invitrogen) and 2 μg of normal mouse antiserum in 5 μl for 30 min at 4 °C followed by centrifugation at 12,000 rpm for 5 min. After preclearing, 2 μg of a Tax monoclonal antibody (4C5), PP2A C-subunit monoclonal antibody, or IKKγ monoclonal antibody were added to each sample. The reactions were preincubated at 4 °C for 1 h. After the addition of 30 μl of protein G-agarose to each reaction, the mixtures were incubated overnight. On the following day, immune complexes were pelleted by centrifugation at 3000 rpm for 5 min at 4 °C, washed 3 times with 0.8 ml of lysis buffer, resuspended in 30 μl of SDS-PAGE loading buffer, heated at 95 °C for 3 min, and centrifuged, and then 15 μl of sample were loaded and resolved on a 12% SDS-polyacrylamide gel containing a 4% stacking gel. Proteins resolved in the gels were transferred to nitrocellulose membranes (Schleicher & Schuell). For Western blotting, membranes were incubated with blocking buffer (50 mm Tris-HCl (pH 8.0), 100 mm NaCl, 0.05% Tween, 0.02% sodium azide, 5% nonfat dry milk) for 3 h and then probed in the same buffer for 2 h with mouse monoclonal antibodies against PP2Ac, IKKγ, or Tax. After washing, the blots were incubated for 1 h in the same buffer containing an anti-mouse horseradish peroxidase-conjugated, secondary antibody (diluted 1:1000, Santa Cruz Biotechnology), washed three times, developed using a chemiluminescent substrate (SuperSignal; Pierce), and exposed to x-ray films. For GST pull-down experiments, ∼500 ng each of purified GST, GST-Tax, or GST-IKKγ was incubated with 300 ng of purified PP2A or PP2A and Tax in 30 μl of 1× binding buffer (25 mm HEPES (pH 7.9), 5 mm KCl, 0.5 mm MgCl2, 0.5 mm EDTA, 1 mg/ml bovine serum albumin, 10% glycerol, 0.15% Nonidet P-40, 0.25 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride) for 30 min at 30 °C. Forty microliters of a 50% slurry of prewashed glutathione-Sepharose 4B (Amersham Biosciences) were added to each binding reaction, and samples were incubated for 1 h with agitation at 4 °C. After incubation, the protein-bound Sepharose beads were washed 3 times with 800 μl of 1× binding buffer, pelleted by centrifugation at 1200 rpm for 5 min, resuspended in 30 μl of SDS-PAGE loading buffer, and heated to 95 °C, and 15 μl from each reaction were resolved by 12% SDS-PAGE followed by immunoblotting, as described. To remove the GST moiety from GST-IKKγ, the fusion protein was proteolysed with 0.2 unit of thrombin (Sigma) overnight at 4 °C, and the treated protein was then incubated with GST-Tax as described above. Under these conditions, GST-IKKγ was converted completely to IKKγ by thrombin, and the residual thrombin in the reaction mixture did not interfere significantly with the binding reactions. To measure IKK activity, 107 MT-4 cells were harvested by centrifugation and extracted in 1 ml of a lysis buffer containing 20 mmTris-HCl (pH 7.6), 20 mm glycerol phosphate, 250 mm NaCl, 3 mm EGTA, 3 mm EDTA, 0.5% Nonidet P-40, 0.1 mm sodium vanadate, 10 μg/ml aprotinin, 2 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, and 10 μg/ml of leupeptin. After a brief centrifugation, extracts were incubated for 2 h with agitation at 4 °C with 0.5 μg of an antibody against IKKγ followed by protein G-Sepharose (Invitrogen) precipitation. Each IKK immune complex reaction was carried out using 1 μg of recombinant GST protein fused to amino acid residues 1–54 of IκBα (GST- IκBα-(1–54)) as the substrate and 10 μm[γ-32P]ATP in a final volume of 30 μl of a reaction buffer containing 30 mm HEPES (pH 7.4), 10 mm MgCl2, and 1 mm dithiothreitol at 25 °C for 30 min. The reactions were terminated by the addition of SDS-PAGE loading buffer. IKK activity as measured by the phosphorylation level of IκBα was evaluated by SDS-PAGE followed by autoradiography. When applicable, 5 ng of PP2A and 0.1–0.6 μg of Tax were added into a 30-μl reaction. Several cellular kinases and regulatory proteins whose activities are regulated by serine/threonine phosphorylation form protein complexes with PP2A (29Andjelkovic N. Zolnierowicz S. Van Hoof C. Goris J. Hemmings B.A. EMBO J. 1996; 15: 7156-7167Crossref PubMed Scopus (63) Google Scholar, 30Westphal R.S. Coffee R.L. Marotta A. Pelech S.L. Wadzinski B.E. J. Biol. Chem. 1999; 274: 687-692Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 31Abraham D. Podar K. Pacher M. Kubicek M. Welzel N. Hemmings B.A. Dilworth S.M. Mischak H. Kolch W. Baccarini M. J. Biol. Chem. 2000; 275: 22300-22304Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 32Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (425) Google Scholar, 33Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (364) Google Scholar, 34Westphal R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (222) Google Scholar). In this manner, PP2A is localized to its targets at once and can affect rapid on/off control of regulatory processes immediately after signaling events are initiated. Because Tax interacts with both PP2A and IKKγ, we wondered if PP2A also forms a complex with IKK. This might explain the rapidity with which the IKK/NF-κB pathway is returned to the inactive state soon after its activation. Furthermore, because Tax constitutively activates IKK, we wondered if, in the context of the IKK-PP2A-Tax complex, the phosphatase activity of PP2A may be inhibited by Tax. Because IKK is activated through serine phosphorylation of its activation loop by upstream kinases, a block in its de-phosphorylation by Tax would maintain phospho-IKK in an active state and can explain the constitutive activation of IKK by Tax. To determine whether IKK, PP2A, and Tax form a ternary complex in vivo, cell extracts were prepared from a Tax-expressing HTLV-I-transformed human T-cell line, MT-4, and a control human T-cell line, Jurkat, and subjected to immunoprecipitation using antibodies against Tax, the catalytic subunit of PP2A (PP2Ac), and IKKγ, respectively. As expected, mouse monoclonal antibody against IKKγ precipitated IKKγ easily (Fig. 1,lanes 3 and 4, upper panel). From Tax-positive MT-4 cell extracts, both the PP2A c-subunit (PP2Ac) and Tax were readily co-precipitated (lane 4, middleand lower panels, respectively). In Jurkat extracts, a low but detectable trace of PP2Ac was also present (lane 3,middle panel). Likewise, Tax monoclonal antibody 4C5 co-precipitated Tax, PP2Ac, and IKKγ (lane 6,lower, middle, and upper panels). The 4C5 antibody is highly specific for Tax. It did not react with either PP2Ac or IKKγ, as indicated by immunoprecipitations done with Tax-null Jurkat extracts (lane 5). Finally, a mouse monoclonal antibody against the COOH-terminal region of PP2Ac co-precipitated IKKγ from both Jurkat and MT-4 extracts. Interestingly and in agreement with the immunoprecipitation performed with anti- IKKγ (lanes 3 and 4), the amount of IKKγ that co-precipitated with PP2Ac is lower when Jurkat extracts were used (compare lanes 7 and 8, upper panel). The PP2Ac antibody failed to co-immunoprecipitate Tax, however (lanes 7 and 8, bottom panel). We think this is due to a disruption of the protein-protein interaction between Tax and PP2Ac by the PP2Ac antibody. The COOH-terminal region of PP2Ac is involved in extensive protein-protein interaction; the carboxyl leucine residue undergoes a methyl esterification (35De B., I Derua R. Janssens V. Van Hoof C. Waelkens E. Merlevede W. Goris J. Biochemistry. 1999; 38: 16539-16547Crossref PubMed Scopus (124) Google Scholar, 36Tolstykh T. Lee J. Vafai S. Stock J.B. EMBO J. 2000; 19: 5682-5691Crossref PubMed Scopus (194) Google Scholar, 37Evans D.R. Hemmings B.A. Mol. Gen. Genet. 2000; 264: 425-432Crossref PubMed Scopus (39) Google Scholar) critical for C subunit assembly with A and B subunits (36Tolstykh T. Lee J. Vafai S. Stock J.B. EMBO J. 2000; 19: 5682-5691Crossref PubMed Scopus (194) Google Scholar, 38Ogris E., Du, X. Nelson K.C. Mak E.K., Yu, X.X. Lane W.S. Pallas D.C. J. Biol. Chem. 1999; 274: 14382-14391Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 39Wei H. Ashby D.G. Moreno C.S. Ogris E. Yeong F.M. Corbett A.H. Pallas D.C. J. Biol. Chem. 2001; 276: 1570-1577Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) to form the core and holoenzymes. It is likely that this region is also important for binding Tax. Together, these data indicate that PP2A interacts with IKK directly. Furthermore, Tax interacts with IKKγ and PP2A in vivo in a stable ternary complex. To characterize further the interaction among Tax, IKKγ, and PP2A, GST pull-down experiments were performed using purified PP2A, consisting principally of both the catalytic C-subunit (PP2Ac) and the regulatory A-subunit, derived from human red blood cells (Upstate Biotechnology) in the presence or absence of Tax protein purified from an Escherichia coli expression system. Care was taken to ensure comparable levels of purified GST, GST-Tax, and GST-IKKγ fusion proteins (Fig. 2 A) were used. In binding reactions containing PP2A only, GST-Tax, GST-IKKγ, and GST-IKKγΔC, the GST fusion of an IKKγ mutant deleted for the COOH-terminal Tax binding domain (amino acid residues 307–419), all displayed efficient binding to PP2Ac (Fig. 2 B, lower panel). Under these experimental conditions, ∼2 and 4% of input PP2Ac became bound to GST-Tax and GST- IKKγ, respectively. The interactions appear specific since under the same binding condition, GST control did not bind PP2Ac. Interestingly, in reactions where both PP2A and Tax were added, the binding of PP2A to GST-IKKγ became significantly enhanced (compare Fig. 2, B and C,GST-IKKγ lanes, lower panel). By contrast, Tax did not increase PP2A binding to GST-IKKγΔC (Fig. 2 C, compare the GST-IKKγ andGST-IKKγΔC lanes in thelower panel) whose Tax binding site is deleted and is unable to bind Tax (upper panel), suggesting that when Tax is bound to IKKγ, it can further stabilize or enhance IKKγ-PP2A interaction. As anticipated, GST-IKKγ interacted with Tax (Fig. 2 C,GST-IKKγ lane, upper panel). In agreement with earlier results, GST-Tax interacted with PP2A (GST-Tax lane, lower panel). Because Tax exists as homodimer, GST-Tax and Tax interaction was also seen (GST-Tax lane, upper panel). The exogenously added Tax (300 ng) did not significantly compete for PP2Ac binding to GST-Tax (500 ng), most likely because the interaction between Tax and PP2Ac was not saturating (only 2% input PP2Ac was pulled down by GST-Tax). No binding of Tax or PP2A to GST was detected. Together, these data support the immunoprecipitation results shown above and indicate that PP2A is associated with IKK. Furthermore, in the presence of Tax, a stable IKK·PP2A·Tax ternary complex can form. Finally, even though IKKγΔC mutant is defective in binding Tax (18Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T.L.H. J. Biol. Chem. 1999; 274: 17402-17405Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 40Xiao G. Harhaj E.W. Sun S.C. J. Biol. Chem. 2000; 275: 34060-34067Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), it is still able to associate with PP2A, much like the full-length IKKγ, suggesting that PP2A binds to a region in IKKγ upstream of the COOH-terminal Tax binding site. If indeed the inhibition of PP2A by Tax in the context of the IKK·PP2A·Tax complex leads to a constitutive activation of IKK, then one might expect that both Tax-PP2A and Tax-IKKγ interactions would be necessary for Tax-mediated activation of IKK and NF-κB. Using yeast 2-hybrid analysis, we have previously mapped the domain critical for PP2Ac binding to the NH2-terminal 100-amino acid residues of Tax (Fig. 3 A).2 This region is distinct from the domain responsible for IKKγ binding previously localized to approximately amino acid residues 100–150 previously (Fig. 3 A) (40Xiao G. Harhaj E.W. Sun S.C. J. Biol. Chem. 2000; 275: 34060-34067Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Two Tax mutants, H43Q and K85N, shown to be defective in binding PP2A earlier,2 and another mutant, H41Q (41Se" @default.
• W2023303889 created "2016-06-24" @default.
• W2023303889 creator A5016458375 @default.
• W2023303889 creator A5018210675 @default.
• W2023303889 creator A5046425547 @default.
• W2023303889 creator A5061735109 @default.
• W2023303889 creator A5080524336 @default.
• W2023303889 date "2003-01-01" @default.
• W2023303889 modified "2023-10-16" @default.
• W2023303889 title "Human T-lymphotropic Virus Type I Tax Activates I-κB Kinase by Inhibiting I-κB Kinase-associated Serine/Threonine Protein Phosphatase 2A" @default.
• W2023303889 cites W1520258702 @default.
• W2023303889 cites W1520425298 @default.
• W2023303889 cites W1553428238 @default.
• W2023303889 cites W1607664081 @default.
• W2023303889 cites W165235925 @default.
• W2023303889 cites W1697605735 @default.
• W2023303889 cites W1829577351 @default.
• W2023303889 cites W1964739066 @default.
• W2023303889 cites W1979217369 @default.
• W2023303889 cites W1979520552 @default.
• W2023303889 cites W1983744843 @default.
• W2023303889 cites W1989844987 @default.
• W2023303889 cites W1993847641 @default.
• W2023303889 cites W1995184275 @default.
• W2023303889 cites W2002674047 @default.
• W2023303889 cites W2006929818 @default.
• W2023303889 cites W2008608024 @default.
• W2023303889 cites W2013202536 @default.
• W2023303889 cites W2022297975 @default.
• W2023303889 cites W2028494050 @default.
• W2023303889 cites W2029278007 @default.
• W2023303889 cites W2032896827 @default.
• W2023303889 cites W2038638713 @default.
• W2023303889 cites W2044481116 @default.
• W2023303889 cites W2062727958 @default.
• W2023303889 cites W2071618706 @default.
• W2023303889 cites W2079523306 @default.
• W2023303889 cites W2084415176 @default.
• W2023303889 cites W2093061708 @default.
• W2023303889 cites W2094900302 @default.
• W2023303889 cites W2125617798 @default.
• W2023303889 cites W2135506398 @default.
• W2023303889 cites W2142541950 @default.
• W2023303889 cites W2147921405 @default.
• W2023303889 cites W2150446853 @default.
• W2023303889 cites W2152044329 @default.
• W2023303889 cites W2154989770 @default.
• W2023303889 cites W2163813144 @default.
• W2023303889 cites W217424672 @default.
• W2023303889 cites W2288796536 @default.
• W2023303889 cites W4240253200 @default.
• W2023303889 cites W95988660 @default.
• W2023303889 doi "https://doi.org/10.1074/jbc.m210631200" @default.
• W2023303889 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12419799" @default.
• W2023303889 hasPublicationYear "2003" @default.
• W2023303889 type Work @default.
• W2023303889 sameAs 2023303889 @default.
• W2023303889 citedByCount "92" @default.
• W2023303889 countsByYear W20233038892012 @default.
• W2023303889 countsByYear W20233038892013 @default.
• W2023303889 countsByYear W20233038892014 @default.
• W2023303889 countsByYear W20233038892015 @default.
• W2023303889 countsByYear W20233038892016 @default.
• W2023303889 countsByYear W20233038892017 @default.
• W2023303889 countsByYear W20233038892018 @default.
• W2023303889 countsByYear W20233038892019 @default.
• W2023303889 countsByYear W20233038892020 @default.
• W2023303889 countsByYear W20233038892021 @default.
• W2023303889 countsByYear W20233038892022 @default.
• W2023303889 crossrefType "journal-article" @default.
• W2023303889 hasAuthorship W2023303889A5016458375 @default.
• W2023303889 hasAuthorship W2023303889A5018210675 @default.
• W2023303889 hasAuthorship W2023303889A5046425547 @default.
• W2023303889 hasAuthorship W2023303889A5061735109 @default.
• W2023303889 hasAuthorship W2023303889A5080524336 @default.
• W2023303889 hasBestOaLocation W20233038891 @default.
• W2023303889 hasConcept C153911025 @default.
• W2023303889 hasConcept C159047783 @default.
• W2023303889 hasConcept C178666793 @default.
• W2023303889 hasConcept C181199279 @default.
• W2023303889 hasConcept C184235292 @default.
• W2023303889 hasConcept C185592680 @default.
• W2023303889 hasConcept C2775880066 @default.
• W2023303889 hasConcept C2776414213 @default.
• W2023303889 hasConcept C55493867 @default.
• W2023303889 hasConcept C86803240 @default.
• W2023303889 hasConcept C90934575 @default.
• W2023303889 hasConcept C97029542 @default.
• W2023303889 hasConcept C99405784 @default.
• W2023303889 hasConceptScore W2023303889C153911025 @default.
• W2023303889 hasConceptScore W2023303889C159047783 @default.
• W2023303889 hasConceptScore W2023303889C178666793 @default.
• W2023303889 hasConceptScore W2023303889C181199279 @default.
• W2023303889 hasConceptScore W2023303889C184235292 @default.
• W2023303889 hasConceptScore W2023303889C185592680 @default.
• W2023303889 hasConceptScore W2023303889C2775880066 @default.
• W2023303889 hasConceptScore W2023303889C2776414213 @default.
• W2023303889 hasConceptScore W2023303889C55493867 @default.

• next