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• W1975509142 abstract "For the orphan nuclear receptor subfamily that includes Nur77 (NGFI-B), Nurr1, and NOR-1, no transcriptional coregulators have been identified thus far. In this report, we found that Ca2+/calmodulin-dependent protein kinase IV enhances Nur77 transactivation in cotransfections either alone or in synergy with AF2dependent coactivator ASC-2, whereas corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) is repressive. Interestingly, Nur77 interacted with SMRT but did not directly bind ASC-2, and accordingly, the putative AF2 core domain of Nur77 did not affect the Nur77 transactivation. SMRT harbors transferable repression domains that associate with various histone deacetylases. Surprisingly, histone deacetylase inhibitor trichostatin A was unable to block the repressive effect of SMRT while dramatically stimulating the Nur77 transactivation. These results suggest that SMRT and ASC-2 are specific coregulators of Nur77 and that SMRT may dynamically compete with a putative adaptor molecule, which links ASC-2 to Nur77, for the identical binding sites within Nur77 in vivo. For the orphan nuclear receptor subfamily that includes Nur77 (NGFI-B), Nurr1, and NOR-1, no transcriptional coregulators have been identified thus far. In this report, we found that Ca2+/calmodulin-dependent protein kinase IV enhances Nur77 transactivation in cotransfections either alone or in synergy with AF2dependent coactivator ASC-2, whereas corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) is repressive. Interestingly, Nur77 interacted with SMRT but did not directly bind ASC-2, and accordingly, the putative AF2 core domain of Nur77 did not affect the Nur77 transactivation. SMRT harbors transferable repression domains that associate with various histone deacetylases. Surprisingly, histone deacetylase inhibitor trichostatin A was unable to block the repressive effect of SMRT while dramatically stimulating the Nur77 transactivation. These results suggest that SMRT and ASC-2 are specific coregulators of Nur77 and that SMRT may dynamically compete with a putative adaptor molecule, which links ASC-2 to Nur77, for the identical binding sites within Nur77 in vivo. ligand-binding domain activation function-2 cAMP-response element-binding protein CREB-binding protein steroid receptor coactivator-1 activating signal cointegrator-2 nuclear receptor corepressor silencing mediator for retinoid and thyroid hormone receptors histone deacetylase calmodulin Ca2+/CaM-dependent protein kinase glutathione S-transferase nerve growth factor-inducible gene B NGFI-B response element Nur77 response element trichostatin A SMRT/HDAC1 associated repressor protein retinoid-related orphan receptor chicken ovalbumin upstream transcription factor The nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in the promoters of target genes (for a review, see Ref. 1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6088) Google Scholar). The superfamily includes receptors for a variety of small hydrophobic ligands, such as steroids, T3, and retinoids, as well as a large number of related proteins that do not have known ligands referred to as orphan nuclear receptors. Functional analysis of nuclear receptors has shown that the ligand-binding domain (LBD)1 exhibits ligand-dependent transcriptional activation function referred to as activation function-2 (AF2). Notably, this region has been shown to play a critical role in mediating transactivation by serving as a ligand-dependent interaction interface with many different coactivators (for reviews, see Refs. 2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar and 3Lee J.W. Lee Y.C. Na S.-Y. Jung D.J. Lee S.-K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar). Transcriptional coactivators either bridge transcription factors and the components of the basal transcriptional apparatus and/or remodel the chromatin structures. In particular, cAMP-response element-binding protein (CREB)-binding protein (CBP) and its functional homologue p300, steroid receptor coactivator-1 (SRC-1) and its family members, and activating signal cointegrator-2 (ASC-2) were shown to be essential for the activation of transcription by a large number of regulated transcription factors, including many members of the nuclear receptor superfamily (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar, 3Lee J.W. Lee Y.C. Na S.-Y. Jung D.J. Lee S.-K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar). Interestingly, SRC-1 and its family member activator of thyroid and retinoic acid receptors (ACTR) along with CBP and p300 were recently shown to contain histone acetyltransferase activities and associate with another histone acetyltransferase protein p300/CBP-associated factor (P/CAF) (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar, 3Lee J.W. Lee Y.C. Na S.-Y. Jung D.J. Lee S.-K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar). In contrast, nuclear receptor corepressor (N-CoR) and its homologue silencing mediator of retinoid and thyroid hormone receptors (SMRT) harbor transferable repression domains that can associate with various histone deacetylases (HDACs) (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar, 3Lee J.W. Lee Y.C. Na S.-Y. Jung D.J. Lee S.-K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar). These results are consistent with the notion that acetylation of histones destabilizes nucleosomes and relieves transcriptional repression by allowing transcription factors access to recognition elements, whereas deacetylation of the histones stabilizes the repressed state. A distinctive structural feature of the AF2-dependent coactivators is the presence of L XXLL signature motifs (i.e. NR box) (4Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1772) Google Scholar, 5Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1107) Google Scholar). The AF2 core region (helix 12) was recently shown to undergo a major restructuring upon ligand binding, forming part of a “charged clamp” that accommodates coactivators within a hydrophobic cleft of the receptor LBD through direct contacts with these NR boxes (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar). Interestingly, the N-CoR/SMRT nuclear receptor interaction motifs exhibited a consensus sequence of (I/L)XX(I/H)I (i.e. CoRNR box, in which H indicates hydrophobic residues) (6Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (526) Google Scholar, 7Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (425) Google Scholar), which was able to interact with specific residues in the same receptor pocket required for coactivator binding. Thus, discrimination of the subtle differences between the coactivator and corepressor interaction helices by the nuclear receptor AF2 core may provide the molecular basis for the exchange of coactivators for corepressors with ligand-dependent formation of the charged clamp that stabilizes NR box binding and inhibits interaction with the CoRNR box helix. The orphan nuclear receptors Nur77 (NGFI-B), Nurr1, and NOR-1 constitute the NGFI-B subfamily within the nuclear receptor superfamily (for a review, see Ref. 8Maruyama K. Tsukada T. Ohkura N. Bandoh S. Hosono T. Yamaguchi K. Int. J. Oncol. 1998; 12: 1237-1243PubMed Google Scholar). Nur77 was originally isolated as an immediate early gene rapidly expressed in response to serum or phorbol ester stimulation of quiescent fibroblasts. Other diverse signals, such as membrane depolarization and nerve growth factor, also increase Nur77 expression. Inactivation of a Nur77-related protein results in agenesis of mesencephalic dopamine neurons (9Zetterstrom R.H. Williams R. Perlmann T. Olson L. Brain Res. Mol. Brain Res. 1996; 41: 111-120Crossref PubMed Scopus (281) Google Scholar). Nur77 is also involved in the regulation of apoptosis in different cell types. It is rapidly induced during apoptosis of immature thymocytes and T cell hybridoma (10Woronicz J.D. Calnan B. Ngo V. Winoto A. Nature. 1994; 367: 277-281Crossref PubMed Scopus (507) Google Scholar, 11Liu Z.G. Smith S.W. McLaughlin K.A. Schwartz L.M. Osborne B.A. Nature. 1994; 367: 281-284Crossref PubMed Scopus (504) Google Scholar), lung cancer cells treated with synthetic retinoid (12Li Y. Lin B. Agadir A. Liu R. Dawson M.I. Reed J.C. Fontana J.A. Bost F. Hobbs P.D. Zheng Y. Chen G.Q. Shroot B. Mercola D. Zhang X.K. Mol. Cell. Biol. 1998; 18: 4719-4731Crossref PubMed Scopus (175) Google Scholar), and prostate cancer cells treated with different apoptosis inducers (13Young C.Y. Murtha P.E. Zhang J. Oncol. Res. 1994; 6: 203-210PubMed Google Scholar, 14Uemura H. Chang C. Endocrinology. 1998; 139: 2329-2334Crossref PubMed Google Scholar). Calmodulin (CaM), the most ubiquitous and abundant Ca2+-binding protein in cells, is an essential protein that serves as a receptor to sense changes in calcium concentrations and, in this fashion, mediates the second messenger role of this ion (for a review, see Ref. 15Means A.R. Mol. Endocrinol. 2000; 14: 4-13Crossref PubMed Scopus (159) Google Scholar). Calcium binds to CaM by means of a structural motif called an EF-hand, and a pair of these structures is located in both globular ends of the protein. CaM binds to and activates target enzymes. These Ca2+/CaM-dependent protein kinases (CaMKs) include CaM kinase kinase, CaMKI, and CaMKIV, which are phosphorylated and activated by CaM kinase kinase, and CaMKII. In particular, CaMKIV is a monomeric multifunctional enzyme that is expressed only in subanatomical portions of the brain, T lymphocytes, and postmeiotic male germ cells. CaMKIV is present in the nucleus of the cells in which it is expressed and has been implicated in the regulation of transcription of a number of genes including those encoding interleukin-2, members of the immediate early gene family such as c-fos; tumor necrosis family members such as CD40L, Fas ligand, and tumor necrosis factor; the neurotrophin brain-derived neurotrophic factor (BDNF); an Epstein-Barr virus gene involved in the switch to the lytic cycle called BZLF1; and orphan members of the steroid receptor superfamily such as retinoid-related orphan receptor (ROR) and chicken ovalbumin upstream transcription factor (COUP-TF) (15Means A.R. Mol. Endocrinol. 2000; 14: 4-13Crossref PubMed Scopus (159) Google Scholar). However, the only direct substrates for CaMKIV involved in transcription that have been defined to date are CREB, cAMP response element modulator (CREM), and the NFκB component p65 (16Schulman H. Braun A. Carafoli E. Klee C. Calcium as a Cellular Regulator. Oxford University Press, New York1999: 311-343Google Scholar, 17Means A.R Ribar T.J. Kane C.D. Hook S.S. Anderson K.A. Recent Prog. Horm. Res. 1997; 52: 389-407PubMed Google Scholar, 18Jang M.K. Goo Y.H. Sohn Y.C. Kim Y.S. Lee S.-K. Cheong J. Lee J.W. J. Biol. Chem. 2001; 276: 20005-20010Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), although CBP has also been indirectly implicated as a possible substrate (19Chawla S. Hardingham G.E. Quinn D.R. Bading H. Science. 1998; 281: 1505-1509Crossref PubMed Scopus (379) Google Scholar). In this work, we show that SMRT and ASC-2 are transcriptional coregulatory proteins of Nur77. We further present experimental evidence that CaMKIV stimulates the Nur77 transactivation, likely through modulation of various functional aspects of SMRT and ASC-2. Coupling of these two distinct signaling pathways (i.e.CaMKIV and Nur77) may play an important role in a subset of tissues that express both Nur77 and CaMKIV, such as brain and T lymphocytes. The polymerase chain reaction-amplified fragments for Nur77ΔAF2, Nur77-TRAF2, the SMRT residues 1074–1291 and 1292–1495 (i.e. SMRT-D/N and SMRT-D/C, respectively), the SMRT residues 1060–1478 (i.e. SMRT-DΔS), and SMRT-Dm and SMRT-DmΔS, which are identical to SMRT-D (the SMRT residues 1060–1495) and SMRT-DΔS, respectively, except for mutations in the CoRNR motifs, were subcloned into EcoRI and XhoI restriction sites of the B42 fusion vector pJG4-5, the in vitro translation and mammalian expression vector pcDNA3, the LexA fusion vector pEG202PL, and the glutathione S-transferase (GST) fusion vector pGEX4T1 (Amersham Pharmacia Biotech). Expression vectors for Nur77; ASC-2; constitutively active forms of CaMKI, CaMKII, and CaMKIV; E1A and E1A(Δ2–36); Gal4 fusions to Nur77, ASC-2, ASC-2-AD2/3, and SMRT-D; VP16 fusions to SMRT-D and Nur77; B42 fusion to SMRT-D; LexA fusion to the LBD of Nur77 (i.e. LexA/Nur77); GST fusions to SMRT-D and ASC2-2C; and the reporter constructs NGFI-B response element (NBRE)-Luc, Nur77 response element (NurRE)-Luc, Gal4-Luc, and LexA-β-Gal were as described previously (18Jang M.K. Goo Y.H. Sohn Y.C. Kim Y.S. Lee S.-K. Cheong J. Lee J.W. J. Biol. Chem. 2001; 276: 20005-20010Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 20Lee S.-K. Anzick S.L. Choi J.-E. Bubendorf L. Guan X.-Y. Jung Y.-K. Kallioniemi O.-P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J. Lee Y.C. Meltzer P. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 21Lee S.-K. Na S.-Y. Jung S.-Y. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Crossref PubMed Scopus (60) Google Scholar, 22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 23Caira F. Antonson P. Pelto-Huikko M. Treuter E. Gustafsson J.A. J. Biol. Chem. 2000; 275: 5308-5317Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). HeLa and CV-1 cells (5×104 cells/well) were grown in 24-well plates with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 24 h and transiently transfected using SuperFect (Qiagen, Valencia, CA) according to the instructions of the manufacturer. 12 h later, cells were washed and refed with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum either in the presence or absence of 100 nmtrichostatin A (TSA). The cells were harvested 24 h later, and normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions as described previously (24Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Crossref PubMed Scopus (128) Google Scholar). For coimmunoprecipitation, expression vectors for Nur77, SMRT, and CaMKIVc, a constitutively active mutant form of CaMKIV that consists of the CaMKIV residues 1–313, were transfected into 293T cells, and the resulting nuclear extracts were subjected to immunoprecipitation against SMRT monoclonal antibody (the kind gift of Dr. Dean Edwards at University of Colorado Health Sciences Center) and blotted with polyclonal antibody against Nur77 (Santa Cruz Biotechnology, Santa Cruz, CA) as described previously (24Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Crossref PubMed Scopus (128) Google Scholar). The cotransformation and β-galactosidase assay in yeast were done as described previously (24Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Crossref PubMed Scopus (128) Google Scholar). For each experiment, at least three to six independently derived colonies expressing chimeric proteins were tested. Equal amounts of GST alone or GST fusions to SMRT-D, SMRT-D/N, SMRT-D/C, and ASC2-2C, expressed in Escherichia coli and purified, were bound to glutathione-Sepharose 4B beads and incubated in reaction buffer (20 mm NaCl, 25 mm Hepes (pH 7.9), 20% glycerol, 0.2 mm EDTA, 1 mm dithiothreitol, and 1.5% bovine serum albumin) with labeled Nur77 expressed by in vitro translation by using the TNT-coupled transcription-translation system with conditions as described by the manufacturer (Promega, Madison, WI). Specifically bound proteins were eluted from beads with 40 mm reduced glutathione in 50 mm Tris (pH 8.0) and were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography as described previously (24Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Crossref PubMed Scopus (128) Google Scholar). CaMKIV has recently been found to stimulate transactivation by a subset of orphan nuclear receptors such as ROR and COUP-TF (15Means A.R. Mol. Endocrinol. 2000; 14: 4-13Crossref PubMed Scopus (159) Google Scholar). These results led us to examine whether CaMKIV similarly modulates the Nur77 transactivation. In CV-1 cells, coexpressed CaMKIVc stimulated the Nur77 transactivation with two different reporter constructs driven by upstream Nur77 binding sites (23Caira F. Antonson P. Pelto-Huikko M. Treuter E. Gustafsson J.A. J. Biol. Chem. 2000; 275: 5308-5317Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) (Fig.1 A). NBRE was identified by genetic selection in yeast, and NurRE is a naturally occurring response element identified in the pro-opiomelanocortin promoter (23Caira F. Antonson P. Pelto-Huikko M. Treuter E. Gustafsson J.A. J. Biol. Chem. 2000; 275: 5308-5317Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Nur77 binds NBRE and NurRE as a monomer and a homodimer, respectively. Similarly, Gal4/Nur77-directed transactivation of the Gal4-Luc reporter construct was significantly enhanced by coexpressed CaMKIVc in a dose-dependent manner (results not shown). Interestingly, the C-terminally deleted, constitutive active form of CaMKI, but not CaMKII, showed similar stimulatory effects on the Nur77 transactivation (Fig. 1 B). Treatment of HeLa cells with ionomycin, the CaMK activator, either in the absence or presence of coexpressed CaMKIV, also led to stimulation of the Nur77 transactivation, whereas the CaMKIVc-stimulated Nur77 transactivation was impaired by KN-93, the CaMK inhibitor, in a dose-dependent manner (results not shown). From these results, we concluded that CaMKI and CaMKIV are capable of stimulating the Nur77 transactivation. Despite a large volume of work with conventional nuclear receptors (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar, 3Lee J.W. Lee Y.C. Na S.-Y. Jung D.J. Lee S.-K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar), the transcriptional coregulatory proteins required for Nur77 and its close relatives have not been identified thus far. ASC-2, also known as RAP250/TRBP/PRIP/NRC, is a tumor-amplified transcriptional coactivator molecule of nuclear receptors and many other transcription factors (20Lee S.-K. Anzick S.L. Choi J.-E. Bubendorf L. Guan X.-Y. Jung Y.-K. Kallioniemi O.-P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J. Lee Y.C. Meltzer P. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 21Lee S.-K. Na S.-Y. Jung S.-Y. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Crossref PubMed Scopus (60) Google Scholar, 22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 25Zhu Y. Kan L. Qi C. Kanwar Y.S. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 2000; 275: 13510-13516Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 26Mahajan M.A. Samulels H.H. Mol. Cell. Biol. 2000; 20: 5048-5063Crossref PubMed Scopus (96) Google Scholar, 27Kang H.J. Song M.R. Lee S.K. Shin E.C. Choi Y.H. Kim S.J. Lee J.W. Lee M.O. Exp. Cell Res. 2000; 256: 545-554Crossref PubMed Scopus (42) Google Scholar, 28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Assoc., New York1995Google Scholar). Interestingly, ASC-2 strongly synergized with CaMKIVc to stimulate the Nur77 transactivation, although ASC-2 alone had no significant effect (Fig.1 C). In accordance with these results, we further found that CaMKIVc dramatically potentiates the previously described autonomous transactivation function of ASC-2 (20Lee S.-K. Anzick S.L. Choi J.-E. Bubendorf L. Guan X.-Y. Jung Y.-K. Kallioniemi O.-P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J. Lee Y.C. Meltzer P. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Assoc., New York1995Google Scholar) (Fig.2 A). CaMKIVc stimulated the transcriptional activities of the Gal4-Luc reporter construct mediated by Gal4 fusion to the full-length ASC-2 or a fragment of ASC-2 containing the previously described autonomous transactivation functions AD2 and AD3 (20Lee S.-K. Anzick S.L. Choi J.-E. Bubendorf L. Guan X.-Y. Jung Y.-K. Kallioniemi O.-P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J. Lee Y.C. Meltzer P. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Assoc., New York1995Google Scholar). In contrast, CaMKIVc was inert with Gal4 alone or Gal4 fusion to the corepressor SMRT (Fig. 2 A). We and others have recently shown that the autonomous transactivation function of ASC-2 requires CBP/p300 and can be down-regulated by CBP-neutralizing viral oncoprotein E1A (22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Assoc., New York1995Google Scholar). Consistent with an idea that the autonomous transactivation function of ASC-2 is indeed essential for the Nur77 transactivation, E1A but not E1A(Δ2–36), which is unable to bind CBP/p300, repressed the Nur77 transactivation (Fig. 2 B). Similar results were also obtained with the CaMKIV-stimulated Nur77 transactivation. Notably, E1A(Δ2–36) still exhibited significant repressive effects, suggesting that the repressive action of E1A may also involve proteins other than CBP. Surprisingly, ASC-2, a typical AF2-dependent coactivator of many nuclear receptors (20Lee S.-K. Anzick S.L. Choi J.-E. Bubendorf L. Guan X.-Y. Jung Y.-K. Kallioniemi O.-P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J. Lee Y.C. Meltzer P. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 21Lee S.-K. Na S.-Y. Jung S.-Y. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Crossref PubMed Scopus (60) Google Scholar, 22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 25Zhu Y. Kan L. Qi C. Kanwar Y.S. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 2000; 275: 13510-13516Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 26Mahajan M.A. Samulels H.H. Mol. Cell. Biol. 2000; 20: 5048-5063Crossref PubMed Scopus (96) Google Scholar, 27Kang H.J. Song M.R. Lee S.K. Shin E.C. Choi Y.H. Kim S.J. Lee J.W. Lee M.O. Exp. Cell Res. 2000; 256: 545-554Crossref PubMed Scopus (42) Google Scholar, 28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Assoc., New York1995Google Scholar), did not interact with Nur77 (Fig. 4 B and results not shown). Accordingly, deletion of the putative AF2 core region of Nur77 or incorporation of the AF2 sequences of thyroid hormone receptor β into Nur77 did not affect the Nur77 transactivation (Fig. 2 C). These results strongly suggest the presence of an AF2-independent coactivator molecule of Nur77 that may function as an adaptor that links Nur77 to the autonomous transactivation potential of ASC-2.Figure 4SMRT as a corepressor of Nur77. A and C, CV-1 cells were transfected with LacZ expression vector and a reporter gene and various expression vectors as indicated. Cells were grown for 24 h before harvest. The results were expressed as -fold activation (n-fold) over the value obtained with a reporter alone, and the error bars are as indicated. Similar results were also obtained with HeLa cells (results not shown). B, the full-length Nur77 was labeled with [35S]methionine by in vitro translation and incubated with glutathione beads containing GST alone and GST-SMRT-D, -SMRT-D/N, -SMRT-D/C, and -ASC2-2C as indicated. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-polyacrylamide gel electrophoresis. Approximately 20% of the total reaction mixture was loaded as input. Similar results were also obtained with the LBD of Nur77 (results not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT) While examining other known coregulatory proteins of nuclear receptors, we found that SMRT directly interacts with Nur77. SMRT-D encodes the C-terminal subregion of SMRT (i.e. the SMRT residues 1060–1495) that contains two CoRNR boxes (6Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (526) Google Scholar, 7Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (425) Google Scholar) as summarized in Fig.3 A. SMRT-Dm is identical to SMRT-D except for mutations in both CoRNR motifs ((L/I)XX(I/H)I, where H represents hydrophobic residues, to A XXAA) that abolished interactions with thyroid hormone receptor β and retinoic acid receptor α in the GST pull-down assays as expected (results not shown). SMRT-D, but not SMRT-Dm, fused to transcriptional activation domain B42 further stimulated the constitutive transcriptional activities directed by the LexA fusion to the LBD of Nur77 in yeast (Fig. 3 B). The Nur77-interacting region was further localized to SMRT-D/C (i.e. the SMRT residues 1292–1495) that contains the second CoRNR motif. The B42 fusion to the LBD of Nur77 also stimulated transactivation by the LexA fusion to SMRT-D. Similar results were also obtained with the mammalian two-hybrid-based assays (Fig. 4 A). In addition, the full-length Nur77 and its LBD alone, produced and radiolabeled by in vitro translation, specifically bound the GST fusion to SMRT-D, SMRT-D/N, and SMRT-D/C (Fig. 4 B and results not shown). Consistent with the yeast results (Fig. 3 B), however, SMRT-D/C, but not SMRT-D/N, showed specific interactions with Nur77 under more stringent conditions (i.e. 100–150 mm NaCl instead of 20 mm) (results not shown). Notably, Nur77 did not show any interaction with GST fusions to ASC2-2C and other ASC-2 fragments (Fig. 4 B and results not shown). Confirming the functional significance of these interactions, cotransfected SMRT repressed the Nur77 transactivation, either in the absence or presence of CaMKIVc, in a dose-dependent manner (Fig. 4 C and results not shown). Interestingly, SMRT-D, but not SMRT-Dm, was also sufficient to repress the Nur77 transactivation. Notably, SMRT-Dm still exhibited significant repression, suggesting that SMRT-D may contain other cryptic receptor or Nur77 binding sites. In addition, the deletion of 17 residues from the C-terminal region of SMRT, which was recently shown to be a binding site for a novel corepressor SMRT/HDAC1-associated repressor protein (SHARP) (29Shi Y. Downes M. Xie W. Kao H.Y. Ordentlich P. Tsai C.C. Hon M. Evans R.M. Genes Dev. 2001; 15: 1140-1151Crossref PubMed Scopus (268) Google Scholar), abolished the repressive effect of SMRT-D (Fig. 4 C). These results suggest that SMRT acts as a specific corepressor of Nur77 via direct interactions with Nur77, and the corepressor SHARP (29Shi Y. Downes M. Xie W. Kao H.Y. Ordentlich P. Tsai C.C. Hon M. Evans R.M. Genes Dev. 2001; 15: 1140-1151Crossref PubMed Scopus (268) Google Scholar) could be essential at least for the SMRT-D-mediated repression of Nur77. We have recently demonstrated that CaMKIV results in a novel translocation of SMRT from the nucleus to the cytoplasm (18Jang M.K. Goo Y.H. Sohn Y.C. Kim Y.S. Lee S.-K. Cheong J. Lee J.W. J. Biol. Chem. 2001; 276: 20005-20010Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Accordingly, αSMRT-mediated immunoprecipitates of cells cotransfected with expression vectors encoding Nur77 and SMRT retained Nur77 less efficiently in the presence of coexpressed CaMKIVc (Fig.5 A). In addition, the Nur77 transactivation was tremendously increased in the presence of 100 nm TSA, an inhibitor of HDAC (Fig. 5 B), as expected from the previously suggested role of SMRT as an adaptor molecule (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar, 3Lee J.W. Lee Y.C. Na S.-Y. Jung D.J. Lee S.-K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar) between HDAC and target transcription factors such as Nur77. Interestingly, the TSA-stimulated level of the Nur77 transactivation was impaired by cotransfected SMRT in a dose-dependent manner. Under this condition, it was noted that SMRT-D was a much stronger inhibitor (Fig. 5 B). Overall, these results suggest that SMRT may share identical binding surfaces of Nur77 with a putative Nur77 coactivator, such as the missing adaptor molecule between Nur77 and ASC-2, and these two coregulators (i.e. SMRT versus ASC-2/adaptor) could be in a dynamic equilibrium to occupy Nur77 in vivo. For instance, with the arrival of the CaMKIV-activating signal, this equilibrium can be driven toward the ASC-2/adaptor-bound form because of CaMKIV-mediated stimulation of the ASC-2 function as well as relocation of SMRT to the cytoplasm. Many transcriptional coregulators have been isolated for various members of the nuclear receptor superfamily (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar, 3Lee J.W. Lee Y.C. Na S.-Y. Jung D.J. Lee S.-K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar). However, none has been identified for the NGFI-B subfamily that includes Nur77 (NGFI-B), Nurr1, and NOR-1 (8Maruyama K. Tsukada T. Ohkura N. Bandoh S. Hosono T. Yamaguchi K. Int. J. Oncol. 1998; 12: 1237-1243PubMed Google Scholar). In particular, the AF2-dependent coactivators ASC-2 (20Lee S.-K. Anzick S.L. Choi J.-E. Bubendorf L. Guan X.-Y. Jung Y.-K. Kallioniemi O.-P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J. Lee Y.C. Meltzer P. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 21Lee S.-K. Na S.-Y. Jung S.-Y. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Crossref PubMed Scopus (60) Google Scholar, 22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 25Zhu Y. Kan L. Qi C. Kanwar Y.S. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 2000; 275: 13510-13516Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 26Mahajan M.A. Samulels H.H. Mol. Cell. Biol. 2000; 20: 5048-5063Crossref PubMed Scopus (96) Google Scholar, 27Kang H.J. Song M.R. Lee S.K. Shin E.C. Choi Y.H. Kim S.J. Lee J.W. Lee M.O. Exp. Cell Res. 2000; 256: 545-554Crossref PubMed Scopus (42) Google Scholar, 28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Assoc., New York1995Google Scholar), SRC-1, CBP/p300, and others (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar, 3Lee J.W. Lee Y.C. Na S.-Y. Jung D.J. Lee S.-K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar) alone did not interact with Nur77 and had no effect on the Nur77 transactivation (Figs. 1 C and 4 B and results not shown). In this report, we demonstrated that ASC-2 enhances the Nur77 transactivation in synergy with CaMKIV (Fig. 1 C). CaMKIV also stimulated the previously described autonomous transactivation function of ASC-2 (20Lee S.-K. Anzick S.L. Choi J.-E. Bubendorf L. Guan X.-Y. Jung Y.-K. Kallioniemi O.-P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J. Lee Y.C. Meltzer P. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Assoc., New York1995Google Scholar) (Fig. 2 A). It is noted that CaMKIV also potentiates the autonomous transactivation function of CBP (19Chawla S. Hardingham G.E. Quinn D.R. Bading H. Science. 1998; 281: 1505-1509Crossref PubMed Scopus (379) Google Scholar), which in turn appears to be essential for the action of ASC-2 (22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar, 28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Assoc., New York1995Google Scholar). Based on the results with CBP-neutralizing viral oncoprotein E1A and a mutant protein E1A(Δ2–36) that fails to bind CBP (22Lee S.-K. Jung S.Y. Na S.-Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar), the autonomous transactivation function of ASC-2 is expected to be essential for the Nur77 transactivation both in the absence and presence of CaMKIV (Fig. 2 B). Consistent with the inability of ASC-2 to directly interact with Nur77, mutations of the AF2 core region of Nur77 did not affect the Nur77 transactivation (Fig.2 C). Overall, these results suggest that ASC-2 may function as a CaMKIV-responsive, indirect coactivator molecule of Nur77 via a putative adaptor molecule that recognizes a non-AF2 subregion of Nur77. We recently found that ASC-2, a protein of 250 kDa, exists as a steady-state complex of ∼2 MDa in vivo, 2Y. C. Lee and J. W. Lee, unpublished results. which consists of multiple polypeptides. One of these polypeptides can be an excellent candidate adaptor molecule that links ASC-2 to Nur77, although another unrelated protein may also accomplish this mission. In contrast to ASC-2, SMRT directly bound Nur77, likely through the second CoRNR motif of SMRT (6Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (526) Google Scholar, 7Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (425) Google Scholar), and repressed its transactivation potential in a dose-dependent manner (Figs. 3 and 4). The C-terminal fragment of SMRT (i.e. SMRT-D) that contains the CoRNR motifs and a recently described novel repression domain (29Shi Y. Downes M. Xie W. Kao H.Y. Ordentlich P. Tsai C.C. Hon M. Evans R.M. Genes Dev. 2001; 15: 1140-1151Crossref PubMed Scopus (268) Google Scholar) was sufficient to transmit the repressive effect of the SMRT to Nur77. This C-terminal repression domain associates with SHARP (29Shi Y. Downes M. Xie W. Kao H.Y. Ordentlich P. Tsai C.C. Hon M. Evans R.M. Genes Dev. 2001; 15: 1140-1151Crossref PubMed Scopus (268) Google Scholar), a potent transcriptional repressor whose repression domain interacts directly with SMRT and at least five members of the nucleosome remodeling and histone deacetylase complex (for a review, see Ref. 30Ahringer J. Trends Genet. 2000; 16: 351-356Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), including HDAC1 and HDAC2. As expected from the functional association of SMRT with various HDACs, the repressive effect of SMRT-HDACs on the Nur77 transactivation was relieved in the presence of HDAC inhibitor TSA, resulting in dramatically enhanced transcriptional activities of Nur77 (Fig. 5 B). Surprisingly, however, cotransfected SMRT and SMRT-D were still repressive of the Nur77 transactivation in the presence of TSA. These results strongly suggest that SMRT may bind identical or overlapping binding sites within Nur77 that are also utilized by the putative coactivator/adaptor molecule. Thus, this adaptor molecule may contain a CoRNR-type interaction interface to interact with Nur77. Alternatively, it is also possible that SMRT binding may force Nur77 to adopt a distinct conformation that is incapable of interacting with the putative Nur77 coactivator/adaptor. However, the limited proteolysis experiments indicated that Nur77 conformation is not affected by the presence of SMRT-D (results not shown). From the results presented in this work, we propose a working model (Fig. 5 C) in which ASC-2 is indirectly recruited to Nur77 via unknown adaptor factor(s) in contrast to the direct recruitment of SMRT (Fig. 4 B), and the “transcriptionally active ASC-2 complex” could be in a dynamic equilibrium with the “repressive SMRT complex.” In this model, CaMKIV stimulates both the autonomous transactivation function of ASC-2 and translocation of SMRT to the cytoplasm, leading to a switch in equilibrium toward the active complex and the maximum stimulation of the Nur77 transactivation. Likewise, the model also predicts that the level of SMRT in a given cell type is an important determinant for the maximum level of the Nur77 transactivation that can be achieved. In this regard, it is noted that transcriptional repression by retinoic acid receptor can be either positively or negatively regulated by changes in the levels of N-CoR expression (31Soderstrom M. Vo A. Heinzel T. Lavinsky R.M. Yang W.M. Seto E. Peterson D.A. Rosenfeld M.G. Glass C.K. Mol. Endocrinol. 1997; 11: 682-692Crossref PubMed Scopus (82) Google Scholar). Recently CaMK signaling was shown to promote myogenesis by disrupting MEF2·HDAC complexes and stimulating HDAC nuclear export (32McKinsey T.A. Zhang C.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14400-14405Crossref PubMed Scopus (428) Google Scholar,33McKinsey T.A. Zhang C.L. Lu J. Olson E.N. Nature. 2000; 408: 106-111Crossref PubMed Scopus (877) Google Scholar). Similarly, phosphorylation of SMRT by MEKK-1 was shown to inhibit the ability of SMRT to physically tether to its transcription factor partners and led to a redistribution of the SMRT protein from a nuclear compartment to a more perinuclear or cytoplasmic compartment (34Hong S.H. Privalsky M.L. Mol. Cell. Biol. 2000; 20: 6612-6625Crossref PubMed Scopus (163) Google Scholar). We have also shown that CaMKI and CaMKIV, but not CaMKII, led to the cytoplasmic translocation of SMRT (18Jang M.K. Goo Y.H. Sohn Y.C. Kim Y.S. Lee S.-K. Cheong J. Lee J.W. J. Biol. Chem. 2001; 276: 20005-20010Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), consistent with their activity profile for the Nur77 transactivation (Fig. 1 B). Consistent with these results, the amount of SMRT associated with Nur77 in vivo decreased upon cotransfection of cells with the CaMKIVc expression vector (Fig. 5 A). Close examinations of the SMRT, Nur77, and ASC-2 sequences revealed that these proteins contain the known consensus phosphorylation sequences for CaMKIV, R XX(S/T) (15Means A.R. Mol. Endocrinol. 2000; 14: 4-13Crossref PubMed Scopus (159) Google Scholar). Currently we are examining if these proteins (particularly ASC-2) are a direct phosphorylation substrate for CaMKIV. In T lymphocytes, where both Nur77 and CaMKIV are expressed, the Ca2+-dependent activation of MEF2 appears to lead to programmed cell death by activating the Nur77 transcription factor (35Youn H.D. Sun L. Prywes R. Liu J.O. Science. 1999; 286: 790-793Crossref PubMed Scopus (236) Google Scholar). Thus, our studies suggest that CaMKIV may synergize with Nur77 to induce apoptosis in T lymphocytes, providing a positive feed-in mechanism to stimulate the Nur77 transactivation potential along with this calcium-responsive MEF2-dependent up-regulation of Nur77 expression (35Youn H.D. Sun L. Prywes R. Liu J.O. Science. 1999; 286: 790-793Crossref PubMed Scopus (236) Google Scholar). However, the calcium-dependent control of programmed cell death is complicated and often cell type-dependent. Greenberg and colleagues (36Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (446) Google Scholar) recently found that Ca2+ stimuli that protected neurons from cell death activated MEF2-dependent transcription in a calcineurin-dependent fashion. Presumably the genes that are activated by MEF2 in neurons protect them from cell death. Several reports have also demonstrated that inhibition of CaMK activity is associated with apoptosis and proliferation. Inhibition of CaMK activity with specific inhibitors induces apoptosis in NIH 3T3 cells (37Tombes R.M. Grant S. Westin E.H. Krystal G. Cell Growth Differ. 1995; 6: 1063-1070PubMed Google Scholar) and sensitizes etoposide-resistant cells to apoptotic challenge (38Kawamura K. Grabowski D. Krivacic K. Hidaka H. Ganapathi R. Biochem. Pharmacol. 1996; 52: 1903-1909Crossref PubMed Scopus (30) Google Scholar). Nonetheless, we suspect that coupling of these two distinct signaling pathways (i.e. CaMKIV and Nur77) may play a pivotal role in tissues that express both Nur77 and CaMKIV. In summary, we have shown that SMRT and ASC-2 mediate transcriptional repression and activation by Nur77, respectively. It is important to note that SMRT appears to be in a dynamic equilibrium in vivo with a putative adaptor molecule that may enable Nur77 to indirectly recruit the CaMKIV-responsive transactivation function of ASC-2. Molecular cloning of this putative adaptor molecule will facilitate complete understanding of the Nur77 regulatory circuit, particularly with regard to CaMKIV signaling. We thank Drs. Tony Means and Dean Edwards for various CaMK plasmids and the SMRT antibody." @default.
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• W1975509142 title "Silencing Mediator of Retinoid and Thyroid Hormone Receptors and Activating Signal Cointegrator-2 as Transcriptional Coregulators of the Orphan Nuclear Receptor Nur77" @default.
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• W1975509142 cites W152077667 @default.
• W1975509142 cites W1603742465 @default.
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• W1975509142 cites W1971424198 @default.
• W1975509142 cites W1972108825 @default.
• W1975509142 cites W1978046684 @default.
• W1975509142 cites W1980655244 @default.
• W1975509142 cites W1981364887 @default.
• W1975509142 cites W1984695968 @default.
• W1975509142 cites W1986050686 @default.
• W1975509142 cites W1986437761 @default.
• W1975509142 cites W2014764883 @default.
• W1975509142 cites W2020085602 @default.
• W1975509142 cites W2025175795 @default.
• W1975509142 cites W2027297574 @default.
• W1975509142 cites W2050743111 @default.
• W1975509142 cites W2052158630 @default.
• W1975509142 cites W2052746699 @default.
• W1975509142 cites W2078850134 @default.
• W1975509142 cites W2094331111 @default.
• W1975509142 cites W2102420815 @default.
• W1975509142 cites W2106208437 @default.
• W1975509142 cites W2111254493 @default.
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• W1975509142 cites W2119963896 @default.
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