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• W2096861468 abstract "Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that contains several autonomous repressive domains (RDs). The N-terminal RD acts by recruiting histone deacetylases (HDACs). In a comprehensive proteomic analysis of RIP140 by MS, 11 phosphorylation sites of RIP140 are identified; among them five sites are located in the N-terminal RD including Ser104, Thr202, Thr207, Ser358, and Ser380. The role of phosphorylation of RIP140 in regulating its biological activity and the underlying mechanism are examined using a site-directed mutagenesis approach. Mutations mimicking constitutive phosphorylation or dephosphorylation are introduced. The N-terminal RD phosphorylation, mediated by the mitogen-activated protein kinase (MAPK), enhances its repressive activity through increased recruitment of HDAC. Mutations mimicking constitutive dephosphorylation at Thr202 or Thr207 significantly impair its repressive activity and HDAC recruitment, whereas mutation at Ser358 only slightly affects its HDAC recruitment and the repressive activity. Consistently, mutations mimicking constitutive phosphorylation at either Thr202 or Thr207 convert RIP140 into a more potent repressor, which is less responsive to a disturbance in the MAPK system. Furthermore, constitutive phosphorylation at both Thr202 and Thr207 residues renders RIP140 fully repressive and strongly interacting with HDAC. The activity of this mutant is resistant to the MAPK inhibitor, indicating an essential role for Thr202 and Thr207 in MAPK-mediated modulation of RIP140 function. The study provides insights into the modulation of RIP140 biological activity through a specific cellular signaling pathway that augments phosphorylation at specific residues of RIP140 molecule and alters its cofactor recruitment. Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that contains several autonomous repressive domains (RDs). The N-terminal RD acts by recruiting histone deacetylases (HDACs). In a comprehensive proteomic analysis of RIP140 by MS, 11 phosphorylation sites of RIP140 are identified; among them five sites are located in the N-terminal RD including Ser104, Thr202, Thr207, Ser358, and Ser380. The role of phosphorylation of RIP140 in regulating its biological activity and the underlying mechanism are examined using a site-directed mutagenesis approach. Mutations mimicking constitutive phosphorylation or dephosphorylation are introduced. The N-terminal RD phosphorylation, mediated by the mitogen-activated protein kinase (MAPK), enhances its repressive activity through increased recruitment of HDAC. Mutations mimicking constitutive dephosphorylation at Thr202 or Thr207 significantly impair its repressive activity and HDAC recruitment, whereas mutation at Ser358 only slightly affects its HDAC recruitment and the repressive activity. Consistently, mutations mimicking constitutive phosphorylation at either Thr202 or Thr207 convert RIP140 into a more potent repressor, which is less responsive to a disturbance in the MAPK system. Furthermore, constitutive phosphorylation at both Thr202 and Thr207 residues renders RIP140 fully repressive and strongly interacting with HDAC. The activity of this mutant is resistant to the MAPK inhibitor, indicating an essential role for Thr202 and Thr207 in MAPK-mediated modulation of RIP140 function. The study provides insights into the modulation of RIP140 biological activity through a specific cellular signaling pathway that augments phosphorylation at specific residues of RIP140 molecule and alters its cofactor recruitment. Environmental factors in the extracellular milieu utilize signal transduction pathways to propagate their cues into gene expression (1Pawson T. Scott J.D. Signaling through scaffold, anchoring, and adaptor proteins.Science. 1997; 278: 2075-2080Google Scholar, 2Pawson T. Protein modules and signalling networks.Nature. 1995; 373: 573-580Google Scholar, 3Hunter T. Signaling-2000 and beyond.Cell. 2000; 100: 113-127Google Scholar). Transcriptional factors and their co-regulators are extensively modified at the post-translational level, which usually regulate the critical function and property of the protein (4Fu M. Wang C. Zhang W.X. Pestell R.G. Acetylation of nuclear receptors in cellular growth and apoptosis.Biochem. Pharmacol. 2004; 68: 1199-1208Google Scholar, 5Quivy V. Van Lint C. Regulation at multiple levels of NF-κB-mediated transactivation by protein acetylation.Biochem. Pharmacol. 2004; 68: 1221-1229Google Scholar, 6Chen L. Fischle W. Verdin E. Greene W.C. Duration of nuclear NF-κB action regulated by reversible acetylation.Science. 2001; 293: 1653-1657Google Scholar, 7Dornan D. Eckert M. Wallace M. Shimizu H. Ramsay E. Hupp T.R. Ball K.L. Interferon regulatory factor 1 binding to p300 stimulates DNA-dependent acetylation of p53.Mol. Cell. Biol. 2004; 24: 10083-10098Google Scholar, 8Mailfait S. Belaiche D. Kouach M. Dallery N. Chavatte P. Formstecher P. Sablonniere B. Critical role of tyrosine 277 in the ligand-binding and transactivating properties of retinoic acid receptor α.Biochemistry. 2000; 39: 2183-2192Google Scholar, 9Chuikov S. Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. McKinney K. Tempst P. Prives C. Gamblin S.J. Barlev N.A. Reinberg D. Regulation of p53 activity through lysine methylation.Nature. 2004; 432: 353-360Google Scholar). Deciphering such modifications and relating them to the biological function is referred to as functional proteomics (10Wells L. Vosseller K. Cole R.N. Cronshaw J.M. Matunis M.J. Hart G.W. Mapping sites of O-GlcNaAc modification using affinity tags for serine and threonine post-translational modification.Mol. Cell. Proteomics. 2002; 1: 791-804Google Scholar). A variety of post-translational modifications have been found that regulate protein functions, including phosphorylation, acetylation, methylation, glycosylation, ubiquitination, and sumoylation, etc. (11Kouzarides T. Acetylation: A regulatory modification to rival phosphorylation.EMBO J. 2000; 19: 1176-1179Google Scholar, 12McBride A.E. Silver P.A. State of the arg: Protein methylation at arginine comes of age.Cell. 2001; 106: 5-8Google Scholar, 13Hart G.W. Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins.Annu. Rev. Biochem. 1997; 66: 315-335Google Scholar, 14Wells L. Vosseller K. Hart G.W. Glycosylation of nucleocytoplasmic proteins: Signal transduction and O-GlcNAc.Science. 2001; 291: 2376-2378Google Scholar). Receptor interacting protein 140 (RIP140) 1The abbreviations used are: RIP140, receptor interacting protein 140; RD, repressive domain; HDAC, histone deacetylase; CtBP, carboxyl-terminal binding protein; MAPK, mitogen activated protein kinase; WT, wild type; Mut, mutant; DCC, dextran charcoal; PKC, protein kinase C; CaCal, calcium-calmodulin-dependent protein kinase II; RLU, relative luciferase unit; CN, constitutive negative; CP, constitutive positive. 1The abbreviations used are: RIP140, receptor interacting protein 140; RD, repressive domain; HDAC, histone deacetylase; CtBP, carboxyl-terminal binding protein; MAPK, mitogen activated protein kinase; WT, wild type; Mut, mutant; DCC, dextran charcoal; PKC, protein kinase C; CaCal, calcium-calmodulin-dependent protein kinase II; RLU, relative luciferase unit; CN, constitutive negative; CP, constitutive positive. is a co-regulator for many transcription factors including nuclear receptors (15White R. Leonardsson G. Rosewell I. Jacobs M.A. Milligan S. Parker M.G. The nuclear receptor co-repressor nrip1 (RIP140) is essential for female fertility.Nat. Med. 2000; 6: 1368-1374Google Scholar, 16Cavailles V. Dauvois S. L'Horset F. Lopez G. Hoare S. Kushner P.J. Parker M.G. Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor.EMBO J. 1995; 14: 3741-3751Google Scholar, 17Collingwood T.N. Rajanayagam O. Adams M. Wagner R. Cavailles V. Kalkhoven E. Matthews C. Nystrom E. Stenlof K. Lindstedt G. Tisell L. Fletterick R.J. Parker M.G. Chatterjee V.K. A natural transactivation mutation in the thyroid hormone β receptor: Impaired interaction with putative transcriptional mediators.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 248-253Google Scholar, 18Joyeux A. Cavailles V. Balaguer P. Nicolas J.C. RIP 140 enhances nuclear receptor-dependent transcription in vivo in yeast.Mol. Endocrinol. 1997; 11: 193-202Google Scholar, 19L'Horset F. Dauvois S. Herry D.M. Cavailles V. Parker M.G. RIP-140 interacts with multiple nuclear receptors by means of two distinct sites.Mol. Cell. Biol. 1996; 16: 6029-6036Google Scholar). Extensive studies have been conducted to examine the versatile activity of RIP140 in transcriptional regulation. It is known that RIP140 acts, primarily, as a transcriptional corepressor through different mechanisms (20Chuang F.M. West B.L. Baxter J.D. Schaufele F. Activities in Pit-1 determine whether receptor interacting protein 140 activates or inhibits Pit-1/nuclear receptor transcriptional synergy.Mol. Endocrinol. 1997; 11: 1332-1341Google Scholar, 21Subramaniam N. Treuter E. Okret S. Receptor interacting protein RIP140 inhibits both positive and negative gene regulation by glucocorticoids.J. Biol. Chem. 1999; 274: 18121-18127Google Scholar, 22Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. Receptor-interacting protein 140 directly recruits histone deacetylases for gene silencing.J. Biol. Chem. 2000; 275: 40782-40787Google Scholar). RIP140 is recruited to nuclear receptors through its nine LXXLL motifs and a modified motif LXXML, where X can be any amino acids (23Lee C.H. Chinpaisal C. Wei L.N. Cloning and characterization of mouse RIP140, a corepressor for nuclear orphan receptor TR2.Mol. Cell. Biol. 1998; 18: 6745-6755Google Scholar,24Wei L.N. Farooqui M. Hu X. Ligand-dependent formation of retinoid receptors, receptor-interacting protein 140 (RIP140), and histone deacetylase complex is mediated by a novel receptor-interacting motif of RIP140.J. Biol. Chem. 2001; 276: 16107-16112Google Scholar). With respect to its repressive activity, four autonomous repressive domains (RDs) are known. RD1 is located in the amino-terminal region (amino acids 1–495), RD2 and RD3 are located in the central portion (amino acids 336–1006), and RD4 is located in the carboxyl-terminal region (amino acids 977–1161). These domains function through various mechanisms. The amino-terminal RD acts by recruiting histone deacetylases (HDACs) through an HDAC-interacting domain, which has been mapped to amino acids 78–303 (22Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. Receptor-interacting protein 140 directly recruits histone deacetylases for gene silencing.J. Biol. Chem. 2000; 275: 40782-40787Google Scholar, 25Castet A. Boulahtouf A. Versini G. Bonnet S. Augereau P. Vignon F. Khochbin S. Jalaguier S. Cavailles V. Multiple domains of the receptor-interacting protein 140 contribute to transcription inhibition.Nucleic Acids Res. 2004; 32: 1957-1966Google Scholar). The central region interacts with the carboxyl-terminal binding proteins (CtBP1 and CtBP2) (26Christian M. Tullet J.M.A. Parker M.G. Characterization of four autonomous repression domains in the corepressor receptor interacting protein 140.J. Biol. Chem. 2004; 279: 15645-15651Google Scholar). In terms of its physiological action, RIP140-null mice exhibit female reproductive defects (15White R. Leonardsson G. Rosewell I. Jacobs M.A. Milligan S. Parker M.G. The nuclear receptor co-repressor nrip1 (RIP140) is essential for female fertility.Nat. Med. 2000; 6: 1368-1374Google Scholar). Further studies of these animals indicate that RIP140 could play an important role in the regulation of fat accumulation in adipose tissues (27Leonardsson G. Steel J.H. Christian M. Pocock V. Milligan S. Bell J. So P.W. Medina-Gomez G. Vidal-Puig A. White R. Parker M.G. Nuclear receptor corepressor RIP140 regulates fat accumulation.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 8437-8442Google Scholar). Recently, we initiated a functional proteomic study of RIP140 expressed and purified from insect cells. Through extensive mass spectrometric analyses, we found RIP140 are extensively phosphorylated (28Huq M.D. Khan S.A. Park S.W. Wei L.N. Mapping of phosphorylation sites of nuclear co-repressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.Proteomics. 2005; 5: 2157-2166Google Scholar) and acetylated (29Huq M.D. Wei L.N. Post-translational modification of nuclear co-repressor receptor-interacting protein 140 by acetylation.Mol. Cell. Proteomics. 2005; 4: 975-983Google Scholar). To continue the functional proteomic endeavor, we took a systematic mutagenesis approach to uncover the function and the mechanism of actions of specifically modified residues of RIP140. This study reports our systematic studies of the functional role of phosphorylation on RIP140, specifically with respect to its role in the HDAC-mediated repressive activity of RD1. We previously reported the identification of 10 phosphorylation sites on RIP140 purified from insect cells by LC-ESI-MS/MS analysis (28Huq M.D. Khan S.A. Park S.W. Wei L.N. Mapping of phosphorylation sites of nuclear co-repressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.Proteomics. 2005; 5: 2157-2166Google Scholar). These residues included Ser104, Thr207, Ser358, Ser380, Ser488, Ser519, Ser531, Ser543, Ser672, and Ser1003. One residue, Thr202, appeared ambiguous in our initial analysis, and a reanalysis of the MS/MS data enabled us to verify Thr202 as an additional phosphorylation site on RIP140. We then determined the effects of phosphorylation on the biological activity of RIP140 and explored the mechanism underlying such an effect in this current study. RIP140 employs multiple repressive pathways including HDAC- and CtBP-triggered events for RD1 and RD2/3, respectively. We focus this initial functional proteomic study on the HDAC-mediated RD1 activity. We identified mitogen-activated protein kinase (MAPK)-mediated phosphorylation as the critical pathway for RD1-mediated repressive activity, which was caused by enhanced recruitment of HDAC by phosphorylated RIP140. Mutations mimicking either constitutive phosphorylation or dephosphorylation were generated to determine whether the effects were caused by altered charges brought about by phosphorylation on specific residues or the amino acid residues per se. With this systemic approach, we identified two residues, Thr202 and Thr207, crucial for the repressive activity of RD1 and its ability to recruit HDACs. Furthermore, increased negative charge caused by phosphorylation on these two specific residues is essential. Constructs of RIP140 full length/N-terminal fused to GAL4-BD (20Chuang F.M. West B.L. Baxter J.D. Schaufele F. Activities in Pit-1 determine whether receptor interacting protein 140 activates or inhibits Pit-1/nuclear receptor transcriptional synergy.Mol. Endocrinol. 1997; 11: 1332-1341Google Scholar), RIP140 full length fused to His-epitope (28Huq M.D. Khan S.A. Park S.W. Wei L.N. Mapping of phosphorylation sites of nuclear co-repressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.Proteomics. 2005; 5: 2157-2166Google Scholar), FLAG-epitope (22Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. Receptor-interacting protein 140 directly recruits histone deacetylases for gene silencing.J. Biol. Chem. 2000; 275: 40782-40787Google Scholar), and GAL4-tk-luciferase reporter (23Lee C.H. Chinpaisal C. Wei L.N. Cloning and characterization of mouse RIP140, a corepressor for nuclear orphan receptor TR2.Mol. Cell. Biol. 1998; 18: 6745-6755Google Scholar) have been described previously. To construct pAD-GAL4-HDAC3, coding regions of HDAC3 were released from pG4-hRPD3-2B (22Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. Receptor-interacting protein 140 directly recruits histone deacetylases for gene silencing.J. Biol. Chem. 2000; 275: 40782-40787Google Scholar) by EcoRI/XbaI and ligated to the EcoRI/XbaI site of pVP16 (AD-GAL4). To identify the phosphorylation sites on RIP140, we expressed the His-tagged RIP140 in insect cells as a eukaryotic host for mammalian protein expression. The protein was purified by affinity column over Talon resin with 95% homogeneity. The details of the procedure for RIP140 purification was described previously (28Huq M.D. Khan S.A. Park S.W. Wei L.N. Mapping of phosphorylation sites of nuclear co-repressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.Proteomics. 2005; 5: 2157-2166Google Scholar). The purified RIP140 was subjected to tryptic digestion, and LC-ESI-MS/MS analysis was conducted as already described to identify the modified residues (28Huq M.D. Khan S.A. Park S.W. Wei L.N. Mapping of phosphorylation sites of nuclear co-repressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.Proteomics. 2005; 5: 2157-2166Google Scholar). The technique for culturing COS-1 cells, transfection experiments, and luciferase and lacZ assay were as described previously (23Lee C.H. Chinpaisal C. Wei L.N. Cloning and characterization of mouse RIP140, a corepressor for nuclear orphan receptor TR2.Mol. Cell. Biol. 1998; 18: 6745-6755Google Scholar). Cells were transiently transfected by the calcium phosphate precipitation method with a mixture of pBD-GAL4-RIP140 full length/N-terminal, wild-type (WT)/mutant (Mut) (0.1 μg) or pBD-GAL4 (0.1 μg), GAL4-tk-luciferase (0.5 μg) reporter, and a CMV-lacZ internal control (0.05 μg). Forty h post-transfection, cells were exchanged with fresh medium containing dextran charcoal (DCC)-treated serum and treated for 8 h with either 1 μℳ chelerythrin chloride (protein kinase C (PKC) inhibitor), 0.5 μℳ KN-93 (calcium-calmodulin-dependent protein kinase II (CaCal) inhibitor), 3 μℳ PD98059 (MAPK inhibitor), or 1 μℳ concentration of anisomycin (MAPK activator) (Calbiochem). Forty-eight h post-transfection, total cell extracts were collected and tested for luciferase and lacZ activity. The fold relative luciferase activity was calculated by normalizing relative luciferase units (RLU) activity found in the experimental samples to the RLU activity found in the pBD-GAL4 alone. Reported values are an average of three experiments with triplicate measurement taken in each experiment. 3T3 cells were transfected with FLAG-RIP140 or CMV empty vector. Forty h post-transfection, cells were subjected to a 1 μℳ concentration of MAPK activator (anisomycin) or a 3 μℳ concentration of MAPK inhibitor (PD98059) for 8 h in Dulbecco’s modified Eagle’s medium containing DCC serum. At 48 h post-transfection, cells were harvested, and FLAG-RIP140 was immunoprecipitated by anti-FLAG antibody (Sigma) on protein G-agarose (Sigma) beads (22Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. Receptor-interacting protein 140 directly recruits histone deacetylases for gene silencing.J. Biol. Chem. 2000; 275: 40782-40787Google Scholar). The partially purified FLAG-RIP140 fusion protein was incubated with [35S]methionine-labeled HDAC3 protein for pull-down reaction as described previously (23Lee C.H. Chinpaisal C. Wei L.N. Cloning and characterization of mouse RIP140, a corepressor for nuclear orphan receptor TR2.Mol. Cell. Biol. 1998; 18: 6745-6755Google Scholar). The specific bound proteins were released by resuspending beads in 20 μl of SDS loading buffer, divided in equal amounts, and resolved by SDS-PAGE. One gel was subjected to Western blot analysis probed with anti-FLAG antibody followed by horseradish peroxidase-conjugated secondary antibody and then was detected with ECL (GE Healthcare), which constituted the input. Another gel was fixed, dried, and exposed to a phosphoimager screen (Molecular Dynamics) overnight to detect labeled HDAC protein and visualized by autoradiography. Densitometric analysis was done for labeled HDAC and input. Intensity of labeled HDAC was normalized to input. The expression vector for His-RIP140 was expressed and purified from insect cells in presence of phosphatase inhibitor that ensured phosphorylated RIP140 (28Huq M.D. Khan S.A. Park S.W. Wei L.N. Mapping of phosphorylation sites of nuclear co-repressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.Proteomics. 2005; 5: 2157-2166Google Scholar). This purified protein was then dephosphorylated by alkaline phosphatase (Roche Diagnostics; 1 unit per 5 μg of protein at 37 °C for 1 h) treatment. Both phosphorylated and dephosphorylated protein was pulled down by Metal Talon affinity resin (BD Biosciences) pre-equilibrated by extraction buffer (50 mℳ sodium phosphate buffer, pH 7.0, 300 mℳ NaCl) and then washed extensively with extraction buffer followed by binding buffer and then tested for interaction with [35S]methionine-labeled HDAC3 protein. For input, Western blot analysis was probed with anti-His antibody (Upstate Biotechnology). COS-1 cells were maintained as described previously (23). To test the interaction of RIP140 full-length/N-terminal, WT/Mut constructs with HDAC3, cells were co-transfected with pBD-GAL4-RIP full-length/N-terminal, WT/Mut (0.1 μg) as bait and pAD-GAL4-HDAC3 (0.1 μg) as prey. All co-transfections included GAL4-tk-luciferase (0.5 μg) reporter and a CMV-lacZ internal control (0.05 μg). Forty h post-transfection, cells were exposed to either 1 μℳ MAPK activator (anisomycin) or 3 μℳ MAPK inhibitor (PD98059) for 8 h in Dulbecco’s modified Eagle’s medium containing 10% DCC-treated fetal bovine serum medium. Forty-eight h post-transfection, total cell extracts were collected and tested for luciferase and lacZ activity. The fold relative luciferase activity was calculated by normalizing RLU activity found in the experimental samples to the RLU activity found in the pBD-GAL4-RIP140 full-length/N-terminal WT and pAD-GAL4-HDAC3 co-transfection. Reported values are an average of three experiments with triplicate measurement taken in each experiment. Constitutive negative/positive, point/sequential mutations involving residues Ser104, Thr202, Thr207, Ser315, Ser358, and Ser380 in WT pBD-GAL4-RIP140 full length/N-terminal expression vector as template were made as instructed by QuikChange XL site-directed mutagenesis kit (Stratagene). The mutagenic primers were designed such that they were nearest matched to alanine (A) or glutamic acid (E). While S/T→A point/sequential mutants refer to a constitutively dephosphorylated state, S/T→E may represent a constitutively phosphorylated state (30Wu R.C. Qin J. Yi P. Wong J. Tsai S.Y. Tsai M.J. O'Malley B.W. Selective phosphorylations of the SRC-3/AIB1 coactivator integrate genomic responses to multiple cellular signaling pathways.Mol. Cell. 2004; 15: 937-949Google Scholar). The mutagenic primers employed to generate mutant constructs are: S104A: 5′-CGGAAGAGGCTGGCTGATGCCATCGTGAATTTAAAC-3′ (sense), 5′-GTTTAAATTCACGATGGCATCAGCCAGCCTCTTCCG-3′ (antisense); T202A: 5′-GAAAAGTCAGATCCCGCCCTCCCTGACGTG-3′ (sense), 5′-CACGTCAGGGAGGGCGGGATCTGACTTTTC-3′ (antisense); T207A: 5′-CTCCCTGACGTGGCTCCAAACCTTATC-3′ (sense), 5′-GATAAGGTTTGGAGCCACGTCAGGGAG-3′ (antisense); S315A: 5′-CAGAAGGACGTGGGCGCTTCGCAGCTCTCC-3′ (sense), 5′-GGAGAGCTGCGAAGCGCCCACGTCCTTCTG-3′ (antisense); S358A: 5′-GGTGTTGTCCCTTCCGCCCCCAAAAACACGAGC-3′ (sense), 5′-GCTCGTGTTTTTGGGGGCGGAAGGGACAACACC-3′ (antisense); S380A: 5′-GCAGGCTGCTAATAACGCTCTGCTTTTGCAT-3′ (sense), 5′-ATGCAAAAGCAGAGCGTTATTAGCAGCCTGC-3′ (antisense); T202E: 5′-AAGTCAGATCCCGAACTCCCTGACGTG-3′ (sense), 5′-CACGTCAGGGAGTTCGGGATCTGACTT-3′ (antisense); T207E: 5′-CTCCCTGACGTGGAACCAAACCTTATC-3′ (sense), 5′-GATAAGGTTTGGTTCCACGTCAGGGAG-3′ (antisense); S358E: 5′-GTTGTCCCTTCCGAACCCAAAAACACG-3′ (sense), 5′-CGTGTTTTTGGGTTCGGAAGGGACAAC-3′ (antisense). The positive clones were verified by DNA sequencing. A MS/MS analysis of tryptic phosphopeptides was carried out to identify the modified residues. Previously, we reported 10 phosphorylation sites including Ser104, Thr207, Ser358, Ser380, Ser488, Ser519, Ser531, Ser543, Ser672, and Ser1003 (28). Phosphorylation on Thr202 appeared ambiguous according to the initial analysis of the MS spectrum. To clarify the ambiguity, we reanalyzed the data to verify Thr202 phosphorylation. In the total ion chromatogram, the tryptic monophosphopeptide spanning residues 199–212 (SGPTLPDVTPNLIR) eluted as a doubly charged precursor ion at m/z 780.38 (precursor mass 1558.76 Da) at 47.36 min. Product ion analysis of this precursor analysis suggested that some species of the peptide were phosphorylated at Thr202, whereas some were phosphorylated at Thr207. The MS/MS data clearly showed an 80-amu delta mass shift at y6, y7, y8, and y9 consecutively. The b5 and b6 ions at m/z 456.22 and m/z 533.26 (indicated as b6** in Fig. 1) were identical to those of the unmodified peptide. This clearly indicated Thr207 phosphorylation. Because the peptide precursor ion was attributed to monophosphorylation and eluted as a single peak in the liquid chromatogram, we thought another phosphorylation site was unlikely to be present. However, upon reexamining the product ion of the precursor ion, we found a relatively high abundant y9 ion (indicated as y9* in Fig. 1) at m/z 1024.6 identical to the unmodified peptide. In addition, the y ions caused by loss of ammonia and H2O for this y9 ion were found. This indicated that Thr207 was not phosphorylated in some species of the precursor peptide. Furthermore, the b2 and b3 ions corresponded to the unmodified peptide. Thus the possibility of Ser199 phosphorylation was excluded. The doubly charged a8 ion showed a 40-amu shift, signifying Thr202 phosphorylation. Finally, the protein phosphorylation sites prediction software NetPhos (www.expasy.org) that assisted in the analysis of RIP140 also showed both Thr202 and Thr207 to be potential sites for phosphorylation, Therefore, we conclude that Thr202 can also be phosphorylated in vivo. All the 11 MS-confirmed phosphorylation sites of RIP140 were compared with the consensus motifs of all known protein kinases (31Pearson R.B. Kemp B.E. Protein kinase phosphorylation site sequences and consensus specificity motifs: Tabulations.Methods Enzymol. 1991; 200: 62-81Google Scholar) as listed in Table I. The N-terminal RD contains five phosphorylation sites. Thr202 and Thr207 are potential sites for MAPK phosphorylation, Ser358 can be phosphorylated either by MAPK or PKC, and the other two at Ser104 and Ser380 were unmatched to consensus sequences of known kinases. The remaining six sites in the central and C-terminal RD are potential targets for MAPK, PKC, or CaCal. We then explored the role of these kinases in regulating the repressive activity of RIP140.Table IPhosphorylation sites of RIP140 and the consensus motif-specific kinase for phosphorylationResidueSequencesaSequences adjoining to the residues were compared to consensus motifs of known kinases.Protein kinasebMAPK: X-X-S/T-P, X-P-S/T-X-X; PKC: S/T-X-K/R, K/R-X-X-S/T, K/R-X-S/T; CaCal: R-X-X-S/T; where S, T, K, R, and P are single-letter codes of amino acids, and X can be any amino acid.RD10499KRLSDSIVNLN109Unmatched202197QKSGPTLPDVT207MAPKN-terminal RD207202TLPDVTPNLIR212MAPK(RD1)358353GVVPSSPKNKN363MAPK/PKC380375QAANNSLLLHL385Unmatched488483EDQDTSTNSKL493Unmatched519514VERNASPQDIH524PKC/CaCal/MAPKCentral RD531526DGTKFSPQNYT536PKC/MAPK(RD2 and RD3)543538YTAIESPSTNR548MAPK672667IDRLNSPLLSN677PKC/CaCal/MAPK1003998DHRTFSYPGMV1008PKC/CaCalC-terminal RD (RD4)a Sequences adjoining to the residues were compared to consensus motifs of known kinases.b MAPK: X-X-S/T-P, X-P-S/T-X-X; PKC: S/T-X-K/R, K/R-X-X-S/T, K/R-X-S/T; CaCal: R-X-X-S/T; where S, T, K, R, and P are single-letter codes of amino acids, and X can be any amino acid. Open table in a new tab To evaluate the intrinsic repressive activity of RIP140, a standard trans-repressive assay was conducted where the full-length RIP140 was fused to a GAL4-BD and analyzed using a GAL4-responsive reporter in a standard cellular system, the COS1 cells. Inhibitors of MAPK, PKC, and CaCal were used to determine the effects of these kinase pathways on the trans-repressive activity of RIP140. The fold relative luciferase activity was evaluated to determine the effect on trans-repression. As shown in Fig. 2, both PKC and CaCal inhibitors have no effect on the trans-repressive activity of RIP140, whereas the specific MAPK inhibitor significantly relieves the repression, suggesting a role for MAPK-induced phosphorylation in mediating the repressive activity of RIP140. Our previous study has identified HDAC recruitment as the primary repressive mechanism of RIP140 through RD1 (22Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. Receptor-interacting protein 140 directly recruits histone deacetylases for gene silencing.J. Biol. Chem. 2000; 275: 40782-40787Google Scholar, 25Castet A. Boulahtouf A. Versini G. Bonnet S. Augereau P. Vignon F. Khochbin S. Jalaguier S. Cavailles V. Multiple domains of the receptor-interacting protein 140 contribute to transcription inhibition.Nucleic Acids Res. 2004; 32: 1957-1966Google Scholar). Therefore we further examined the effects of MAPK-induced phosphorylation on the recruitment of HDAC by RIP140. To examine the direct effect of phosphorylation on HDAC recruitment, we employed an in vitro pull-down assay that directly monitors protein-protein interaction. In vivo phosphorylated or hypophosphorylated RIP140 was prepared by transfecting FLAG-RIP140 into a mammalian cell line that has a full capacity for MAPK signaling, 3T3, followed by an 8-h pulse of 1 μℳ concentration of MAPK activator (anisomycin) or 3 μℳ concentration of MAPK inhibitor (PD 98059) prior to harvesting the cells. The phosphorylated or hypophosphorylated FLAG-RIP140 was partially purified through anti-FLAG-coupled protein G beads (Sigma) and incubated with in vitro-translated, 35S-labeled HDAC3. To monitor" @default.
• W2096861468 created "2016-06-24" @default.
• W2096861468 creator A5024957372 @default.
• W2096861468 creator A5034365665 @default.
• W2096861468 creator A5064792308 @default.
• W2096861468 creator A5069176432 @default.
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• W2096861468 date "2005-11-01" @default.
• W2096861468 modified "2023-10-18" @default.
• W2096861468 title "Regulation of Co-repressive Activity of and HDAC Recruitment to RIP140 by Site-specific Phosphorylation" @default.
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