M. D. Mostaqul Huq

Tyrosine Nitration on p65 A Novel Mechanism to Rapidly Inactivate Nuclear Factor-κB

NO is an important factor that induces post-translational modifications of proteins by cellular reduction and oxidation mechanism: cysteinyl-nitrosylation or Tyr nitration. Nuclear factor (NF)-κB activity can be rapidly suppressed by sodium nitroprusside, a NO donor. This effect was effectively reversed by peroxynitrite scavenger deferoxamine, suggesting a Tyr nitration-mediated mechanism. Western blot with nitrotyrosine-specific antibody demonstrated that the p65 subunit of NF-κB was predominantly nitrated on Tyr residues. Tyr nitration of p65 induced its dissociation from p50, its association with IκBα, and subsequent sequestration of p65 in the cytoplasm by IκBα-mediated export. Liquid chromatography-coupled nanoelectrospray mass spectrometry revealed specific nitration on Tyr-66 and Tyr-152 residues of p65. Mutation studies confirmed that both Tyr-66 and Tyr-152 residues were important for the direct effects of NO on p65, which resulted in more p65 export and inactivation of NF-κB activity. This study identified a novel and efficient pathway where NO rapidly inactivated NF-κB activity by inducing Tyr nitration on p65.

Suppression of receptor interacting protein 140 repressive activity by protein arginine methylation

Receptor interacting protein 140 (RIP140), a ligand‐dependent corepressor for nuclear receptors, can be modified by arginine methylation. Three methylated arginine residues, at Arg‐240, Arg‐650, and Arg‐948, were identified by mass spectrometric analysis. Site‐directed mutagenesis studies demonstrated the functionality of these arginine residues. The biological activity of RIP140 was suppressed by protein arginine methyltransferase 1 (PRMT1) due to RIP140 methylation, which reduced the recruitment of histone deacetylases to RIP140 and facilitated its nuclear export by enhancing interaction with exportin 1. A constitutive negative (Arg/Ala) mutant of RIP140 was resistant to the effect of PRMT1, and a constitutive positive (Arg/Phe) mutation mimicked the effect of arginine methylation. The biological activities of the wild type and the mutant proteins were examined in RIP140‐null MEF cells. This study uncovered a novel means to inactivate, or suppress, RIP140, and demonstrated protein arginine methylation as a critical type of modification for corepressor.

Retinoic acid-stimulated sequential phosphorylation, PML recruitment, and SUMOylation of nuclear receptor TR2 to suppress Oct4 expression

We previously reported an intricate mechanism underlying the homeostasis of Oct4 expression in normally proliferating stem cell culture of P19, mediated by SUMOylation of orphan nuclear receptor TR2. In the present study, we identify a signaling pathway initiated from the nongenomic activity of all-trans retinoic acid (atRA) to stimulate complex formation of extracellular signal-regulated kinase 2 (ERK2) with its upstream kinase, mitogen-activated protein kinase kinase (MEK). The activated ERK2 phosphorylates threonine-210 (Thr-210) of TR2, stimulating its subsequent SUMOylation. Dephosphorylated TR2 recruits coactivator PCAF and functions as an activator for its target gene Oct4. Upon phosphorylation at Thr-210, TR2 increasingly associates with promyelocytic leukemia (PML) nuclear bodies, becomes SUMOylated, and recruits corepressor RIP140 to act as a repressor for its target, Oct4. To normally proliferating P19 stem cell culture, exposure to a physiological concentration of atRA triggers a rapid nongenomic signaling cascade to suppress Oct4 gene and regulate cell proliferation.

Regulation of retinal dehydrogenases and retinoic acid synthesis by cholesterol metabolites

Retinoic acid (RA) constitutes the major active ingredient of vitamin A and is required for various biological processes. The tissue RA level is maintained through a cascade of metabolic reactions where retinal dehydrogenases (RALDHs) catalyze the terminal reaction of RA biosynthesis from retinal, a rate‐limiting step. We showed that dietary supplement of cholesterol enhanced the expression of RALDH1 and 2 genes and the cellular RA content in vital organs such as brain, kidney, liver and heart. Consistently, the cholesterol‐lowering agent (pravastatin sodium) downregulated the expression of RALDH1 and 2 genes in several organs especially the liver and in cultured liver cells. Further, cholesterol metabolites, predominantly the oxysterols, the natural ligands for liver X receptor (LXR), induced these genes via upregulation of sterol regulatory element binding protein‐1c (SREBP‐1c) that bound to the regulatory regions of these genes. Knockdown of LXRα/β or SREBP‐1c downregulated the expression of RALDH genes, which could be rescued by re‐expressing SREBP‐1c, suggesting SREBP‐1c as a direct positive regulator for these genes. This study uncovered a novel crosstalk between cholesterol and RA biosynthesis.

Regulation of Co-repressive Activity of and HDAC Recruitment to RIP140 by Site-specific Phosphorylation

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.

Lysine Trimethylation of Retinoic Acid Receptor-α A Novel Means To Regulate Receptor Function

Retinoic acid receptors (RARs) belong to the nuclear receptor superfamily. The mechanism of ligand-dependent activation of RARs is well known. The effect of protein phosphorylation on the activity of RARs has also been demonstrated. However, it is unclear whether other types of modifications exist and if so whether they can affect the activity of RARs. In a mass spectrometric analysis of mouse RARα expressed in insect cells, we identified a trimethylation site on Lys347 in the ligand binding domain. The modification site was verified in mammalian cells, and site-directed mutagenesis studies revealed the functionality of Lys347 methylation in vivo. Constitutive negative mutants, mimicking hypomethylated RARα, were prepared by replacing methylated Lys347 with either alanine or glutamine. A constitutive positive mutant partially mimicking the hypermethylated RARα was generated by replacing the methylated lysine residue with phenylalanine, a bulky hydrophobic amino acid, to introduce a site-specific hydrophobicity similar to that contributed by lysine methylation. Studies of these mutants revealed that trimethylation of Lys347 of RARα facilitated its interactions with cofactors p300/CREB-binding protein-associated factor and receptor-interacting protein 140 as well as its heterodimeric partner retinoid X receptor, suggesting that site-specific hydrophobicity at Lys347 enhanced molecular interaction of RARα with its modulators. This study uncovers the first example of lysine trimethylation on a mammalian non-histone protein that has an important biological consequence. Our finding also provides the evidence for lysine methylation for the family of nuclear receptors for the first time.

Post-translational modifications of nuclear co-repressor RIP140: a therapeutic target for metabolic diseases.

Most proteins undergo post-translational modification (PTM), which is known to play roles in normal physiological processes and the progression of many diseases. In this review, we summarized and discussed the mass spectrometry (MS)-based studies of various PTMs of nuclear co-repressor, receptor interacting protein 140 (RIP140), as well as the significance of these PTMs in modulating the biological activities of RIP140, specifically in adipocytes. Comprehensive analyses of RIP140 by MS identified specific sites of PTMs on RIP140, including that of phosphorylation, acetylation, pyridoxylation, and protein arginine methylation. Studies of these PTMs revealed their combinatorial effects on the activities of RIP140 with respect to the regulation of hormone target genes and fat accumulation in adipocytes. These proteomic studies have presented evidence for the biological significance of specific PTMs of RIP140, and uncovered nutritional and physiological factors that trigger these PTMs in adipocytes. This could provide insights into potential, new therapeutic targets for diseases concerning adipocytes such as metabolic disorders.

Post-translational Modification of Nuclear Co-repressor Receptor-interacting Protein 140 by Acetylation

Receptor-interacting protein 140 (RIP140) is a versatile co-regulator for nuclear receptors and many transcription factors and contains several autonomous repressive domains. RIP140 can be acetylated, and acetylation affects its biological activity. In this study, a comprehensive proteomic analysis using liquid chromatography-tandem mass spectroscopy was conducted to identify the in vivo acetylation sites on RIP140 purified from Sf21 insect cells. Eight acetylation sites were found within the amino-terminal and the central regions, including Lys111, Lys158, Lys287, Lys311, Lys482, Lys529, Lys607, and Lys932. Reporter assays were conducted to examine the effects of acetylation on various domains of RIP140. Green fluorescent protein-tagged fusion proteins were used to demonstrate the effect on nuclear translocation of these domains. A general inhibitor of reversible protein deacetylation was used to enrich the acetylated population of RIP140. The amino-terminal region (amino acids (aa) 1–495) was more repressive and accumulated more in the nuclei under hyperacetylated conditions, whereas hyperacetylation reduced the repressive activity and nuclear translocation of the central region (aa 336–1006). The deacetylase inhibitor had no effect on the carboxyl-terminal region (aa 977–1161) where no acetylation sites were found. Hyperacetylation also enhanced the repressive activity of the full-length protein but triggered its export into the cytosol in a small population of cells. This study revealed differential effects of post-translational modification on various domains of RIP140 through acetylation, including its effects on repressive activity and nuclear translocation of the full-length protein and its subdomains.

Modulation of lysine acetylation-stimulated repressive activity by Erk2-mediated phosphorylation of RIP140 in adipocyte differentiation

Receptor-interacting protein 140 is a co-regulator for many transcription factors. Previous mass spectrometry studies showed that either phosphorylation or lysine acetylation of RIP140 directly enhanced its trans-repressive activity. In this study, we first identified p300 as a specific lysine acetyltransferase, and extracellular-signal-related kinase 2 (Erk2) as a specific kinase for threonine phosphorylation, of RIP140 in vivo. We further determined two specific acetylated lysine residues (Lys(158)/Lys(287)) and phosphorylated threonine residues (Thr(202)/Thr(207)) that were critical for its gene-repressive activity. We then delineated signal transduction from Erk2-mediated phosphorylation of RIP140 that enhanced its recruiting p300 for subsequent lysine acetylation, and demonstrated the kinetics of activation of this signal transduction pathway in differentiating adipocytes. Finally, the physiological significance of this cell signal transduction pathway was illustrated in rescuing experiments where the defect in fat accumulation of RIP140-null cultures was rescued by re-expressing the wild type RIP140 or its phospho-mimetic mutant, but not its acetylation deficient mutant. These results demonstrate the signal transduction pathway, initiated from Erk2 activation for specific threonine phosphorylation, followed by p300 recruitment for lysine acetylation, which ultimately enhances the gene-repressive activity of RIP140 and its functional role in fat accumulation in differentiated adipocytes.

PKCε Stimulated Arginine Methylation of RIP140 for Its Nuclear-Cytoplasmic Export in Adipocyte Differentiation

Background Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that plays roles in diverse metabolic processes including fat accumulation in adipocytes. Previously we identified three methylated arginine residues in RIP140, which rendered its export to the cytoplasm; but it was unclear what triggered RIP140 arginine methylation. Methodology/Principal Findings In this study, we determined the activated PKCε as the specific trigger for RIP140 arginine methylation and its subsequent export. We identified two PKCε–phosphorylated residues of RIP140, Ser-102 and Ser-1003, which synergistically stimulated direct binding of RIP140 by 14-3-3 that recruited protein arginine methyl transferase 1 to methylate RIP140. The methylated RIP140 then preferentially recruited exportin 1 for nuclear export. As a result, the nuclear gene-repressive activity of RIP140 was reduced. In RIP140 null adipocyte cultures, the defect in fat accumulation was effectively rescued by the phosphoylation-deficient mutant RIP140 that resided predominantly in the nucleus, but less so by the phospho-mimetic RIP140 that was exported to the cytoplasm. Conclusions/Significance This study uncovers a novel means, via a cascade of protein modifications, to inactivate, or suppress, the nuclear action of an important transcription coregulator RIP140, and delineates the first specific phosphorylation-arginine methylation cascade that could alter protein subcellular distribution and biological activity.