Lysine methylation of nuclear co-repressor receptor interacting protein 140.

Receptor interacting protein 140 (RIP140) undergoes extensive post-translational modifications (PTMs), including phosphorylation, acetylation, arginine methylation, and pyridoxylation. PTMs affect its subcellular distribution, protein-protein interaction, and biological activity in adipocyte differentiation. Arginine methylation on Arg(240), Arg(650), and Arg(948) suppresses the repressive activity of RIP140. Here, we find that endogenous RIP140 in differentiated 3T3-L1 cells is also modified by lysine methylation. Three lysine residues, Lys(591), Lys(653), and Lys(757), are mapped as potential methylation sites by mass spectrometry. Site-directed mutagenesis study shows that lysine methylation enhances its gene repressive activity. Mutation of lysine methylation sites enhances arginine methylation, while mutation on arginine methylation sites has little effect on its lysine methylation, suggesting a relationship between lysine methylation and arginine methylation. Kinetic analysis of PTMs of endogenous RIP140 in differentiated 3T3-L1 cells demonstrates sequential modifications on RIP140, initiated from constitutive lysine methylation, followed by increased arginine methylation later in differentiation. This study reveals a potential hierarchy of modifications, at least for lysine and arginine methylation, which bidirectionally regulate the functionality of a nonhistone protein.

Modulation of retinoic acid receptor alpha activity by lysine methylation in the DNA binding domain.

Metabolic labeling and detection with a methylated lysine-specific antibody confirm lysine methylation of RAR alpha in mammalian cells. We previously reported Lys (347) trimethylation of mouse retinoic acid receptor alpha (RAR alpha) in the ligand binding domain (LBD) that affected ligand sensitivity of the dissected LBD. Here we report two monomethylated residues, Lys (109) and Lys (171) identified by LC-ESI-MS/MS in the DNA binding domain (DBD) and the hinge region, which affect retinoic acid (RA) sensitivity, coregulator interaction and heterodimerization with retinoid X receptor (RXR) in the context of the full-length protein. Constitutive negative mutation at Lys (109), but not Lys (171), reduces RA-dependent activation. Methylation at Lys (109) plays a more dominant role than trimethylation at Lys (347) in terms of RA activation of the full-length receptor. Lys (109) is located in a homologous sequence (CEGC K GFFRRS) of the DBD in RARs and is conserved in the nuclear receptor superfamily even across the species boundary. This study uncovers a potential role for monomethylation at Lys (109) in coordinating the synergy between DBD and LBD for ligand-dependent activation of RAR alpha.

PKCepsilon 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 PKCepsilon as the specific trigger for RIP140 arginine methylation and its subsequent export. We identified two PKCepsilon-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 phosphorylation-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.

Vitamin B6 conjugation to nuclear corepressor RIP140 and its role in gene regulation.

Pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6, is an important cofactor in amino acid metabolism, and supplementary vitamin B6 has protective effects in many disorders. Other than serving as a cofactor, it can also modulate the activities of steroid hormone receptors and transcription factors. However, the molecular basis of this modulation is unclear. Here, we report that mouse nuclear receptor interacting protein 140 (RIP140) can be modified by PLP conjugation. We mapped the modification site to Lys613 by LC-ESI-MS/MS analysis. This modification enhanced its transcriptional corepressive activity and its physiological function in adipocyte differentiation. We attribute this effect to increased interaction of RIP140 with histone deacetylases and nuclear retention of RIP140. This study uncovers a new physiological role of vitamin B6 in gene regulation by PLP conjugation to a transcriptional coregulator, which represents a new function of an old form of protein post-translational modification that has important biological consequences.

Lysine trimethylation of retinoic acid receptor-alpha: 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 RARalpha expressed in insect cells, we identified a trimethylation site on Lys(347) in the ligand binding domain. The modification site was verified in mammalian cells, and site-directed mutagenesis studies revealed the functionality of Lys(347) methylation in vivo. Constitutive negative mutants, mimicking hypomethylated RARalpha, were prepared by replacing methylated Lys(347) with either alanine or glutamine. A constitutive positive mutant partially mimicking the hypermethylated RARalpha 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 Lys(347) of RARalpha 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 Lys(347) enhanced molecular interaction of RARalpha 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.

Modulation of testicular receptor 4 activity by mitogen-activated protein kinase-mediated phosphorylation.

Testicular receptor 4 (TR4) is an orphan member of the nuclear receptor superfamily. Despite the lack of identified ligands, its functional role has been demonstrated both in animals and cell cultures. However, it remains unclear how the biological activity of TR4 is regulated without specific ligands. In this study, we showed that in the absence of specific ligands the activity of TR4 could be modulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation of its activation function 1 (AF-1) domain. A mass spectrometry-based proteome analysis of TR4 expressed in insect cells revealed three phosphorylation sites in its AF-1 domain, specifically on Ser(19), Ser(55), and Ser(68). Site-directed mutagenesis studies demonstrated the functionality of phosphorylation on Ser(19) and Ser(68) but not Ser(55). We also demonstrated that MAPK-mediated phosphorylation of the AF-1 domain rendered TR4 a repressor, mediated through the preferential recruitment of corepressor RIP140. Dephosphorylation of its AF-1 made TR4 an activator due to its selective recruitment of coactivator, P300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF). The biological effects were validated by using the wild type TR4 and its constitutive negative (dephosphorylated) and constitutive positive (phosphorylated) mutants in the studies of regulation of its natural target gene, apoE. This study uncovered, for the first time, a ligand-independent mechanism underlying the biological activity of TR4 that was mediated by MAPK-mediated receptor phosphorylation of AF-1 domain.

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 LXRalpha/beta 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.

Ligand-independent orphan receptor TR2 activation by phosphorylation at the DNA-binding domain.

In a previous report we demonstrated protein kinase C (PKC)-mediated phosphorylation of the ligand-binding domain (LBD) of orphan nuclear receptor TR2. In this report, we provide the evidence of PKC-mediated phosphorylation of the DNA-binding domain (DBD) of TR2. Two PKC target sites were predicted within the DBD, at Ser-170 and Ser-185, but only Ser-185 was confirmed by MS. Phosphorylation of DBD facilitated DNA binding of the TR2 receptor and its recruiting of coactivator p300/CBP-associated factor (P/CAF). Ser-185 was required for DNA binding, whereas both Ser-170 and Ser-185 were necessary for receptor interaction with P/CAF. The P/CAF-interacting domain of TR2 was located in its DBD. A double mutant (Ser-170 and Ser-185) of TR2 significantly lowered the activation of its target gene RARbeta2. This study provides the first evidence for ligand-independent activation of TR2 orphan receptor through PTM at the DBD, which enhanced its DNA-binding ability and interaction with coactivator P/CAF.

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.

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.

Mapping of phosphorylation sites of nuclear corepressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.

Receptor interacting protein (RIP140) is a versatile coregulator for many nuclear receptors and transcription factors. Analysis by liqid chromatography tandem mass spectroscopy led to the identification of 11 phosphopeptides from tryptic digests of His6-RIP140 purified from Sf21 insect cells. No phosphopeptides were detected on RIP140 expressed in E. coli in a parallel experiment, suggesting that RIP140 phosphorylation occurred specifically only in eukaryotic cells. The tandem mass spectra of the precursor ions of the phosphopeptides were analyzed to map the exact phosphorylation sites on RIP140. All the phosphopeptides displayed intact phosphate containing y- or b-ion signals along with their beta-eliminated product ions, due to neutral loss of phosphoric acid. Phosphorylation occurred specifically on nine serine and a single threonine residues, including Ser-104, Thr-207, Ser-358, Ser-380, Ser-488, Ser-519, Ser-531, Ser-543, Ser-672, and Ser-1003. No tyrosine phosphorylation was found. These data suggested that the central region of RIP140, one major repressive domain, was extensively modified by phosphorylation. These phosphorylation sites can be the targets in future studies addressing post-translational modification of RIP140 with regards to its biological activities.

Retinoic acid-induced chromatin remodeling of mouse kappa opioid receptor gene.

The mouse kappa opioid receptor (KOR) gene is constitutively expressed in P19 embryonic stem cells but is first suppressed and reactivated during retinoic acid (RA)-induced neuronal differentiation. However, no RA response element (RARE) can be found in this gene regulatory region. The suppression and reactivation of the KOR gene in this neuronal differentiation model suggested chromatin remodeling occurred on this gene promoter triggered by RA induction. This study asks whether RA induces alteration in the nucleosomal structure of this gene promoter that has no apparent RARE and, if so, how RA remodels chromatin of this promoter. The results revealed two loose nucleosomes, N1 at -44 (3' boundary) from the transcription initiation site and N2 spanning the transcription initiation site, that are relevant to active transcription. RA formed a repressive chromatin configuration of this promoter by compacting nucleosome N1, followed by nucleosome N2 condensation. Chromatin immunoprecipitation assay demonstrated RA induced replacement of the c-Myc/Max complex with the Max/Mad1 complex on the E box located within nucleosome N1, coinciding with reduced Sp1 binding to GC boxes located within nucleosome N2 and recruitment of chromatin remodeling factor Brahma-related gene 1 (BRG-1) to this promoter. Consistently, histone deacetylation, Lys9 methylation, and hypophosphorylation of RNA polymerase II C-terminal domain were detected on this promoter after RA treatment. It is concluded that RA induces KOR gene suppression, as early neuronal differentiation marker, by inducing substitution of c-Myc/Max with Max/Mad on the E box and by BRG-1 involved nucleosome recruitment and chromatin condensation, thereby abolishing Sp1 binding.

Tyrosine nitration on p65: a novel mechanism to rapidly inactivate nuclear factor-kappaB.

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)-kappaB 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-kappaB was predominantly nitrated on Tyr residues. Tyr nitration of p65 induced its dissociation from p50, its association with IkappaBalpha, and subsequent sequestration of p65 in the cytoplasm by IkappaBalpha-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-kappaB activity. This study identified a novel and efficient pathway where NO rapidly inactivated NF-kappaB activity by inducing Tyr nitration on p65.