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.

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.

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. 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 fulllength 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 fulllength protein and its subdomains. Environmental factors in the extracellular milieu utilize signal transduction pathways to propagate their cues into gene expression (1-3). Often the proteins involved in such signaling pathways undergo post-translational modification. A variety of post-translational modifications, including phosphorylation, acetylation, methylation, and glycosylation, regulate protein functions (4 -7). The study of protein function by identification of proteins along with their post-translational modification has been referred to as "functional proteomics" and is an important step in delineating signal transduction pathways. One major challenge is to identify post-translational modifications on these proteins in vivo (8).Receptor-interacting protein 140 (RIP140) 1 is a co-regulator for many transcription factors (9). Nuclear receptors represent the largest group of transcription factors that interact with RIP140 (10 -13). Human RIP140 was initially characterized as a ligand-dependent co-activator for a chimeric estrogen receptor (10). However, the mouse RIP140 cloned in our laboratory with the ligand-binding domain of an orphan nuclear receptor TR2 as the bait was shown to be a potent corepressor for TR2 in the absence of putative ligand (14). Later many researchers including our group reported RIP140 as a suppressor for nuclear hormone receptors and many other transcription factors (15-17). RIP140 is recruited to nuclear receptors through its nine LXXLL motifs and a modified motif of...

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 (HDACsEnvironmental factors in the extracellular milieu utilize signal transduction pathways to propagate their cues into gene expression (1-3). 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 (4 -9). Deciphering such modifications and relating them to the biological function is referred to as functional proteomics (10). A variety of post-translational modifications have been found that regulate protein functions, including phosphorylation, acetylation, methylation, glycosylation, ubiquitination, and sumoylation, etc. (11-14).Receptor interacting protein 140 (RIP140) 1 is a co-regulator for many transcription factors including nuclear receptors (15)(16)(17)(18)(19). 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 (20 -22). RIP140 is recruited to nuclear receptors through its nine LXXLL motifs and a modified motif LXXML, where X can be any amino acids (23,24). With respect to its repressive activity, four autonomous repressive domains (RDs) are known. RD1 is located in the aminoterminal 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 (22, 25). The central region interacts with the carboxyl-terminal binding proteins (CtBP1 and CtBP2) (26). In terms of its physiological action, RIP140-null mice exhibit female reproductive defects (15). Further studies of these animals indicate that RIP140 could play an important role in the regulation of fat accumulation in adipose tissues (27).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 (28) and acetylated (29). 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

BackgroundReceptor 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 FindingsIn 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/SignificanceThis 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.