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

Huq, M. D. Mostaqul; Li, Wei

doi:10.1074/mcp.m500015-mcp200

Cited by 40 publications

(51 citation statements)

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“…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.…”

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confidence: 99%

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

Gupta

Huq

Khan

et al. 2005

Molecular & Cellular Proteomics

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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

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

Gupta

Huq

Khan

et al. 2005

Molecular & Cellular Proteomics

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“…Moreover, because certain modification sites clearly deviate from the consensus, other factors, such as subcellular environment, local concentration of E3 SUMO ligase activity and/or other covalent modifications, must influence the modification and affect the target site specificity (22). Interestingly, the Lys 111 of RIP140 appears to be acetylated in vivo (17), which may explain its lack of SUMOylation. Overexpression of SUMO E3 ligases PIAS1 and PIAS3 that promoted SUMO-1 modification of wild type RIP140 also brought about weak SUMO-1 modification of the RIP140K756R,K1154R mutant, suggesting that secondary sites, such as Lys 170 , can become SUMOylated under conditions promoting the modification.…”

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confidence: 99%

“…In contrast, arginine methylation at Arg 240 , Arg 650 , and Arg 948 was shown to weaken the corepressor activity because of attenuated recruitment of HDACs and increased nuclear export (16). Huq and Wei identified four acetylation sites in RD1 and three in RD2, and their experiments with an HDAC inhibitor suggested that hyperacetylation enhances the repressive activity of RIP140 (17). According to another study, acetylation of Lys 446 within the N-terminal CtBP-binding motif reduces its interaction with CtBP, thereby attenuating transcription repression by RIP140 (13).…”

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“…RIP140 binds to nuclear receptors through its LXXLL motifs (NR boxes), and because several NR coactivators also possess and utilize the NR boxes, RIP140 may compete with the coactivators of the same binding sites on the ligandbound receptors (2,9). RIP140 has recently been shown to be subject to phosphorylation, arginine methylation, and acetylation (13,(15)(16)(17). Phosphorylation of several sites at the N-terminal region of RIP140 was reported to increase its transrepression activity because of enhanced recruitment of HDACs (15).…”

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SUMOylation Modulates the Transcription Repressor Function of RIP140

Rytinki

Palvimo

2008

Journal of Biological Chemistry

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RIP140/NRIP1 (receptor-interacting protein 140) functions as a corepressor of nuclear receptors. It plays an important role in the transcriptional control of energy metabolism and female fertility. RIP140 contains four distinct repression domains (RD1-RD4), and the repressive activity of RIP140 involves complex mechanisms. The function of both RD1 and RD2 is linked to recruitment of histone deacetylases and C-terminal binding protein, respectively, but the mechanism of repression for RD3 and RD4 has remained elusive. Because covalent modification by small ubiquitin-like modifiers (SUMO-1, -2, and -3; SUMOylation) is often associated with transcriptional repression, we studied whether SUMOylation is involved in the repressive activity of RIP140. We show that two conserved lysines, Lys 756 and Lys 1154 , located in RD3 and RD4, respectively, are subject to reversible SUMOylation, with SUMO-1 being more efficiently conjugated than SUMO-2. Interestingly, mutations of the RIP140 SUMOylation sites compromised the transcription repressor function of RIP140 and blunted its capacity to repress estrogen receptor ␣-dependent transcription. Conjugation of SUMO-1 also influenced the subnuclear distribution pattern of RIP140. In sum, our demonstration that the function of RIP140 repression domains 3 and 4 can be modulated by reversible SUMO modification thus adds a novel level to the regulation of RIP140 activity, which may have ramifications in the control of gene networks exerted by RIP140.RIP140/NRIP1 (receptor-interacting protein 140) is a transcriptional corepressor for nuclear receptors. It appears to be capable of interacting with several, maybe most, members of the nuclear receptor (NR) 2 superfamily, including estrogen receptor (ER), estrogen-related receptors, peroxisome proliferator-activated receptors, glucocorticoid receptors, and thyroid receptors (1-5). RIP140 has an important role in the control of gene networks regulating female fertility and energy homeostasis. Studies with mice lacking RIP140 indicate that the corepressor is necessary for oocyte release during ovulation and for maintaining normal fat accumulation (6 -9). The corepressor function of RIP140 is mediated by four distinct repression domains (RD1-RD4) that are conserved in vertebrates (10, 11). The repression by RD1 involves recruitment of histone deacetylases (HDACs) (11, 12), and RD2 harbors interaction motifs for C-terminal binding protein (CtBP) that in turn may also recruit HDACs (10, 11, 13). However, the function of individual RDs and the role of HDAC activity in the context of whole RIP140 molecule appear to be complex. The HDAC enzymatic activity has been reported to be both dispensable and indispensable for the repressive activity of full-length RIP140 (10 -12, 14). RIP140 binds to nuclear receptors through its LXXLL motifs (NR boxes), and because several NR coactivators also possess and utilize the NR boxes, RIP140 may compete with the coactivators of the same binding sites on the ligandbound receptors (2, 9). RIP140 has recently be...

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“…Site-directed mutagenesis studies demonstrated the functionality of phosphorylation on Ser 19 and Ser 68 but not Ser…”

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Modulation of Testicular Receptor 4 Activity by Mitogen-activated Protein Kinase-mediated Phosphorylation

Huq¹,

Gupta²,

Tsai

et al. 2006

Molecular & Cellular Proteomics

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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 Testicular receptor 4 (TR4)1 (also known as TAK1 and Nr2c2) remains an orphan member among the nuclear receptor superfamily (1, 2). Based upon sequence alignment, TR4 is closely related to some other orphan receptors such as retinoid X receptors, chicken ovalbumin upstream promotertranscription factor, and hepatocyte nuclear factor 4 (3). The expression of TR4 was found to be ubiquitous in both embryonic and adult stages (4 -6). Due to the lack of identified ligands, its physiological roles have been speculated. However, recent gene knock-out studies revealed its potential roles in animal behavior, coordination, and reproduction (7-9).In classical studies, it was shown that TR4 could bind to direct repeat (DR) AGGTCA with variable spacer nucleotides in the promoters of putative target genes (10 -13). These target genes could be activated or repressed. Among the potential target sequences, the DR1 sequence of the apolipoprotein E/C-I/C-II gene cluster was confirmed in the TR4 gene knock-out studies (13). In addition, TR4 was also shown to be an important regulator of other nuclear receptor signaling pathways, such as in pathways involving retinoic acid receptor, retinoid X receptor, peroxisome proliferator-activated receptor (PPAR), vitamin D 3 receptor, thyroid hormone receptor, estrogen receptor, and another closely related orphan member, TR2 (14). Together these studies suggested TR4 as an important member of nuclear receptors. Despite the widely demonstrated transcriptional regulatory activity of TR4, it remains unclear how the activity of TR4, as an activator or a repressor, is triggered without the involvement of specific ligands.Our recent proteomics studies of another orphan receptor, TR2, revealed interesting regulatory mechanisms mediated by receptor phosphorylation that could modulate its biological activities (15, 16). We thus set out to determine whether TR4 could also be regulated by protein modification such as phosphorylation. A systematic mass spectrometry-based proteomics analysis of TR4 was initiated by examining modifications of TR4 protein expressed in insect cells, a widely used eukaryotic system for protein expression. In this endeavor, we identified three mitogen-activated protein kinase-mediated phosphorylation sites on the activation function 1 (AF-1) domain of TR4. To validate the biological effects of these modified residue...

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

Cited by 40 publications

References 42 publications

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

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

SUMOylation Modulates the Transcription Repressor Function of RIP140

Modulation of Testicular Receptor 4 Activity by Mitogen-activated Protein Kinase-mediated Phosphorylation

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