Acetylation of nuclear hormone receptor-interacting protein RIP140 regulates binding of the transcriptional corepressor CtBP - PubMed (original) (raw)

Acetylation of nuclear hormone receptor-interacting protein RIP140 regulates binding of the transcriptional corepressor CtBP

N Vo et al. Mol Cell Biol. 2001 Sep.

Abstract

CtBP (carboxyl-terminal binding protein) participates in regulating cellular development and differentiation by associating with a diverse array of transcriptional repressors. Most of these interactions occur through a consensus CtBP-binding motif, PXDLS, in the repressor proteins. We previously showed that the CtBP-binding motif in E1A is flanked by a Lys residue and suggested that acetylation of this residue by the p300/CBP-associated factor P/CAF disrupts the CtBP interaction. In this study, we show that the interaction between CtBP and the nuclear hormone receptor corepressor RIP140 is regulated similarly, in this case by p300/CBP itself. CtBP was shown to interact with RIP140 in vitro and in vivo through a sequence, PIDLSCK, in the amino-terminal third of the RIP140 protein. Acetylation of the Lys residue in this motif, demonstrated in vivo by using an acetylated RIP140-specific antibody, dramatically reduced CtBP binding. Mutation of the Lys residue to Gln resulted in a decrease in CtBP binding in vivo and a loss of transcriptional repression. We suggest that p300/CBP-mediated acetylation disrupts the RIP140-CtBP complex and derepresses nuclear hormone receptor-regulated genes. Disruption of repressor-CtBP interactions by acetylation may be a general mode of gene activation.

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Figures

FIG. 1

FIG. 1

RIP140 contains a consensus CtBP-binding site. The nine LXXLL motifs present in RIP140 are represented by the vertical bars. The fragment of RIP140 (aa 403 to 521) isolated from an E9.5 mouse embryo cDNA library is indicated by the gray bar. Fragments of RIP140 used in GST pull-down assays are also depicted. Mutations in the consensus CtBP-binding motif (aa 440 to 446) are indicated.

FIG. 2

FIG. 2

RIP140 binds to hCtBP1 in vitro. (A) Equimolar amounts of GST-RIP140 (1-495), GST-RIP140 (623-951), GST-RIP140 (977-1158), and GST alone were incubated with in vitro-translated hCtBP1. The samples were electrophoresed on a 10% polyacrylamide gel and analyzed by autoradiography. (B) Mutation of the consensus hCtBP1 binding motif abolishes the RIP140-hCtBP1 interaction. Equimolar concentrations of GST-RIP140 (1-495) wild type (WT), PIDL→AIAL, PIDL→AAAA, and GST alone were incubated with recombinant, His-tagged hCtBP1. The samples were electrophoresed on a 10% polyacrylamide gel and analyzed by Western blotting using a monoclonal His tag antibody.

FIG. 3

FIG. 3

RIP140 interacts with hCtBP1 in vivo. COS7 cells were transiently transfected (+) or mock transfected (−) with FLAG-hCtBP1. Immunoprecipitations were performed using an M2 FLAG affinity matrix. Western analyses were performed using a polyclonal RIP140 (left panel) or FLAG (right panel) antibody, as indicated at the top of the figure.

FIG. 4

FIG. 4

RIP140 is acetylated at lysine 446. (A) Eleven nanomoles of recombinant GST-RIP140 (1-495) wild type or K446Q mutant was incubated with full-length, baculovirus-purified p300 (1 pmol) in the presence of [3H]AcCoA. Reactions were analyzed by SDS-PAGE and visualized by fluorography. (B) Recombinant GST-RIP140 (1-495) (25 and 300 ng) was acetylated or mock acetylated with p300. Samples were electrophoresed on a 10% polyacrylamide gel and subjected to Western analysis using an antibody directed against acetylated (top) or unacetylated (bottom) RIP140. (C) In the top panels, endogenous RIP140 was immunoprecipitated from COS7 and HepG2 cells with a RIP140 antibody and subjected to Western analysis using the acetylated K446 RIP140 antibody. Middle panels show cells transfected with full-length CBP before immunoprecipitation. Total RIP140 proteins immunoprecipitated from these cells are shown in the bottom panels.

FIG. 5

FIG. 5

Acetylation of RIP140 disrupts its interaction with hCtBP1. (A) Mutation or acetylation of K446 disrupts the interaction of RIP140 with hCtBP1. GST pull-down assays were performed using acetylated or mock-acetylated GST-RIP140 (1-495) wild type (WT) or K446Q. Samples were incubated with in vitro-translated hCtBP1, electrophoresed on a polyacrylamide gel, and analyzed by autoradiography. (B) Peptide competition assays. GST-RIP140 (1-495) was incubated with recombinant, His-tagged hCtBP1 (11.6 nmol) in the presence or absence of 20 or 40 μM nonacetylated (NonAc) or acetylated (Ac) peptides. Bound hCtBP1 was analyzed by Western blotting using a His tag antibody. −, binding in the absence of peptide competitor. (C) K446Q mutation attenuates the RIP140-hCtBP1 interaction in a mammalian two-hybrid assay. Gal4-RIP140 (386-470) and VP16-hCtBP1 were cotransfected with either (Gal4)5-E1b-luc or (Gal4)5-TK-luc reporter constructs into HepG2 cells. Luciferase activity was measured 48 h posttransfection.

FIG. 6

FIG. 6

RIP140 represses transcription by binding to hCtBP1. Gal4-RIP140 fusion protein containing the CtBP interaction motif was analyzed for the ability to repress a Gal4-SV40-CAT reporter. The wild type (WT) and the K446Q and AIAL mutants were compared to Gal4-ZEB (700-776). COS7 and HepG2 cells were transiently transfected with 300 ng of reporter, 700 ng of Gal4 fusion constructs, and 20 ng of SV40-lacZ. CAT protein produced in the absence of repressor (−) is indicated. The total amount of DNA was kept constant using empty pcDNA3 vector. Protein levels of the RIP140 constructs are shown in the insets. CAT protein production was measured 48 h after transfection using an enzyme-linked immunosorbent assay system (Roche Molecular Biochemicals), and values are normalized to β-galactosidase activity.

FIG. 7

FIG. 7

RIP140 represses estrogen receptor-dependent transcription. The ability of full-length RIP140, wild type and mutants, to repress transcription of an (ERE)2-pS2-CAT reporter gene was analyzed in HepG2 cells treated with 10 nM estradiol. Cells were transfected with 100 ng of CAT reporter, 20 ng of SV40-lacZ, and 100 ng of pEFRIP140 wild type and mutants in the absence or presence of pCMV-hCtBP1. The total concentration of DNA was kept constant by addition of empty pcDNA3 vector. CAT protein production was measured 48 h after transfection, and values are normalized to β-galactosidase activity.

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