LIM protein Ajuba functions as a nuclear receptor corepressor and negatively regulates retinoic acid signaling

Abstract

Corepressors play an essential role in nuclear receptor-mediated transcriptional repression. In general, corepressors directly bind to nuclear receptors via CoRNR boxes (L/I-X-X-I/V-I) in the absence of ligand and appear to act as scaffolds to further recruit chromatin remodeling complexes to specific target genes. Here, we describe the identification of the multiple LIM domain protein Ajuba as a unique corepressor for a subset of nuclear hormone receptors. Ajuba contains functional nuclear-receptor interacting motifs and selectively interacts with retinoic acid receptors (RARs) and rexinoid receptor (RXRs) subtypes in a ligand-dependent manner. Simultaneous mutation of these motifs abolishes RAR binding and concomitantly leads to loss of repression on RARE reporter genes. P19 cells depleted of Ajuba are highly sensitized to all-trans retinoic acid (atRA)-induced transcription and differentiation. In the absence of atRA, Ajuba can be readily found at the RARE control elements of RAR endogenous target genes. Stimulation of cells with atRA results in the dissociation of Ajuba from these regions. Moreover, we observed that coexpression of the known Ajuba binding partner Prmt5 (protein arginine methyltransferase-5) inhibited the Ajuba/RAR interaction. The high-affinity Ajuba-RAR/RXR interaction site overlaps the region responsible for Ajuba/Prmt5 binding, and thus binding appears to be mutually exclusive, providing a potential mechanism for these observations. Identification of Ajuba as a unique corepressor for nuclear receptors sheds new light on mechanisms for nuclear receptor-mediated repression and provides a unique target for developing more effective therapeutics to modulate this important pathway.

Fig. 1.

Ajuba interacts with RARα. (A) Ajuba contains conserved nuclear-receptor binding motifs. Ajuba is characterized by the C-terminal tandem LIM domains and the glycine-proline rich N-terminal preLIM regions. The potential nuclear-receptor motifs are designated as NR1 to NR4. The second motif, NR2, serves as the Prmt5 binding site. The numbers correspond to the locations of the amino acid residues within the Ajuba protein. (B) Ajuba interacts with RARα when coexpressed in 293 cells. Myc-Ajuba and Flag-RARα were transiently expressed in 293 cells. Whole-cell lysates were immunoprecipitated with α-Myc antibody and Western blot was performed with α-Flag antibody. *, nonspecific band. (C) The endogenous Ajuba and RARα proteins interact in P19 cells. Nuclear extracts were prepared from exponentially growing P19 cells treated with/without atRA (1μM) for 8 h and were precleared with protein A/G beads. Coimmunoprecipitation assays were carried out with α-RARα antibody, and rabbit IgG was used a control. Western blot was performed with an α-Ajuba antibody. (D) In vitro interaction of Ajuba and RARα. Bacterially expressed and purified full-length GST-RARα and in vitro-translated full-length Ajuba, which was labeled with S³⁵, were used for in vitro binding assays. atRA were added at concentration of 1 μM, 10 μM, or 100 μM into the reactions and incubated for 1 h.

Fig. 2.

Ajuba selectively interacts with nuclear receptor subtypes. (A) Ajuba interacts with RARα, RARγ, and RXRγ when coexpressed in 293 cells. *, nonspecific band. (B) Diagram shows the Ajuba/Zyxin family members. (C) RARα interacts with Ajuba, Limd1, and WTIP. (D) The interaction between Ajuba and RARα, RARγ, and RXRγ were attenuated by atRA treatment. (E) The interaction between RARα and Ajuba, Limd1, and WTIP were also attenuated by atRA. Plasmids were transiently transfected into 293 cells. Transfected cells were treated with atRA at 1 μM for 24 h before harvest.

Fig. 3.

The putative nuclear receptor binding motifs are necessary for RARα binding. (A) A diagram shows the Ajuba mutants. Ajuba mutants were generated from full-length Ajuba cDNA tagged with 6× Myc epitopes at the N terminus using a site-directed mutagenesis method. (B) Simultaneous mutations of the nuclear receptor binding motifs NR2, -3, and -4 abolish its interaction with RARα. Plasmids were transiently transfected into 293 cells, and co-IP assays were performed using an α−Flag antibody. Western blot was performed using an α-Myc antibody. (C) Colocalization of Ajuba and RARα in P19 cells. Cells were treated with atRA at 1 μM for 2 h, fixed, and immunofluorecent assays were performed. The images were taken using confocal microscopy.

Fig. 4.

Ajuba negatively regulates the RA target gene in P19 cells. (A) Ajuba represses the activity of a RARE-driven promoter. The luciferase reporter construct driven by RAREs (DR5-Luc) and Ajuba were cotransfected into P19 cells and the resulting luciferase activity was normalized to β-galactosidase activity. (B) The Ajuba mutant failed to interact with RARα and loses repression to DR5-luc. (C) Similar to Ajuba, Limd1 and WTIP repress DR5-Luc activity. (D) Western blot shows shRNA-mediated depletion of Ajuba protein in P19 cells. P19 cells were infected with viral vectors containing shRNAs targeting murine Ajuba or luciferase genes and the stable cells were established using puromycin. (E) Dose-response of P19 cells to atRA. Exponentially growing cells were treated with atRA at different doses in media containing 10% FBS for 24 h and total mRNA was extracted. The expression of the RA target genes was determined using RT-PCR. The PCR-amplification for Hox genes is 30 cycles, and 24 cycles for GAPDH. (F) PCR analysis of the immunoprecipitated DNA fragments from P19 cells. ChIP assays were performed in the P19-siLuc and P19-siAjuba cells treated with atRA at 1 μM or DMSO for 8 h. The primers flanking the RAREs in Hoxa1 and Hoxb1 were used for PCR amplifications.

Fig. 5.

Modulation of Ajuba in P19 cells results in the cells sensitized to atRA-induced differentiation. (A) atRA induces differentiation in P19-siLuc and P19-siAjuba cells. (B) Immunofluorecent assays show the expression of TROMA-1 in P19 cells. (C) Western blot shows the induction of TROMA-1 in P19 cells in the presence or absence of Ajuba. (D) Temporal expression of TROMA-1 in P19 cell variants stimulated with atRA.

Fig. 6.

Prmt5 inhibits the interaction between Ajuba and RARα. (A) Prmt5 inhibits the repression of Ajuba on DR5-Luc activity. (B) Prmt5 inhibits the interaction between Ajuba and RARα when coexpressed in 293 cells. (C) In vitro competition assays between Prmt5 and RARα. Bacterially expressed and purified GST-Ajuba (244–350aa) and in vitro-translated Prmt5 and RARα were used for in vitro binding assays. (D) Western blots show the patterns of the RARα, Ajuba or Prmt5 proteins eluted from superose-6 sizing column. Whole-cell extract (8 mg) was prepared from 293 cells overexpressing Myc-Ajuba, Flag- RARα, and Flag-Prmt5 and was loaded onto a superpose-6 gel-filtration column. (E) Model illustrating the roles of Ajuba in retinoic acid-mediated gene expression.