Vinexin β Interacts with the Non-phosphorylated AF-1 Domain of Retinoid Receptor γ (RARγ) and Represses RARγ-mediated Transcription

Abstract

Nuclear retinoic acid receptors (RARs) are ligand-dependent transcription factors that regulate the expression of retinoic acid target genes. Although the importance of RAR phosphorylation in their N-terminal domain is clearly established, the underlying mechanism for the phosphorylation-dependent transcriptional activity of the receptors had not been elucidated yet. Here, using a yeast two-hybrid system, we report the isolation of vinexin ␤ as a new cofactor that interacts with the N-terminal A/B domain of the RAR␥ isotype. Vinexin ␤ is a multiple SH3 motif-containing protein associated with the cytoskeleton and also present in the nucleus. We demonstrate that vinexin ␤ colocalizes with RAR␥ in the nucleus and interacts with the non-phosphorylated form of the AF-1 domain of RAR␥. We also show that this interaction is prevented upon phosphorylation of the AF-1 domain. Using F9 cells stably overexpressing vinexin ␤ or vinexin knockdown by RNA interference, we demonstrate that vinexin ␤ is an inhibitor of RAR␥-mediated transcription. We propose a model in which phosphorylation of the AF-1 domain controls RAR␥-mediated transcription through triggering the dissociation of vinexin ␤.Retinoic acid (RA), 1 the most potent biologically active metabolite of vitamin A, influences the proliferation, differentiation, and apoptosis of a variety of cell types through modifications of expression of subsets of RA target genes (1-3). The effects of RA are mediated by two classes of nuclear receptors, the retinoic acid receptors (RAR␣, RAR␤, and RAR␥) and the retinoid X receptors (RXR␣, RXR␤, and RXR␥), which function as ligand-dependent heterodimeric RAR/RXR transcription activators (4 -6). RARs and RXRs exhibit a conserved modular structure (see Fig. 1A) with a central DNA-binding domain and two activation domains (AF-1 and AF-2) that synergize for the activation of RA target genes.Ligand-induced conformational changes in the AF-2 domain of RARs bound at cognate response elements (RA response elements) located in the promoter of target genes cause the dynamic, coordinated, and combinatorial recruitment of coactivators and large complexes with chromatin-modifying and chromatin-remodeling activity, which will decompact repressive chromatin to allow positioning of the transcription machinery at the promoter (2, 7). Other proteins are also recruited and serve as connections with the transcription machinery. In line with this, RARs interact with the general transcription factor TFIIH (8, 9). This results in the phosphorylation of one residue located in their N-terminal AF-1 domain (Ser 77 in RAR␣1, Ser 79 in RAR␥1, and Ser 68 in RAR␥2) (see Fig. 1A) by the Cdk7 subunit of TFIIH, which has cyclin H-dependent kinase activity. This phosphorylation process, which has been extensively studied especially in the case of RAR␣ (10), plays a critical role in the response to RA.However, in the particular case of the RAR␥ isotype, phosphorylation by TFIIH, although necessary, is not sufficient. Indeed, to be transcriptionally active, RAR␥ needs...

Cyclin H binding to the RARα activation function (AF)-2 domain directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7

“…COS-1 cells were grown and transiently transfected in six-well plates by using DM-RIE-C reagent (Invitrogen) as in ref. 19. Whole-cell extracts were prepared, resolved by 10% SDS͞PAGE, electrotransferred to nitrocellulose membranes, and immunoprobed as described in ref.…”

“…19. Chloramphenicol acetyltransferase (CAT) assays were performed by using the ELISA method (19). Luciferase activities were determined according to standard procedures.…”

“…Whole-cell extracts were prepared, resolved by 10% SDS͞PAGE, electrotransferred to nitrocellulose membranes, and immunoprobed as described in ref. 19. Chloramphenicol acetyltransferase (CAT) assays were performed by using the ELISA method (19).…”

“…19. The primers were as follows: 36B4, 5Ј-GAAGTCACTGTGC-CAGCCCA-3Ј and 5Ј-GAAGGTGTAATCCGTCTCCA-3Ј; RAR␣2, 5Ј-CGAGGGGAAAGATGTACGAG-3Ј and 5Ј-CCTTCTCTGG GAGCAAACAG-3Ј; cyclin H, 5Ј-GCATT-GACGGATGCTTACCT-3Ј and 5Ј-TGACATCGCTCCAACT-TCTG-3Ј; RAR␣1, 5Ј-TGTCTGCCTCCCTTCTGACT-3Ј and 5Ј-GGGGATGGTGTGCTATATCC-3Ј.…”

“…Equimolar amounts of GST and GST fusion proteins expressed in Escherichia coli were purified onto gluthatione-Sepharose beads (Amersham Pharmacia Biosciences) and incubated with recombinant cyclin H, cdk7, MAT1, or the purified cdk7͞cyclin H and CAK complexes in the presence or absence of RA (10 Ϫ7 M) as described in ref. 19. Bound proteins were resolved by SDS͞PAGE and detected by immunoblotting.…”