TFIIH Interacts with the Retinoic Acid Receptor γ and Phosphorylates Its AF-1-activating Domain through cdk7

Julie Bastien,Sylvie Adam-Stitah,Thilo Riedl,Cécile Rochette-Egly,Pierre Chambon,Jean-Marc Egly

doi:10.1074/jbc.m001985200

Abstract

Retinoic acid receptor gamma (RARgamma) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RARgamma1 and serines 66 and 68 in RARgamma2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7.cyclin H. MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RARgamma is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RARgamma phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RARgamma transcriptional activity in a response gene-dependent manner.

Highlights

The pleiotropic effects of retinoids are transduced by two nuclear receptor families, the retinoic acid receptors (RARs)1 and the retinoid X receptors (RXRs), that are ligand-dependent transregulators belonging to the nuclear receptor superfamily [1,2,3,4]
Two-dimensional phosphoamino acid and tryptic phosphopeptide separations were carried out on thin layer cellulose plates using the Hunter thin-layer electrophoresis (HTLE) system as described [15]. Both Human and Mouse RAR␥ Overexpressed in COS-1 Cells Are Phosphorylated in Their N-terminal A/B Region, whereas Mouse RAR␥ Is Phosphorylated in Its F Region—To determine whether wild type human Retinoic acid receptor ␥ (RAR␥)1 is a phosphoprotein, COS-1 cells were transfected with the corresponding expression vector and labeled with [32P]orthophosphate in the absence or presence of RA (10Ϫ7 M)
RAR␥ Is Phosphorylated in Vitro by cdk7 Present in the CAK Complex and TFIIH—We investigated whether like RAR␣ [14], RAR␥ could be phosphorylated by cdk7 contained in either CAK or the general transcription/DNA repair factor TFIIH [17]

Summary

Introduction

The pleiotropic effects of retinoids are transduced by two nuclear receptor families, the retinoic acid receptors (RARs)1 and the retinoid X receptors (RXRs), that are ligand-dependent transregulators belonging to the nuclear receptor superfamily [1,2,3,4]. Transfection in COS-1 cells of a chimeric construct expressing the A/B region of hRAR␥1 fused to the DNA binding domain (C) of the human estrogen receptor (ER) showed that the corresponding chimeric protein, hRAR␥1(AB)ER(C), was phosphorylated (Fig. 2A, lane 8) and yielded the three phosphopeptides a, b, and c (Fig. 2C, panel 2).

Results

Conclusion