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1.
EMBO J ; 28(1): 34-47, 2009 Jan 07.
Article in English | MEDLINE | ID: mdl-19078967

ABSTRACT

The nuclear retinoic acid (RA) receptor alpha (RARalpha) is a transcriptional transregulator that controls the expression of specific gene subsets through binding at response elements and dynamic interactions with coregulators, which are coordinated by the ligand. Here, we highlighted a novel paradigm in which the transcription of RARalpha target genes is controlled by phosphorylation cascades initiated by the rapid RA activation of the p38MAPK/MSK1 pathway. We demonstrate that MSK1 phosphorylates RARalpha at S369 located in the ligand-binding domain, allowing the binding of TFIIH and thereby phosphorylation of the N-terminal domain at S77 by cdk7/cyclin H. MSK1 also phosphorylates histone H3 at S10. Finally, the phosphorylation cascade initiated by MSK1 controls the recruitment of RARalpha/TFIIH complexes to response elements and subsequently RARalpha target gene activation. Cancer cells characterized by a deregulated p38MAPK/MSK1 pathway, do not respond to RA, outlining the essential contribution of the RA-triggered phosphorylation cascade in RA signalling.


Subject(s)
Gene Expression Regulation , Promoter Regions, Genetic , Receptors, Retinoic Acid/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Line , Cyclin-Dependent Kinases/metabolism , Histones/metabolism , Mice , Models, Biological , Phosphorylation , Protein Binding , Retinoic Acid Receptor alpha , Transcription Factor TFIIH/metabolism
2.
Proc Natl Acad Sci U S A ; 103(25): 9548-53, 2006 Jun 20.
Article in English | MEDLINE | ID: mdl-16769902

ABSTRACT

Nuclear retinoic acid receptors (RARs) work as ligand-dependent heterodimeric RAR/retinoid X receptor transcription activators, which are targets for phosphorylations. The N-terminal activation function (AF)-1 domain of RARalpha is phosphorylated by the cyclin-dependent kinase (cdk) 7/cyclin H complex of the general transcription factor TFIIH and the C-terminal AF-2 domain by the cAMP-dependent protein kinase A (PKA). Here, we report the identification of a molecular pathway by which phosphorylation by PKA propagates cAMP signaling from the AF-2 domain to the AF-1 domain. The first step is the phosphorylation of S369, located in loop 9-10 of the AF-2 domain. This signal is transferred to the cyclin H binding domain (at the N terminus of helix 9 and loop 8-9), resulting in enhanced cyclin H interaction and, thereby, greater amounts of RARalpha phosphorylated at S77 located in the AF-1 domain by the cdk7/cyclin H complex. This molecular mechanism relies on the integrity of the ligand-binding domain and the cyclin H binding surface. Finally, it results in higher DNA-binding efficiency, providing an explanation for how cAMP synergizes with retinoic acid for transcription.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Receptors, Retinoic Acid/metabolism , Amino Acid Sequence , Animals , Cell Line , Chlorocebus aethiops , Cyclin H , DNA/metabolism , Dimerization , Humans , Models, Molecular , Molecular Sequence Data , Phosphorylation , Phosphoserine/metabolism , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/genetics , Retinoic Acid Receptor alpha , Signal Transduction
3.
Cell Signal ; 17(10): 1229-39, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16038797

ABSTRACT

The retinoid response is mediated by two classes of nuclear receptors, the retinoic acid receptors (RARalpha, beta, and gamma) and the retinoid X receptors (RXRalpha, beta, and gamma) which act as ligand-dependent heterodimeric RAR/RXR transcription activators. Like most transcription factors, RARs and RXRs are regulated by phosphorylation processes. Here, we report that stress agents induce RXRalpha phosphorylation, subsequently to the activation of the stress-activated protein kinases cascade (JNKs). This phosphorylation process concerns three residues located in the N-terminal AF-1 domain of RXRalpha and one located in the omega loop of the Ligand Binding Domain. To decipher how stress-induced RXRalpha phosphorylation influences the transcription of RA-target genes, we used a ribotoxic stress agent, anisomycin, which activates signaling kinases without promoting DNA or protein damages, at subinhibitory concentrations. Taking advantage of vectors expressing recombinant RXRalpha mutated at its phosphorylation sites and of F9 cell lines re-expressing the same RXRalpha mutants in an RXRalpha null background, we provide evidence that stress signaling modulates RAR/RXRalpha-mediated transcription, through the phosphorylation of RXRalpha at the residue located in the Omega loop, in a promoter context-dependent manner.


Subject(s)
Promoter Regions, Genetic/genetics , Retinoid X Receptor alpha/physiology , Tretinoin/pharmacology , Animals , Anisomycin/pharmacology , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Cytochrome P-450 Enzyme System/genetics , Gene Expression/drug effects , Gene Expression/genetics , Gene Expression Regulation , Homeodomain Proteins/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mutation , Phosphorylation/drug effects , Receptors, Retinoic Acid/agonists , Receptors, Retinoic Acid/genetics , Retinoic Acid 4-Hydroxylase , Retinoic Acid Receptor alpha , Retinoid X Receptor alpha/agonists , Retinoid X Receptor alpha/metabolism , Serine/metabolism , Signal Transduction/physiology , Transcription Factors/genetics , Transfection , p38 Mitogen-Activated Protein Kinases/metabolism , Retinoic Acid Receptor gamma
4.
J Biol Chem ; 280(17): 17027-37, 2005 Apr 29.
Article in English | MEDLINE | ID: mdl-15734736

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 beta as a new cofactor that interacts with the N-terminal A/B domain of the RARgamma isotype. Vinexin beta is a multiple SH3 motif-containing protein associated with the cytoskeleton and also present in the nucleus. We demonstrate that vinexin beta colocalizes with RARgamma in the nucleus and interacts with the non-phosphorylated form of the AF-1 domain of RARgamma. We also show that this interaction is prevented upon phosphorylation of the AF-1 domain. Using F9 cells stably overexpressing vinexin beta or vinexin knockdown by RNA interference, we demonstrate that vinexin beta is an inhibitor of RARgamma-mediated transcription. We propose a model in which phosphorylation of the AF-1 domain controls RARgamma-mediated transcription through triggering the dissociation of vinexin beta.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Muscle Proteins/metabolism , Receptors, Retinoic Acid/chemistry , Transcription, Genetic , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , COS Cells , Cell Nucleus/metabolism , Chloramphenicol O-Acetyltransferase/metabolism , Cloning, Molecular , DNA, Complementary/metabolism , Glutathione Transferase/metabolism , Humans , Immunoprecipitation , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Muscle Proteins/chemistry , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , RNA/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Time Factors , Transcriptional Activation , Transfection , Tretinoin/metabolism , Two-Hybrid System Techniques , beta-Galactosidase/metabolism , src Homology Domains , Retinoic Acid Receptor gamma
5.
Oncogene ; 24(14): 2277-88, 2005 Mar 31.
Article in English | MEDLINE | ID: mdl-15688020

ABSTRACT

Arsenite trioxide (As2O3) induces apoptosis in several cell lines by disturbing key signal transduction pathways through its oxidative properties. Here, we report that As2O3 also induces the phosphorylation of the retinoid receptor RXRalpha, subsequent to oxidative damages and the activation of the stress-activated protein kinases cascade (JNKs). We also report that RA amplifies both As2O3-induced phosphorylation of RXRalpha and apoptosis. Taking advantage of 'rescue' F9 cell lines expressing RXRalpha mutated at its phosphorylation sites, in an RXRalpha null background, we provide evidence that RXRalpha is a key element involved in that potentiating effect. Finally, we demonstrate that As2O3 also abrogates the transactivation of RA-target genes.


Subject(s)
Apoptosis/drug effects , Arsenicals/pharmacology , Oxides/pharmacology , Retinoid X Receptor alpha/metabolism , Tretinoin/pharmacology , Animals , Arsenic Trioxide , Base Sequence , Cell Line , Cell Line, Tumor , DNA Primers , Dimerization , Mice , Phosphorylation , Plasmids , Reverse Transcriptase Polymerase Chain Reaction
6.
EMBO J ; 21(14): 3760-9, 2002 Jul 15.
Article in English | MEDLINE | ID: mdl-12110588

ABSTRACT

The nuclear retinoic acid receptor RAR gamma 2 undergoes proteasome-dependent degradation upon ligand binding. Here we provide evidence that the domains that signal proteasome-mediated degradation overlap with those that activate transcription, i.e. the activation domains AF-1 and AF-2. The AF-1 domain signals RAR gamma 2 degradation through its phosphorylation by p38MAPK in response to RA. The AF-2 domain acts via the recruitment of SUG-1, which belongs to the 19S regulatory subunit of the 26S proteasome. Blocking RAR gamma 2 degradation through inhibition of either the p38MAPK pathway or the 26S proteasome function impairs its RA-induced transactivation activity. Thus, the turnover of RAR gamma 2 is linked to transactivation.


Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Receptors, Retinoic Acid/metabolism , Transcription Factors , Transcriptional Activation/drug effects , Tretinoin/pharmacology , ATPases Associated with Diverse Cellular Activities , Animals , COS Cells , Cysteine Endopeptidases/metabolism , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins , Ligands , Multienzyme Complexes/metabolism , Phosphorylation , Proteasome Endopeptidase Complex , Receptors, Retinoic Acid/genetics , Ubiquitin/metabolism , p38 Mitogen-Activated Protein Kinases , Retinoic Acid Receptor gamma
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