Lysine trimethylation of retinoic acid receptor-alpha: a novel means to regulate receptor function.
Retinoic acid receptors (RARs) belong to the nuclear receptor superfamily. The mechanism of ligand-dependent activation of RARs is well known. The effect of protein phosphorylation on the activity of RARs has also been demonstrated. However, it is unclear whether other types of modifications exist and if so whether they can affect the activity of RARs. In a mass spectrometric analysis of mouse RARalpha expressed in insect cells, we identified a trimethylation site on Lys(347) in the ligand binding domain. The modification site was verified in mammalian cells, and site-directed mutagenesis studies revealed the functionality of Lys(347) methylation in vivo. Constitutive negative mutants, mimicking hypomethylated RARalpha, were prepared by replacing methylated Lys(347) with either alanine or glutamine. A constitutive positive mutant partially mimicking the hypermethylated RARalpha was generated by replacing the methylated lysine residue with phenylalanine, a bulky hydrophobic amino acid, to introduce a site-specific hydrophobicity similar to that contributed by lysine methylation. Studies of these mutants revealed that trimethylation of Lys(347) of RARalpha facilitated its interactions with cofactors p300/CREB-binding protein-associated factor and receptor-interacting protein 140 as well as its heterodimeric partner retinoid X receptor, suggesting that site-specific hydrophobicity at Lys(347) enhanced molecular interaction of RARalpha with its modulators. This study uncovers the first example of lysine trimethylation on a mammalian non-histone protein that has an important biological consequence. Our finding also provides the evidence for lysine methylation for the family of nuclear receptors for the first time.
Huq
M D Mostaqul
MD
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Tsai
Nien-Pei
NP
Khan
Shaukat Ali
SA
Wei
Li-Na
LN
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D021241:Spectrometry, Mass, Electrospray Ionization; D018411:Spodoptera; D053719:Tandem Mass Spectrometry; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; C000607072:Rara protein, mouse; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D008239:Lysine