Mapping of phosphorylation sites of nuclear corepressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.
Receptor interacting protein (RIP140) is a versatile coregulator for many nuclear receptors and transcription factors. Analysis by liqid chromatography tandem mass spectroscopy led to the identification of 11 phosphopeptides from tryptic digests of His6-RIP140 purified from Sf21 insect cells. No phosphopeptides were detected on RIP140 expressed in E. coli in a parallel experiment, suggesting that RIP140 phosphorylation occurred specifically only in eukaryotic cells. The tandem mass spectra of the precursor ions of the phosphopeptides were analyzed to map the exact phosphorylation sites on RIP140. All the phosphopeptides displayed intact phosphate containing y- or b-ion signals along with their beta-eliminated product ions, due to neutral loss of phosphoric acid. Phosphorylation occurred specifically on nine serine and a single threonine residues, including Ser-104, Thr-207, Ser-358, Ser-380, Ser-488, Ser-519, Ser-531, Ser-543, Ser-672, and Ser-1003. No tyrosine phosphorylation was found. These data suggested that the central region of RIP140, one major repressive domain, was extensively modified by phosphorylation. These phosphorylation sites can be the targets in future studies addressing post-translational modification of RIP140 with regards to its biological activities.
Huq
M D Mostaqul
MD
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, 55455, USA.
Khan
Shaukat Ali
SA
Park
Sung Wook
SW
Wei
Li-Na
LN
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D019476:Insect Proteins / Q000187:drug effects; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D000078723:Nuclear Receptor Interacting Protein 1; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D018411:Spodoptera / Q000737:chemistry / Q000166:cytology; D013912:Threonine / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D019476:Insect Proteins; D009687:Nuclear Proteins; D000078723:Nuclear Receptor Interacting Protein 1; D010748:Phosphopeptides; D012097:Repressor Proteins; D013912:Threonine; D012694:Serine; D014357:Trypsin