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Mapping of phosphorylation sites of nuclear corepressor receptor interacting protein 140 by liquid chromatography‐tandem mass spectroscopy
Author(s) -
Mostaqul Huq M. D.,
Khan Shaukat Ali,
Park Sung Wook,
Wei LiNa
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401090
Subject(s) - corepressor , phosphorylation , chemistry , tandem , mass spectrometry , tandem mass spectrometry , chromatography , nuclear receptor , computational biology , biology , biochemistry , materials science , transcription factor , composite material , gene
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 β‐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.