Mapping of phosphorylation sites of nuclear corepressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy

M. D Mostaqul Huq, Shaukat Ali Khan, Sung Wook Park, Li-Na Wei

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

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 RIP 140. 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.

Original languageEnglish (US)
Pages (from-to)2157-2166
Number of pages10
JournalProteomics
Volume5
Issue number8
DOIs
StatePublished - May 2005

Keywords

  • Gene regulation
  • Mass spectroscopy
  • Phosphorylation
  • Receptor interacting protein 140

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