Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem-loop-binding proteins that contributes to high-affinity RNA binding

Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3094-9. doi: 10.1073/pnas.0511289103. Epub 2006 Feb 21.

Abstract

The stem-loop-binding protein (SLBP) is involved in multiple aspects of histone mRNA metabolism. To characterize the modification status and sites of SLBP, we combined mass spectrometric bottom-up (analysis of peptides) and top-down (analysis of intact proteins) proteomic approaches. Drosophilia SLBP is heavily phosphorylated, containing up to seven phosphoryl groups. Accurate M(r) determination by Fourier transform ion cyclotron resonance (FTICR)-MS and FTICR-MS top-down experiments using a variety of dissociation techniques show there is removal of the initiator methionine and acetylation of the N terminus in the baculovirus-expressed protein, and that T230 is stoichiometrically phosphorylated. T230 is highly conserved; we have determined that this site is also completely phosphorylated in baculovirus-expressed mammalian SLBP and extensively phosphorylated in both Drosophila and mammalian cultured cells. Removal of the phosphoryl group from T230 by either dephosphorylation or mutation results in a 7-fold reduction in the affinity of SLBP for the stem-loop RNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster / chemistry
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism
  • Humans
  • Mass Spectrometry
  • Molecular Sequence Data
  • Molecular Weight
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Nucleic Acid Conformation
  • Phosphorylation
  • Phosphothreonine / metabolism
  • Protein Binding
  • Proteomics / methods*
  • RNA / chemistry
  • RNA / metabolism*
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Spectroscopy, Fourier Transform Infrared
  • mRNA Cleavage and Polyadenylation Factors / chemistry*
  • mRNA Cleavage and Polyadenylation Factors / genetics
  • mRNA Cleavage and Polyadenylation Factors / metabolism*

Substances

  • Drosophila Proteins
  • Nuclear Proteins
  • RNA-Binding Proteins
  • SLBP protein, Drosophila
  • SLBP protein, human
  • mRNA Cleavage and Polyadenylation Factors
  • Phosphothreonine
  • RNA