Quantitative analysis of the human spindle phosphoproteome at distinct mitotic stages

J Proteome Res. 2009 Oct;8(10):4553-63. doi: 10.1021/pr9003773.

Abstract

During mitosis, phosphorylation of spindle associated proteins is a key regulatory mechanism for spindle formation, mitotic progression, and cytokinesis. In the recent past, mass spectrometry has been applied successfully to identify spindle proteomes and phosphoproteomes, but did not address their dynamics. Here, we present a quantitative comparison of spindle phosphoproteomes prepared from different mitotic stages. In total, we report the identification and SILAC based relative quantitation of 1940 unique phosphorylation sites and find that late mitosis (anaphase, telophase) is correlated with a drastic alteration in protein phosphorylation. Further statistical cluster analyses demonstrate a strong dependency of phosphorylation dynamics on kinase consensus patterns, thus, linking subgroups of identified phosphorylation sites to known key mitotic kinases. Surprisingly, we observed that during late mitosis strong dephosphorylation occurred on a significantly larger fraction of phospho-threonine than phospho-serine residues, suggesting a substrate preference of phosphatases for phospho-threonine at this stage. Taken together, our results constitute a large quantitative data resource of phosphorylation abundances at distinct mitotic stages and they provide insight into the systems properties of phosphorylation dynamics during mitosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cluster Analysis
  • Consensus Sequence
  • Culture Media
  • HeLa Cells
  • Humans
  • Mitosis / physiology*
  • Phosphoproteins / analysis*
  • Phosphorylation
  • Phosphoserine
  • Phosphothreonine
  • Phosphotransferases
  • Proteome / analysis*
  • Reproducibility of Results
  • Spindle Apparatus / metabolism*
  • Time Factors

Substances

  • Culture Media
  • Phosphoproteins
  • Proteome
  • Phosphothreonine
  • Phosphoserine
  • Phosphotransferases