A Phospho-Proteomic Screen Identifies Substrates of the Checkpoint Kinase Chk1

Genome Biol. 2011 Aug 18;12(8):R78. doi: 10.1186/gb-2011-12-8-r78.

Abstract

Background: The cell-cycle checkpoint kinase Chk1 is essential in mammalian cells due to its roles in controlling processes such as DNA replication, mitosis and DNA-damage responses. Despite its paramount importance, how Chk1 controls these functions remains unclear, mainly because very few Chk1 substrates have hitherto been identified.

Results: Here, we combine a chemical genetics approach with high-resolution mass spectrometry to identify novel Chk1 substrates and their phosphorylation sites. The list of targets produced reveals the potential impact of Chk1 function not only on processes where Chk1 was already known to be involved, but also on other key cellular events such as transcription, RNA splicing and cell fate determination. In addition, we validate and explore the phosphorylation of transcriptional co-repressor KAP1 Ser473 as a novel DNA-damage-induced Chk1 site.

Conclusions: By providing a substantial set of potential Chk1 substrates, we present opportunities for studying unanticipated functions for Chk1 in controlling a wide range of cellular processes. We also refine the Chk1 consensus sequence, facilitating the future prediction of Chk1 target sites. In addition, our identification of KAP1 Ser473 phosphorylation as a robust readout for Chk1 activity could be used to explore the in vivo effects of Chk1 inhibitors that are being developed for clinical evaluation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line
  • Checkpoint Kinase 1
  • Co-Repressor Proteins / genetics
  • Co-Repressor Proteins / metabolism
  • DNA Damage
  • DNA Replication
  • Humans
  • Mitosis
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Kinases / genetics*
  • Protein Kinases / metabolism*
  • Proteomics / methods*
  • Repressor Proteins / metabolism
  • Tripartite Motif-Containing Protein 28

Substances

  • Cell Cycle Proteins
  • Co-Repressor Proteins
  • Repressor Proteins
  • TRIM28 protein, human
  • Tripartite Motif-Containing Protein 28
  • Protein Kinases
  • CHEK1 protein, human
  • Checkpoint Kinase 1