TY - JOUR

T1 - A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1

AU - Blasius, Melanie

AU - Forment, Josep V

AU - Thakkar, Neha

AU - Wagner, Sebastian A

AU - Choudhary, Chuna Ram

AU - Jackson, Stephen P

PY - 2011

Y1 - 2011

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Cell Cycle Proteins

KW - Cell Line

KW - Co-Repressor Proteins

KW - DNA Damage

KW - DNA Replication

KW - Humans

KW - Mitosis

KW - Molecular Sequence Data

KW - Phosphorylation

KW - Protein Kinases

KW - Proteomics

KW - Repressor Proteins

U2 - 10.1186/gb-2011-12-8-r78

DO - 10.1186/gb-2011-12-8-r78

M3 - Journal article

C2 - 21851590

VL - 12

SP - R78

JO - Genome Biology (Online Edition)

JF - Genome Biology (Online Edition)

SN - 1474-7596

IS - 8

ER -