Exp Mol Med.  2007 Apr;39(2):205-212.

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif

• Kim MA
• Kim HJ
• Brown AL
• Lee MY
• Bae YS
• Park JI
• Kwak JY
• Chung JH
• Yun J

Abstract

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1alpha were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1alpha were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.

Keyword

checkpoint kinase 1; checkpointkinase 2; DNA damage; phosphorylation; substrate specificity

MeSH Terms

Amino Acid Motifs
Amino Acid Sequence
*Consensus Sequence
Genome, Human/*genetics
Heat-Shock Proteins/chemistry/metabolism
Histone Deacetylases/chemistry/metabolism
Humans
Molecular Sequence Data
Peptide Fragments/chemistry/metabolism
Phosphorylation
Phosphoserine/metabolism
Protein Kinases/*metabolism
Protein-Serine-Threonine Kinases/*metabolism
Substrate Specificity
Transcription Factors/chemistry/metabolism