Interaction with checkpoint kinase 1 modulates the recruitment of nucleophosmin to chromatin.
The Checkpoint kinase 1 (Chk1) plays a central role in the cellular response to DNA damage and also contributes to the efficacy of DNA replication in the absence of genomic stress. However, we have only limited knowledge regarding the molecular mechanisms that regulate differential Chk1 function in the absence and presence of DNA damage. To address this, we used vertebrate cells with compromised Chk1 function to analyze how altered Chk1 activity influences protein interactions in chromatin. Avian and mammalian cells with compromised Chk1 activity were used in combination with genomic stress, induced by UV, and DNA-associated proteomes were analyzed using 2-DE/MS proteomics and Western-blot analysis. Only one protein, the histone chaperone nucelophosmin, was altered consistently in line with changes in chromatin-associated Chk1 and increased in response to DNA damage. Purified Chk1 and NPM were shown to interact in vitro and strong in vivo interactions were implied from immunoprecipitation analysis of chromatin extracts. During chromatin immunoprecipitation, coassociation of the major cell cycle regulator proteins p53 and CDC25A with both Chk1 and NPM suggests that these proteins are components of complex interaction networks that operate to regulate cell proliferation and apoptosis in vertebrate cells.
Chen
Songbi
S
Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Maya-Mendoza
Apolinar
A
Zeng
Kang
K
Tang
Chi W
CW
Sims
Paul F G
PF
Loric
Josip
J
Jackson
Dean A
DA
D000818:Animals; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002645:Chickens; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004249:DNA Damage / Q000528:radiation effects; D015180:Electrophoresis, Gel, Two-Dimensional; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000090243:Nucleophosmin; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D015398:Signal Transduction; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D014466:Ultraviolet Rays
D002843:Chromatin; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D020543:Proteome; D000090243:Nucleophosmin; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D019311:Staurosporine