title: Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control.
abstract: Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated G(2)/M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression.
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences and The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21231, USA.
authors.forename: Deborah
authors.initials: D
authors.lastname: Wilsker
authors.forename: Eva
authors.initials: E
authors.lastname: Petermann
authors.forename: Thomas
authors.initials: T
authors.lastname: Helleday
authors.forename: Fred
authors.initials: F
authors.lastname: Bunz
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D018385:Centrosome / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D016195:G2 Phase / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D008677:Metaphase / Q000502:physiology*; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D040641:Quantitative Trait Loci / Q000502:physiology
chemical_list: D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1