Importance of a C-terminal conserved region of Chk1 for checkpoint function.
BACKGROUND
The protein kinase Chk1 is an essential component of the DNA damage checkpoint pathway. Chk1 is phosphorylated and activated in the fission yeast Schizosaccharomyces pombe when cells are exposed to agents that damage DNA. Phosphorylation, kinase activation, and nuclear accumulation are events critical to the ability of Chk1 to induce a transient delay in cell cycle progression. The catalytic domain of Chk1 is well-conserved amongst all species, while there are only a few regions of homology within the C-terminus. A potential pseudosubstrate domain exists in the C-terminus of S. pombe Chk1, raising the possibility that the C-terminus acts to inhibit the catalytic domain through interaction of this domain with the substrate binding site.


RESULTS
To evaluate this hypothesis, we characterized mutations in the pseudosubstrate region. Mutation of a conserved aspartic acid at position 469 to alanine or glycine compromises Chk1 function when the mutants are integrated as single copies, demonstrating that this domain of Chk1 is critical for function. Our data does not support, however, the hypothesis that the domain acts to inhibit Chk1 function as other mutations in the amino acids predicted to comprise the pseudosubstrate do not result in constitutive activation of the protein. When expressed in multi-copy, Chk1D469A remains non-functional. In contrast, multi-copy Chk1D469G confers cell survival and imposes a checkpoint delay in response to some, though not all forms of DNA damage.


CONCLUSIONS
Thus, we conclude that this C-terminal region of Chk1 is important for checkpoint function and predict that a limiting factor capable of associating with Chk1D469G, but not Chk1D469A, interacts with Chk1 to elicit checkpoint activation in response to a subset of DNA lesions.
Palermo
Carmela
C
Department of Pharmacology, University of Medicine and Dentistry, New Jersey (UMDNJ), Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
Hope
Justin C
JC
Freyer
Greg A
GA
Rao
Hui
H
Walworth
Nancy C
NC
D000483:Alleles; D002166:Camptothecin / Q000494:pharmacology; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004271:DNA, Fungal / Q000187:drug effects; D009154:Mutation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D002166:Camptothecin