Regulation of Chk1 by its C-terminal domain.
Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of approximately 200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration.
Kosoy
Ana
A
Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA.
O'Connell
Matthew J
MJ
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects; D001224:Aspartic Acid / Q000235:genetics; D000071877:Checkpoint Kinase 1; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000187:drug effects; D016195:G2 Phase / Q000187:drug effects; D016153:Genes, Suppressor; D018698:Glutamic Acid / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D013329:Structure-Activity Relationship; D013696:Temperature
D050505:Mutant Proteins; D029702:Schizosaccharomyces pombe Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe