Role of the N-terminal forkhead-associated domain in the cell cycle checkpoint function of the Rad53 kinase.
Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases. Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks. Here we show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1). This phenotype was highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro. Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain resulted in an increased DNA damage sensitivity in vivo. Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1).
St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.
B L
BL
Pike
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A
Hammet
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Heierhorst
D000483:Alleles; D001426:Bacterial Proteins / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002455:Cell Division; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000235:genetics; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors; D015151:Immunoblotting; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D015003:Yeasts / Q000378:metabolism
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D051858:Forkhead Transcription Factors; D009687:Nuclear Proteins; D010455:Peptides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae