title: The Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 directly influence the DNA damage-dependent functions of Rad53.
abstract: In this study, we mutated autophosphorylation sites in Rad53 based on their conservation with previously identified autophosphorylation sites in the mammalian Rad53 ortholog, Chk2. As with wild-type Rad53, the autophosphorylation mutant, rad53-TA, undergoes Mec1/Tel1-dependent interactions with Rad9 and Dun1 in response to genotoxic stress. Whereas rad53-TA in vitro kinase activity is severely impaired, the rad53-TA strains are not completely deficient for cell-cycle checkpoint functions, indicating that the mutant kinase retains a basal level of function. We describe a genetic interaction among Rad53, Dun1, and the 14-3-3 proteins Bmh1 and Bmh2 and present evidence that 14-3-3 proteins directly facilitate Rad53 function in vivo. The data presented account for the previously observed checkpoint defects associated with 14-3-3 mutants in Saccharomyces pombe and Saccharomyces cerevisiae. The 14-3-3 functional interaction appears to modulate Rad53 activity, reminiscent of 14-3-3's effect on human Raf1 kinase and distinct from the indirect mode of regulation by 14-3-3 observed for Chk1 or Cdc25.
authors.affiliation: Laboratory of Chromosome Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Usui
authors.forename: John H J
authors.initials: JH
authors.lastname: Petrini
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D018628:Gene Dosage; D047468:Immunoprecipitation; D050505:Mutant Proteins / Q000378:metabolism; D010641:Phenotype; D010748:Phosphopeptides / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013489:Suppression, Genetic
chemical_list: D048948:14-3-3 Proteins; C075852:BMH1 protein, S cerevisiae; C097164:BMH2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D050505:Mutant Proteins; D010748:Phosphopeptides; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae