title: Mutation of serine 1333 in the ATR HEAT repeats creates a hyperactive kinase.
abstract: Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR) causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Jessica W
authors.initials: JW
authors.lastname: Luzwick
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Nam
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Runxiang
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Cortez
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D064007:Ataxia Telangiectasia Mutated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000528:radiation effects; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D011839:Radiation, Ionizing; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; D012694:Serine; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; D006918:Hydroxyurea