title: Structural basis of Rad53 kinase activation by dimerization and activation segment exchange.
abstract: The protein kinase Rad53 is a key regulator of the DNA damage checkpoint in budding yeast. Its human ortholog, CHEK2, is mutated in familial breast cancer and mediates apoptosis in response to genotoxic stress. Autophosphorylation of Rad53 at residue Thr354 located in the kinase activation segment is essential for Rad53 activation. In this study, we assessed the requirement of kinase domain dimerization and the exchange of its activation segment during the Rad53 activation process. We solved the crystal structure of Rad53 in its dimeric form and found that disruption of the observed head-to-tail, face-to-face dimer structure decreased Rad53 autophosphorylation on Thr354 in vitro and impaired Rad53 function in vivo. Moreover, we provide critical functional evidence that Rad53 trans-autophosphorylation may involve the interkinase domain exchange of helix αEF via an invariant salt bridge. These findings suggest a mechanism of autophosphorylation that may be broadly applicable to other protein kinases.
authors.affiliation: The Arthur and Sonia Labatt Brain Tumour Research Centre and Program in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada. Electronic address: leanne.wybenga.groot@utoronto.ca.
authors.forename: Leanne E
authors.initials: LE
authors.lastname: Wybenga-Groot
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Electronic address: cynthia.sw.ho@gmail.com.
authors.forename: Cynthia S
authors.initials: CS
authors.lastname: Ho
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada; Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: frederic.sweeney@gmail.com.
authors.forename: Frédéric D
authors.initials: FD
authors.lastname: Sweeney
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Electronic address: ceccarelli@lunenfeld.ca.
authors.forename: Derek F
authors.initials: DF
authors.lastname: Ceccarelli
authors.affiliation: The Arthur and Sonia Labatt Brain Tumour Research Centre and Program in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada. Electronic address: jmcglade@sickkids.ca.
authors.forename: C Jane
authors.initials: CJ
authors.lastname: McGlade
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada; Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: durocher@lunenfeld.ca.
authors.forename: Daniel
authors.initials: D
authors.lastname: Durocher
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada; Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: sicheri@lunenfeld.ca.
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation
chemical_list: D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; C067022:RAD53 protein, S cerevisiae
keywords: CHEK2; Checkpoint kinase 2; Chk2