title: Chk1 Activation Protects Rad9A from Degradation as Part of a Positive Feedback Loop during Checkpoint Signalling.
abstract: Phosphorylation of Rad9A at S387 is critical for establishing a physical interaction with TopBP1, and to downstream activation of Chk1 for checkpoint activation. We have previously demonstrated a phosphorylation of Rad9A that occurs at late time points in cells exposed to genotoxic agents, which is eliminated by either Rad9A overexpression, or conversion of S387 to a non-phosphorylatable analogue. Based on this, we hypothesized that this late Rad9A phosphorylation is part of a feedback loop regulating the checkpoint. Here, we show that Rad9A is hyperphosphorylated and accumulates in cells exposed to bleomycin. Following the removal of bleomycin, Rad9A is polyubiquitinated, and Rad9A protein levels drop, indicating an active degradation process for Rad9A. Chk1 inhibition by UCN-01 or siRNA reduces Rad9A levels in cells synchronized in S-phase or exposed to DNA damage, indicating that Chk1 activation is required for Rad9A stabilization in S-phase and during checkpoint activation. Together, these results demonstrate a positive feedback loop involving Rad9A-dependend activation of Chk1, coupled with Chk1-dependent stabilization of Rad9A that is critical for checkpoint regulation.
authors.affiliation: Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, Ontario K7L 3N6, Canada.|Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: William F
authors.initials: WF
authors.lastname: Osorio-Zambrano
authors.affiliation: Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, Ontario K7L 3N6, Canada.|Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario K7L 3N6, Canada.|Department of Oncology, Queen's University, Kingston, Ontario K7L 3N6, Canada.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Scott
authors.initials: S
authors.lastname: Davey
mesh_terms: D001761:Bleomycin / Q000494:pharmacology; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004789:Enzyme Activation / Q000187:drug effects; D025461:Feedback, Physiological; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D018797:Cell Cycle Proteins; D007976:Leupeptins; D034741:RNA, Small Interfering; D001761:Bleomycin; D025821:Polyubiquitin; C069930:rad9 protein; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D046988:Proteasome Endopeptidase Complex; D019311:Staurosporine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde