title: Grp/DChk1 is required for G2-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable.
abstract: Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-dependent pathways in Drosophila are not understood in detail. Here, we report the function of these checkpoint kinases, referred to as Grp/DChk1 and Dmnk/DChk2 in Drosophila Schneider's cells, and identify an upstream regulator as well as downstream targets of Grp/DChk1. First, we demonstrate that S2 cells are a suitable model for G(2)/M checkpoint studies. S2 cells display Grp/DChk1-dependent and Dmnk/DChk2-independent cell-cycle-checkpoint activation in response to hydroxyurea and ionizing radiation. S2 cells depleted for Grp/DChk1 using RNA interference enter mitosis in the presence of impaired DNA integrity, resulting in prolonged mitosis and mitotic catastrophe. Grp/DChk1 is phosphorylated in a Mei-41/DATR-dependent manner in response to hydroxyurea and ionizing radiation, indicating that Mei-41/ATR is an upstream component in the Grp/DChk1 DNA replication and DNA-damage-response pathways. The level of Cdc25(Stg) and phosphorylation status of Cdc2 are modulated in a Grp/DChk1-dependent manner in response to hydroxyurea and irradiation, indicating that these cell-cycle regulators are downstream targets of the Grp/DChk1-dependent DNA replication and DNA-damage responses. By contrast, depletion of Dmnk/DChk2 by RNA interference had little effect on checkpoint responses to hydroxyurea and irradiation. We conclude that Grp/DChk1, and not Dmnk/DChk2, is the main effector kinase involved in G(2)/M checkpoint control in Drosophila cells.
authors.affiliation: Department of Radiation and Stress Cell Biology, Division Cell Biology, Faculty of Medical Sciences, University of Gröningen, Ant. Deusinglaan 1, Building 3215, 9713 AV Groningen, The Netherlands.
authors.forename: Hilda I
authors.initials: HI
authors.lastname: de Vries
authors.forename: Lyle
authors.initials: L
authors.lastname: Uyetake
authors.forename: Willy
authors.initials: W
authors.lastname: Lemstra
authors.forename: Jeanette F
authors.initials: JF
authors.lastname: Brunsting
authors.forename: Tin Tin
authors.initials: TT
authors.lastname: Su
authors.forename: Harm H
authors.initials: HH
authors.lastname: Kampinga
authors.forename: Ody C M
authors.initials: OC
authors.lastname: Sibon
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D049109:Cell Proliferation; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005434:Flow Cytometry; D016195:G2 Phase; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008938:Mitosis; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012313:RNA / Q000737:chemistry; D034622:RNA Interference; D011839:Radiation, Ionizing; D013997:Time Factors; D014981:Xenopus; D029867:Xenopus Proteins
chemical_list: D018076:DNA, Complementary; D029721:Drosophila Proteins; D029867:Xenopus Proteins; D012313:RNA; D004247:DNA; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; C000605877:grp protein, Drosophila; D016203:CDC2 Protein Kinase; D006918:Hydroxyurea