Grp/DChk1 is required for G(2)-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

Hilda I. de Vries, Lyle Uyetake, Willy Lemstra, Jeanette F. Brunsting, Tin Tin Su, Harm H. Kampinga, Ody C. M. Sibon

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    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 G2/M checkpoint control in Drosophila cells.
    Original languageEnglish
    Pages (from-to)1833-1842
    Number of pages10
    JournalJournal of Cell Science
    Volume118
    Issue number9
    DOIs
    Publication statusPublished - 1-May-2005

    Keywords

    • Chk1
    • Chk2
    • hydroxyurea
    • ionizing radiation
    • RNA interference
    • DNA-DAMAGE CHECKPOINT
    • REPLICATION CHECKPOINT
    • PROTEIN-KINASE
    • MIDBLASTULA TRANSITION
    • FISSION YEAST
    • CYCLE REGULATION
    • CHK1
    • PATHWAYS
    • ATM
    • PHOSPHORYLATION

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