title: The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses.
abstract: DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.
authors: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455 takad003@umn.edu.
authors: Saeko
authors: S
authors: Takada
authors: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455.
authors: Eric R
authors: ER
authors: Collins
authors: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455.
authors: Kayo
authors: K
authors: Kurahashi
mesh_terms: D000818:Animals; D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000378:metabolism*; D059006:Chromosomes, Insect; D004249:DNA Damage; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis; D009154:Mutation; D010766:Phosphorylation; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D021382:Protein Sorting Signals; D064447:Checkpoint Kinase 2; C112435:lok protein, Drosophila