An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63.
Activation of the protein kinases ATM and ATR following chromosomal breakage prevents initiation of DNA replication and entry into mitosis. However, the effects of ATM and ATR activation in cells already progressing through mitosis are poorly understood. Here we report that ATM and ATR activation induced by DNA double-strand breaks (DSBs) inhibits centrosome-driven spindle assembly in Xenopus laevis mitotic egg extract and somatic cells, delaying mitotic progression. Using a cDNA expression library to screen for ATM and ATR substrates, we identified centrosomal protein CEP63 as an ATM and ATR target required for normal spindle assembly. ATM and ATR phosphorylate Xenopus CEP63 (XCEP63) on Ser 560 and promote its delocalization from the centrosome. Suppression of ATM and ATR activity or mutation of XCEP63 Ser 560 to Ala prevented spindle assembly defects. Consistently, inactivation of the CEP63 gene in avian DT40 cells impaired spindle assembly and prevented ATM- and ATR-dependent effects on mitosis. These data indicate that ATM and ATR control mitotic events in vertebrate cells by targeting CEP63 and centrosome dependent spindle assembly.
Smith
Eloise
E
Genome Stability Unit, London Research Institute, Clare Hall Laboratories, South Mimms, London EN6 3LD, UK.
Dejsuphong
Donniphat
D
Balestrini
Alessia
A
Hampel
Martin
M
Lenz
Christof
C
Takeda
Shunichi
S
Vindigni
Alessandro
A
Costanzo
Vincenzo
V
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002645:Chickens; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D010063:Ovum / Q000166:cytology / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D008941:Spindle Apparatus / Q000201:enzymology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; C539485:Cep63 protein, Xenopus; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases