Protein phosphatase-1alpha regulates centrosome splitting through Nek2.
ATM is a central mediator of the cellular response to the DNA damage produced by ionizing radiation. We recently showed that protein phosphatase 1 (PP1) is activated by ATM. Because Nek2 is activated by autophosphorylation, and because its dephosphorylation is catalyzed by PP1, we asked if the radiation damage signal to Nek2 was mediated by PP1. Overexpression of Nek2 induces premature centrosome splitting probably by phosphorylating centrosome cohesion proteins C-Nap1 and Rootletin. In this study, we show isoform specificity of PP1 binding and regulation of Nek2. Although both PP1alpha and PP1gamma coimmunoprecipitated with Nek2, only PP1alpha regulated Nek2 function. Ionizing radiation inhibited Nek2 activity, and this response was dependent on ATM and on PP1 binding to Nek2 and coincident with Thr(320) dephosphorylation of PP1. Radiation-induced inhibition of centrosome splitting was abrogated in cells expressing Nek2 mutated in the PP1-binding motif outside the kinase domain. Conversely, cells depleted of PP1alpha by small interfering RNA showed enhanced centrosome splitting and loss of radiation-induced inhibition of centrosome splitting. The identification of a PP1-specific isoform mediating a checkpoint response opens up the possibility of selectively targeting phosphatases as novel radiation sensitizers.
Mi
Jun
J
Department of Radiation Oncology, University of Virginia Health System, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Guo
Changyue
C
Brautigan
David L
DL
Larner
James M
JM
D000818:Animals; D019556:COS Cells; D018385:Centrosome / Q000378:metabolism / Q000502:physiology* / Q000528:radiation effects; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D000072379:NIMA-Related Kinases; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology* / Q000528:radiation effects
D007527:Isoenzymes; C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1