VRK1 regulates AurKB activity and localization in early mitosis

Abstract

[Introduction]: The regulation of cell cycle relies on the activity of several protein kinases. These kinases control the correct transmission of genetic information to the newly-formed cells, DNA replication, chromosome segregation, and chromatin condensation, between innumerous other mechanisms. Between these kinases, we can found two different serine-threonine kinases, the Aurora Kinase B (AurKB) and the Vaccinia-related Kinase 1 (VRK1). VRK1 is a nuclear kinase implicated in p53 regulation and DNA damage repair, as well as in the chromatin condensation through histone H3 phosphorylation, mainly, in Thr3. Besides, VRK1 regulates CCND1 gene and correlates positively with proliferation markers such as the Ki67. In turn, AurKB is a component of the Chromosomal Passenger Complex (CPC) necessary for accurate spindle-kinetochore attachments during chromosome segregation and cytokinesis. This kinase contributes also to chromatin condensation, phosphorylating the histone H3Ser10. [Objectives]: We hypothesize that VRK1 and Aurora B might form a complex, and that the establishment of this interaction affects the activity of AurKB. Moreover, we hypothesize that VRK1 may affect the centromeric localization of AurKB, through modulation of histone H3T3 phosphorylation. [Methods]: VRK1 and Aurora B interaction was assessed by reciprocal immunoprecipitations in HEK 293T. The cell cycle-dependent interaction, between both kinases was analyzed performing the arrested of HeLa or U2OS cells in G1/S, after double Thymidine block or in G2/M, after Thymidine-Nocodazole block. The same protocol was used to assess the co-localization of VRK1 and AurKB by immunofluorescence. In vitro kinase assay, in presence of cold ATP or Kinase assay (32P[ATP]) was performed to evaluate histone H3Ser10 phosphorylation by GST-AurKB, after incubation with active or inactive GST-VRK1. Finally, sub-cellular localization of AurKB was assessed by immunofluorescence after VRK1 knockdown. [Results]: Herein, we showed that VRK1 interact with AurKB. This interaction was observed, between transfected VRK1 and AurKB, and also, among endogenous proteins. The interaction seemed to occur mainly in mitosis, when VRK1 and AurKB levels were increased. However, we could not find a possible co-localization between VRK1 and AurKB during mitosis. The effect of this interaction results in the inhibition of AurKB activity. On the other hand, VRK1 downregulation affects the centromeric localization of AurKB-dependent on the H3T3 phosphorylation levels. [Conclusions]: VRK1 interacts with AurKB during mitosis, and this interaction impairs the kinase activity of AurKB. VRK1 regulates AurKB localization in early phases of mitosis.