Figure 4.

NEK8 suppresses cyclin A-associated CDK activity to prevent DSB formation.

(A) Nek8^−/− MEFs have higher levels of cyclin A-associated CDK activity. Immortalized MEFs were UV-irradiated as indicated and allowed to recover for 1 h, then cyclin A was immunoprecipitated and used in an in vitro kinase assay with histone H1 as a substrate. The CDK1/2 inhibitor (CDK1/2i) was used to demonstrate specificity. An autoradiograph of ³²P-labeled histone H1 is shown. The kinase activity was quantified by densitometry. Each data point represents the mean of three experimental replicates, and error bars represent standard error of mean (SEM, n=3) (B) MEFs were treated with indicated concentrations of aphidicolin and either DMSO or CDK1/2i (200 nM) for 24 h. Surviving clones were scored 9 days later. Each data point represents the mean of three experimental replicates and cell viability was analyzed as in Figure 1G. (C) A neutral comet assay was performed in MEFs treated with or without APH (400 nM) and/or CDK1/2i (200 nM) for 18 h. Boxes and whiskers represent the 25-75 and 10-90 percentile, respectively. Horizontal lines mark the medians. Dots represent the data not included between the whiskers. Differences between distributions were assessed with the Mann-Whitney rank sum test (***p < 0.0001). (D & E) Knockdown of MUS81 rescues DNA damage induced by NEK8 loss. HeLa cells were transfected with the indicated siRNAs for 72 h then γH2AX level was assessed by immunofluorescence (D). Each data point represents the mean of three experimental replicates. Error bars represent standard error of mean (SEM, n=3). The neutral comet assay was performed and analyzed (E) as described in (C). (F) NEK8 and WEE1 act in independent pathways to suppress the accumulation of DNA damage. γH2AX levels were analyzed by immunofluorescence in MEFs treated with APH (400 nM) and/or WEE1 inhibitor (WEE1 i, 10 μM) for 18 h. The mean γH2AX intensity for each cell is plotted in box and whisker format with the same statistical analysis as in (C). (G) NEK8 interacts with cyclin A-CDK2 and WEE1. EYFP-tagged NEK8 was co-expressed with FLAG-tagged CDK1, CDK2, cyclin A or WEE1 in HEK293T cells. The cells were lysed 48 h later, and FLAG-tagged proteins and associated proteins were immunoprecipitated using M2 beads, followed by western blotting as indicated. (H) NEK8 regulates cyclin A and CDK2 protein levels. Extracts from Nek8^+/+ or Nek8^−/− MEFs were collected and cyclin A, cyclin B, CDK1 or CDK2 levels were analyzed by western blotting. All the results are from three independent experiments. AU, arbitrary units. See also Figure S4.