Fig 1: RNAi studies of VRK kinase function in mitosis. (A) Kinome-wide RNAi screen for kinases involved in H3T3 phosphorylation in HeLa cells. The standard score reflects the intensity of H3T3ph. Note that negative values indicate a decline in H3T3ph intensity. The ranking of Haspin (red), VRK1, VRK2, VRK3 (blue) and PASK (black) are shown. Some results of this screen were previously published65. (B) RNAi depletion of VRK1 from HeLa cells does not cause a significant loss of H3T3ph as detected by immunoblotting. The original blots are presented in Supplementary Fig. 7. (C) RNAi depletion of VRK1 does not cause a significant loss of H3T3ph or H3S10ph in individual mitotic HeLa cells, as detected by immunofluorescence microscopy. Scale bar, 10 µm. (D) Quantification of the proportion of HeLa cells in (C) with H3S10ph (i.e. mitotic cells) that also stain for H3T3ph.
Fig 2: RNAi of VRK2 does not have a detectable influence on H3T3ph. Control and VRK1 KO HAP1 cells were treated with control or VRK2 siRNAs and subjected to immunoblotting with the stated antibodies. Note that the top 3 panels are from a different immunoblot from the one in the bottom 2 panels. The original blots are presented in Supplementary Fig. 7.
Fig 3: Immunofluorescence microscopy of histone H3 phosphorylation in HAP1 cells. (A) Loss of Haspin, but not VRK1, eliminates H3T3ph in individual mitotic HAP1 cells, as detected by immunofluorescence microscopy. H3S10ph appears unaffected in both cases. Scale bar, 10 µm. (B) Quantification of the intensity of H3T3ph staining in cells stained as in (A). Black symbols represent individual cells. Grey, red and blue symbols show the average values for each of 3 independent experiments, normalised to control HAP1 cells. ***p = 0.0019 (two-tailed t test using non-normalised data; n = 3). (C) Quantification of the intensity of H3S10ph staining in cells as in (B). Two-tailed t tests using non-normalised data revealed no significant differences at p = 0.05 (n = 3). (D) Quantification of the ratio of H3T3ph intensity to H3S10ph intensity in cells as in (B). *p = 0.0496 (two-tailed t test using non-normalised data; n = 3).
Fig 4: Phosphorylation of Histone H3 and BAF by recombinant human Haspin or VRK1. Phosphorylation of H3T3 by Haspin and VRK1 on (A) H3(1–21) peptides, and (B) recombinant nucleosome substrates, detected by H3T3ph antibodies. Error bars show means ± SD, n = 3 independent experiments. In (C), phosphorylation of recombinant BAF or H3 by various preparations of recombinant VRK1 were monitored by [γ32P]-incorporation, SDS-PAGE and phosphorimaging analysis. The integrity and purity of the proteins was validated by SDS-PAGE and silver-staining. The original gel is presented in Supplementary Fig. 7.
Fig 5: KiPIK screens for H3T3 kinase activity in mitotic HeLa cell lysates using the kinase profiling datasets of (A) Anastassiadis et al.66, (B) Davis et al.68, (C,D) Gao et al.67, (E) Federov et al.69 and (F) the differential scanning fluorimetry (DSF) method of Elkins et al.99. Some results of the Elkins DSF screen were previously published65. The ranking of Haspin (red), VRK1, VRK2, VRK3 (blue) and PASK (black) are shown for all datasets in which they appear.
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