Figure 7.

Plk1 controls Eg5 phosphorylation at the Nek6 site Ser1033. Both Ser1033 and the CDK1 site Thr926 phosphorylation are necessary for prophase centrosome separation and Eg5 recruitment. (A) HeLa cells were arrested in mitosis by either nocodazole (ND) treatment or RNAi against Plk1 or Eg5 (24 h transfection), collected after mitotic shake off and cell extracts were analysed by western blot (WB) using the indicated antibodies. Mitotic arrest was confirmed by FACS (not shown) and the phosphorylatin state of Cdc27. Untreated cells (Exp) are shown in the first lane as a control. Asterisks mark protein bands with altered mobility due to phosphorylation. (B) HeLa cells were transfected with either control or Eg5 siRNAs, after 16 h retransfected with expression plasmids for the indicated Myc-tagged proteins (cDNAs rendered resistant to the siRNA by several silent point mutations), fixed and stained with antibodies against Myc, γ-tubulin and DAPI. The percentage of Myc-positive prophase cells showing two unseparated centrosomes (together), two centrosomes separated <2 μm (<2 μm) or fully separated centrosomes (>2 μm) is shown in the upper graphic (mean±s.e.m. of three independent experiments; ∼40 cells counted in each experiment). Levels of endogenous and recombinant Eg5 as determined by WB are shown in Supplementary Figure S6C. (C) Cells transfected and processed as in (B). Representative examples of the observed phenotypes (Myc–Eg5, green) are shown below (bar, 5 μm). Insets show the same field stained with γ-tubulin (red) plus DAPI (blue). Centrosomal accumulation of Eg5 is noted with arrows.