Figure 6.

Phosphorylation of S326 regulates Plk1 function during mitosis. (a) HeLa cells were transfected with GFP-Plk1 (WT or S326A). At 1 day post-transfection, cells were stained with a phospho-histone H3 (p-H3) antibody. (b, c) Cells were synchronized with the double thymidine block and transfected with GFP-Plk1 or GFP-Plk1-S326A during the 8-h interval. Upon release for the times indicated, cells were stained with a phospho-histone H3 antibody (b) or an α-tubulin antibody (c). In (b), only GFP-positive cells were counted. In (c), only the cells with mid-body structure were counted. (d, e) Cells were transfected with GFP-Plk1 (d) or Flag-Plk1 (e) and treated with nocodazole. The M-phase cells were collected by mechanic shake off, released for different times and stained with a phospho-histone H3 antibody (d) or analysed by western blot (e). In (d), only GFP-positive cells were counted. (f, g) At 1 day post-transfection with GFP-Plk1, cells were stained with a γ-tubulin antibody. (h, i) Cells were transfected with siRNA to deplete BubR1 or MAD2 (Tang et al., 2006a), then synchronized and transfected with GFP-Plk1-S326A as in (b). Upon release for the times indicated, cells were analysed by western blot (h) or stained with a phospho-histone H3 antibody (i). (j–l) Cells were transfected with siRNA to deplete Plk1 (j), p38a (k) or MK2 (l), then subjected to the double thymidine block with transfection of GFP fusion Plk1 during the 8-h interval. Cells were released from the block for 16 h and stained with a phospho-histone H3 antibody. (m, n) COS-7 cells were transfected with Flag-Plk1 (WT, S326A or S326E), treated with 200 ng/ml nocodazole for 12 h and harvested. Lysates were subjected to anti-Flag immunoprecipitation (IP)/kinase assays using a purified GST-topoisomerase IIα fragment as a substrate. The quantification results were shown in (n).