Polo-Like Kinase 1 Directs Assembly of the HsCyk-4 RhoGAP/Ect2 RhoGEF Complex to Initiate Cleavage Furrow Formation (original) (raw)
Figure 1
Inhibition of Plk1 prevents association of HsCyk-4 with Ect2–BRCT.
(A) HeLa cells transfected with H2O (control) or CMV-Myc-Ect2-BRCT (myc-BRCT) were synchronized in anaphase using an MG132 arrest/release protocol. Following treatment with 100 nM BI-2536 or DMSO, cells were fixed and stained with antibodies to Myc and α-tubulin, and DNA was stained with DAPI. (B) HeLa cells, released into anaphase from an MG132 block, were treated with 100 nM BI-2536 or DMSO and harvested at the time points indicated above the lanes. Lysates and Ect2-BRCT–bound fractions were probed with antibodies to HsCyk-4 and Mklp1. The percentages of anaphase and telophase cells and of cells with ingressing furrows are indicated below the corresponding lanes. (C) HeLa cells arrested in prometaphase with nocodazole were treated with DMSO, 22.5 µM purvalanol A, 100 nM BI-2536, or 22.5 µM purvalanol A + 100 nM BI-2536 for 30 min. Lysates (∼5%) and Ect2-BRCT bound fractions were probed with antibodies to HsCyk-4, cyclin B1, and α-tubulin. (D) Lysates were prepared from HeLa cells synchronized by nocodazole block, incubated with the indicated immobilized derivatives of Ect2-BRCT, washed, and separated on SDS-PAGE. Lysates (∼5%) and Ect2-BRCT bound fractions were probed with antibodies to HsCyk-4 (upper blots). Sequence alignment showing conservation of phosphate coordinating residues in the indicated BRCT-domain containing proteins (lower sequences). (E) HeLa cells were transfected with vector (control), CMV-Myc-Ect2-BRCT (wt), or CMV-Myc-Ect2-BRCT T153A/K195M (TA/KM). Cells were fixed 8 h after release from thymidine, and stained with DAPI and antibodies to Myc and α-tubulin. Scale bars in this and all other figures represent 10 µm.