Subcellular localization of endogenous PRK2 during the cell cycle. (A) HeLa S3 cells were fixed and stained with anti-PRK2 antibody and Hoechst. (B) PRK2 siRNA-transfected cells were fixed and stained with anti-PRK2 antibody.

Images were taken every 10 min and representative frames are shown. The experiment was repeated five times. For PRK2 siRNA, 85% of binucleate cells arose from a late cytokinesis defect at abscission and 15% arose from an early cleavage furrow defect. For Ect2 siRNA, 17% of binucleate cells arose from an abscission defect and 83% arose from a cleavage furrow defect. (E) PRK2 localization is dependent on Ect2. Control or Ect2 siRNA-transfected cells were fixed after 48 h and visualized with anti-PRK2 and anti-Ect2 antibodies.

PRK2 is phosphorylated during mitosis in a Rho GTPase- and Cdk1-dependent manner. (A) HeLa S3 cells were synchronized with a double thymidine block and released into fresh medium. Whole cell extracts were prepared at the indicated time points and analysed by SDS–PAGE and Western blotting using anti-PRK2, anti-PRK1, anti-Ect2, anti-Cdc25B, anti-P-Cdk1, anti-Cdk1, anti-cyclin A, anti-cyclin B1, anti-P-Histone3 and anti-tubulin antibodies. PRK proteins are modified during mitosis. (B) Endogenous PRK2 was precipitated from lysates of cells synchronized with a double thymidine block and released for 0 or 10 h. PRK2 precipitates from the 10 h samples were either left untreated or incubated with or without λ phosphatase for 30 min at 30°C. Samples were subjected to SDS–PAGE and Western analysis with anti-PRK2 antibody. (C) HeLa S3 cells were synchronized with a double thymidine block and released into fresh medium. After 6 h, Toxin B was added at the indicated concentrations and whole cell extracts were prepared 10 h after release from the thymidine block and analysed by SDS–PAGE and Western blotting using anti-PRK2 and anti-tubulin antibodies. (D) Control and Ect2 siRNA-transfected cells were synchronized with a double thymidine block and released into fresh medium. Whole cell extracts were prepared at the indicated time points and analysed by SDS–PAGE and Western blotting using anti-Ect2, anti-PRK2, anti-cyclin A, anti-P-Histone3 and anti-tubulin antibodies. (E) HeLa S3 cells were synchronized with a double thymidine block and released into fresh medium. Roscovitine (25 μM) was added after 8 h and whole cell extracts were prepared 10 h after release from the thymidine block and analysed by SDS–PAGE and Western blotting using anti-PRK2 and anti-tubulin antibodies. (F) Control and Cdc25B siRNA-transfected cells were synchronized with a double thymidine block and released into fresh medium. Whole cell extracts were prepared at the indicated time points and analysed by SDS–PAGE and Western blotting using anti-Cdc25B, anti-PRK2, anti-P-Cdk1, anti-cyclin A, anti-P-Histone3 and anti-tubulin antibodies.

Dual role of Rho GTPases and PRK2 during mitosis. See Discussion for further details.

Depletion of PRK2 from HeLa S3 cells leads to the formation of binucleated cells. (A) Unsynchronized control, PRK2 A and PRK1 siRNA-transfected cells were subjected to FACS analysis and the percentage of ⩾4n DNA cells was determined. The averages±standard deviation for at least two independent experiments are shown. (B) Unsynchronized control, PRK2 A, PRK2 B and PRK1 siRNA-transfected cells were fixed after 72 h and stained with Hoechst. PRK2 A and PRK2 B siRNA-transfected cells result in the accumulation of binucleated cells (arrowheads). (C) Unsynchronized control, PRK2 A, PRK2 B and PRK1 siRNA-transfected cells were fixed after 72 h and stained with Hoechst. The percentage of binucleated cells was determined. The averages of at least three independent experiments±standard deviation are shown. (D) Unsynchronized control, PRK2 A, PRK2 B and PRK1 siRNA-transfected cells were lysed after 72 h and cell extracts were analysed by Western blotting using anti-PRK2, anti-PRK1 and anti-tubulin antibodies. (E) Control and myc PRK2-transfected cells were fixed after 72 h and stained with anti-myc antibody and Hoechst. Overexpression of myc PRK2 results in the accumulation of binucleated cells (arrowheads). (F) Control and myc PRK2-transfected cells were fixed after 72 h, stained with anti-myc antibody and Hoechst, and the percentage of binucleated cells determined. The averages of at least three independent experiments±standard deviation are shown. (G) PRK1 protein is expressed at low levels in HeLa S3 cells. Recombinant Gst-PRK1 and Gst-PRK2 proteins (5–50 ng) together with total cell extract (10+20 μl) were analysed by SDS–PAGE and Western blotting using anti-PRK1 and anti-PRK2 antibodies.

PRK2-depleted cells have an abscission defect. Live-cell imaging of control cells (A); PRK2 A siRNA-transfected cells (B); and Ect2 siRNA-transfected cells (C, D).

PRK2 depletion delays cell cycle progression. (A) Control, PRK2 A siRNA- and Ect2 siRNA-transfected cells were fixed after 72 h and visualized with Hoechst. PRK2-depleted cells are binucleated, whereas Ect2-depleted cells are multinucleated. (B) Control, PRK2 A siRNA- and Ect2 siRNA-transfected cells were observed by time-lapse imaging, and cell cycle lengths, that is, the time between two consecutive mitosis, were calculated. For PRK2 A siRNA- and Ect2 siRNA-transfected cells, the cell cycle duration was determined for cells that entered mitosis and then failed cytokinesis. Average cell cycle lengths±standard deviation for 20–30 cells from independent experiments are shown. (C) Control cells and PRK2 A siRNA-transfected cells were synchronized with a double thymidine block and released into fresh medium containing BrdU. Cells were fixed after 6 h and stained with anti-BrdU antibody, and the percentage of cells with incorporated BrdU was determined. The average percentages from four independent experiments±standard deviation are shown. (D) PRK2-depleted cells show a delay in G2/M progression. Control cells and PRK2 A siRNA-transfected cells were synchronized with a double thymidine block and released into fresh medium. Whole cell extracts prepared at the indicated time points were analysed by SDS–PAGE and Western blotting using anti-PRK2, anti-Cdc25B, anti-P-Cdk1, anti-Cdk1, anti-cyclin A, anti-cyclin B1, anti-P-Histone3 and anti-tubulin antibodies.

Cdc25B depletion causes a cell cycle delay and a cytokinesis defect. (A) Unsynchronized control and Cdc25B siRNA-transfected cells were lysed after 72 h and whole cell extracts were subjected to SDS–PAGE and Western analysis using anti-Cdc25B and anti-PRK2 antibodies. (B) Unsynchronized control and Cdc25B siRNA-transfected cells were fixed after 72 h and visualized with Hoechst. Cdc25B-depleted cells are binucleated. (C) Unsynchronized control, Cdc25B siRNA- and PRK2 siRNA-transfected cells were fixed after 72 h and visualized with Hoechst. The percentages of binucleated cells were determined. The averages of at least three independent experiments±standard deviation are shown. (D) Control, Cdc25B siRNA- and PRK2 siRNA-transfected cells were observed by time-lapse imaging, and the cell cycle lengths, that is, the time between two consecutive mitosis, were calculated. For Cdc25B siRNA- and PRK2 siRNA-transfected cells, the cell cycle duration was determined for cells that entered mitosis and then failed cytokinesis. Average cell cycle lengths±standard deviation are shown.