CDK1-mediated mitotic phosphorylation of PBK is involved in cytokinesis and inhibits its oncogenic activity

Abstract

PDZ-binding kinase (PBK) plays a major role in proliferation and in safeguarding mitotic fidelity in cancer cells. Frequently upregulated in many cancers, PBK drives tumorigenesis and metastasis. PBK has been shown to be phosphorylated in mitosis by cyclin-dependent kinase 1 (CDK1)/cyclin B, however, no studies have been done examining PBK mitotic phosphorylation in oncogenesis. Additionally to the previously identified Threonine-9 phosphorylation, we found that Threonine-24, Serine-32, and Serine-59 of PBK are also phosphorylated. PBK is phosphorylated in vitro and in cells by CDK1 during antimitotic drug-induced mitotic arrest and in normal mitosis. We demonstrated that mitotic phosphorylation of Threonine-9 is involved in cytokinesis. The non-phosphorylatable mutant PBK-T9A augments tumorigenesis to a greater extent than wild type PBK in breast cancer cells, suggesting that PBK mitotic phosphorylation inhibits its tumor promoting activity. The PBK-T9A mutant also transforms and increases the proliferation of immortalized breast epithelial cells. Collectively, this study reveals that CDK1-mediated mitotic phosphorylation of PBK is involved in cytokinesis and inhibits its oncogenic activity.

Keywords: CDK1; Cytokinesis; Mitotic phosphorylation; Oncogenesis; PBK.

Figure 1. Phosphorylation of PBK by CDK1 during mitotic arrest

A, HeLa cells were treated with DMSO, taxol (100 nM for 16 h) or nocodazole (100 ng/ml for 16 h). Total cell lysates were probed with the indicated antibodies on Phos-tag or regular SDS-polyacrylamide gels. B, HeLa cells were treated with nocodazole as indicated and cell lysates were further treated with (+) or without (−) λ-phosphatase (ppase). Total cell lysates were probed with anti-PBK antibody. o marks a non-specific band. C, HeLa cells were treated with nocodazole together with or without various kinase inhibitors as indicated. RO3306 (5 μM), Purvalanol A (10 μM), BI2536 (100 nM), VX680 (2 μM), MK2206 (10 μM), SB203580 (10 μM), SB203580 (20 μM), SP600125 (20 μM), U0126 (20 μM), and SB216763 (10 μM) were used. Inhibitors were added 1.5 h before harvesting the cells (with MG132 to prevent cyclin B degradation and consequent mitotic exit). Total cell lysates were subjected to Western blotting with the indicated antibodies. * marks the two lanes in which the PBK mobility-shift was inhibited.

Figure 2. CDK1 phosphorylates PBK in vitro

A, in vitro kinase assays with purified CDK1/cyclin B complex and recombinant GST-PBK. RO3306 (5 μM) was used to inhibit CDK1 kinase activity. B, Conservation of PBK’s mitotic phosphorylation sites. C, in vitro kinase assays with purified CDK1/cyclin B complex to phosphorylate recombinant GST-PBK or GST-PBK-4A. D, In vitro kinase assays were done as in (A) except anti-phospho-PBK T9, T24, S32 and S59 antibodies were used.

Figure 3. CDK1 phosphorylates PBK in cells

A, HeLa cells were treated with nocodazole or taxol for 16 hours. The T24 phospho-antibody was incubated for 16 hours at 4°C with either PBS (no peptide), peptide, or phospo-peptide used for immunizing rabbits, before Western blotting. B, HeLa cells were treated with nocodazole or taxol for 16 hours before Western blotting was performed with the indicated antibodies. C, HeLa cells were transfected with scrambled siRNA (control) or siRNA against PBK for 48 h and were further treated with (+) or without (−) taxol for 16 hours. The total cell lysates were subjected to Western blotting with the indicated antibodies. * marks a non-specific band. D, HeLa cells were nocodazole together with or without various kinase inhibitors as indicated. Inhibitors were added with MG132 (to halt cyclin B degradation and prevent mitotic exit) for 1.5 h before harvesting the cells. Total cell lysates were subjected to Western blotting with the indicated antibodies.

Figure 4. CDK1 phosphorylates PBK in normal mitosis and during mitotic arrest

A, HeLa cells were treated with nocodazole or taxol for 16 hours and then fixed. Before the cells were immunostained with the T24 phospho-antibody, the antibody was incubated for 16 hours at 4°C with either PBS (no peptide), peptide, or phospo-peptide. White and yellow arrows mark some of the metaphase cells and the interphase cells, respectively. B, HeLa cells were synchronized by the double thymidine block and release method. Cells were stained with antibodies against p-PBK T24, β-tubulin, and with DAPI. C, A low power (40X objective) lens was used to view various phases of the cells in a field from (B). D, HeLa cells were synchronized by a double thymidine block and release method. Total cell lysates were harvested at the specified time points after release and subjected to Western blotting analysis with the indicated antibodies.

Figure 5. Generation and characterization of PBK knockout (KO) U2OS cell line

A, Western blot of parental and PBK knockout U2OS cell lines. Equal amount of U2OS control and PBK knockout cell lysates were probed with anti-PBK antibody. B, U2OS control and PBK knockout cells were used to quantify the percentage of cells in cytokinesis. C, Immunofluorescence confocal images showing the difference in the amount of cytokinesis between the U2OS control and PBK knockout cell lines. β-tubulin is shown in green and nuclei is stained with DAPI (blue). The arrows point to cytokinetic bridges. D, U2OS KO cells were stably transduced with vector, PBK, or PBK-T9A. Total cell lysates from U2OS cell lines which were treated with taxol and probed with the indicated antibodies. E, The indicated U2OS cell lines were used to quantify the percentage of cells in cytokinesis as in (B). F, The specified U2OS cell lines were labeled with PI and flow cytometry analysis was performed to determine the percentage of tetra-ploidy cells in the whole population. G, U2OS control and PBK knockout cell whole lysates were probed with the indicated YAP antibodies. Numbers below the p-S397 YAP blot indicate the quantification of relative band intensity from three independent experiments. H, U2OS control and PBK knockout cell whole lysates were probed with the indicated cell cycle-related antibodies. Numbers below the Cyclin E1 blot indicate the quantification of relative band intensity from three independent experiments. **:p<0.01;***:p<0.001 (t-test).

Figure 6. Overexpression of PBK elicits EMT in normal cells

A, PBK protein expression in various normal and breast cancer cell lines. B, MCF10A (an immortalized breast epithelial cell line) cells transduced with vector control, PBK, or PBK-T9A and probed with the indicated YAP antibodies. C, MCF10A cells transduced with vector control, PBK, or PBK-T9A and probed with the indicated EMT marker antibodies. D, 10x microscope images displaying the morphology differences between MCF10A vector control, PBK, and PBK-T9A. E,F Cell migration (wound healing) assays with cell lines established in (B). G, Cell proliferation assays with cell lines established in (B). H, The cell lines established in (B) were used to quantify the percentage of cells in anaphase/telophase. **:p<0.01;***:p<0.001 (t-test).

Figure 7. Mitotic phosphorylation of PBK inhibits its oncogenic activity in cells

A, Western blot of PBK mono-allelic knockout T47D cell line and transduced PBK and PBK-T9A cell lines. B, C, Invasion assays with cell lines established in (A). D, E, Cell migration (wound healing) assays with cell lines established in (A). F, Cell proliferation assays with cell lines established in (A). *:p<0.05; **:p<0.01; ***:p<0.001 (t-test).