Critical roles of T-LAK cell-originated protein kinase in cytokinesis

Abstract

We previously reported up-regulation of T-LAK cell-originated protein kinase (TOPK), a novel mitotic kinase, in the great majority of breast cancers. Here we report its critical roles in mitosis, especially in cytokinesis. We found that protein phosphatase 1 alpha (PP1alpha) inactivation by cyclin-dependent kinase 1 (CDK1)/cyclin B1 caused enhancement of autophosphorylation of TOPK and resulted in its activation at an early stage of mitosis. Then TOPK interacted with and phosphorylated p97, a member of the AAA+ family of ATPase proteins, through an interaction with p47 protein as an adaptor protein. Interestingly, knockdown of TOPK or p97 in breast cancer cells caused the mitotic failures in the abscission process. This mitotic failure could be rescued by additional exogenous introduction of wild-type TOPK protein, but not by that of its kinase-dead form. Our findings suggest that TOPK is indispensable for cancer cell cytokinesis throughout phosphorylation on p97.

Figure 1

Autophosphorylation of T‐LAK cell‐originated protein kinase (TOPK) protein in mitotic cells. (a) TOPK was phosphorylated in mitotic cells. The isolated cells at mitosis (M) or the interphase (I) were analyzed by FACS analysis, and cell lysates were treated with lambda protein phosphatase (λPPase) before immunoblotting with TOPK monoclonal antibody. (b) Autophosphorylation of TOPK in mitotic cells. T47D cells were transfected with wild‐type TOPK (WT), alanine‐substituted mutant at Thr9 (T9A), kinase‐dead (KD), and double mutant (T9A/KD), respectively, and immunoblotting was performed using anti‐HA monoclonal antibody. Both WT and T9A, but neither KD nor T9A/KD, were phosphorylated and showed a slowly migrating band (arrow). (c) Phosphorylation of TOPK was induced by treatment of okadaic acid (OA). Left panels: T47D cells were treated with 50, 100, and 200 nm of OA, and cells were harvested at 3 h after treatment of OA. Right panels: The phosphorylated band, which appeared after treatment with 200 nm of OA for 3 h, was verified by λPPase assay. (d) Interaction of TOPK and protein phosphatase 1 alpha (PP1α). COS‐7 cells were co‐transfected with GST‐fused PP1α (PP1α‐GST), HA‐tagged TOPK (HA‐TOPK), and pulled‐down with equilibrated glutathione sepharose 4B beads or immunoprecipitated with anti‐HA monoclonal antibody, followed by immunoblotting analysis using anti‐GST or HA monoclonal antibodies. (e) The recombinant TOPK (left panel) and endogenous TOPK from mitotic cell lysates (right panel) were dephosphorylated by treatment of recombinant PP1α protein. λPPase served as a positive control for clarification of the band‐shift of TOPK protein.

Figure 2

T‐LAK cell‐originated protein kinase (TOPK) was activated by cyclin‐dependent kinase 1 (CDK1) through inactivation of protein phosphatase 1 alpha (PP1α). (a) FACS analysis of M‐phase arrested cells after treatment with CDK1 inhibitor. T47D cells were treated with nocodazole for 16 h, followed by incubation with 25 nmol/L of CDK1 inhibitor (CGP74514A) from 0 to 4 h before FACS analysis. The population (%) of each cell cycle in the indicated time points was graphed. Grey bar, G1 phase; white bar, S phase; black bar, G2/M phase. (b) Expression of TOPK‐related proteins after treatment with CDK1 inhibitor. Equal amounts of total protein were immunoblotted with anti‐TOPK and anti‐total‐Rb monoclonal antibodies and with anti‐phospho‐PP1α (Thr320), anti‐total‐PP1α, and anti‐phospho‐Rb (Ser807/811) polyclonal antibodies. The arrow indicates the phosphorylated TOPK protein. (c) FACS analysis as mitosis progression. Only mitotic T47D cells were isolated and released from mitosis (see the Materials and Methods), followed by FACS analysis. The population (%) of each cell cycle is graphed. Grey bar, G1 phase; white bar, S phase; black bar, G2/M phase. (d) Expression of TOPK‐related proteins during mitosis progression. Equal amounts of total protein were immunoblotted with monoclonal antibodies of anti‐cyclin B1 and anti‐CDK1. The arrow indicates the phosphorylated TOPK protein.

Figure 3

Cytokinetic failure induced by T‐LAK cell‐originated protein kinase (TOPK)‐depletion. (a,b) Two days after transfection with si‐enhanced green fluorescent protein (EGFP) (a) or si‐TOPK (b) in T47D cells, the duration of cell mitosis was measured by time‐lapse microscopy. White and yellow arrows indicate the cells under mitosis and the intercellular bridge, respectively. (c) RNAi rescue experiments. T47D cells were transfected with wild‐type (WT) or kinase‐dead of HA‐tagged TOPK‐expression vectors, and subsequently were transfected with si‐TOPK. Forty‐eight hours after transfection of siRNA, immunocytochemistry was performed. The exogenously expressed TOPK proteins were immunostained with anti‐HA monoclonal antibody (green). The wild‐type of TOPK restored cell morphology of T47D (white arrows), whereas the kinase‐dead did not but still showed intercellular bridges (yellow arrows). The actin structure was stained with Alexa Fluor 594 phalloidin (red), and nuclei were counter‐stained with DAPI (blue). The percentage of cytokinetic defects in the cells expressing the exogenous wild‐type or kinase‐dead of TOPK protein was graphed after counting 50 cells.

Figure 4

T‐LAK cell‐originated protein kinase (TOPK) phosphorylates p97 at the M‐phase. (a) p47/p97 complex interacts with TOPK in the mitotic cells. GST‐pulldown assay was performed with the cell lysates from T47D treated with/without nocodazole. The protein pools co‐precipitated with GST or GST‐TOPK were immunoblotted with anti‐GST and anti‐TOPK monoclonal antibodies, and with anti‐p47 and anti‐p97 polyclonal antibodies. (b) In vitro kinase assay of recombinant TOPK and p97 proteins using non‐labeled ATP. Each of 5 μg of recombinant p97 protein was incubated with 1 μg of active TOPK for 2 h at 30°C. After reaction, the protein samples were loaded on SDS‐PAGE gel followed by Coomassie Brilliant Blue (CBB) staining and immunoblotting with anti phospho‐Thr polyclonal antibody. The asterisk indicates autophosphorylation of TOPK. (c) Mitotic phosphorylation of p97. The isolated T47D cells at interphase (I) or mitosis (M) were analyzed by FACS analysis, and cell lysates were immunoblotted with anti‐TOPK monoclonal antibody (left) or phospho‐Thr specific polyclonal antibody (right) after treatment with lambda protein phosphatase (λPPase). β‐actin and total p97 served as loading controls. (d) TOPK‐depletion attenuates mitotic phosphorylation of p97. After transfection with si‐EGFP or si‐TOPK, T47D cells were incubated for 12 h and then treated with/without 0.3 μg/mL of nocodazole for additional 18 h before cell collection. The population (%) of each cell cycle is graphed. Grey bar, G1 phase; white bar, S phase; black bar, G2/M phase. (e) Equal amounts of total protein were immunoblotted with anti‐TOPK monoclonal antibody to show knockdown and phosphorylation (arrow) of TOPK protein induced by si‐TOPK and nocodazole, respectively. After in vitro binding and pulldown assays with GST‐tagged p47 protein, equal amounts of endogenous p97 proteins were co‐precipitated with sepharose 4B beads and immunoblotted with phospho‐Thr specific polyclonal antibody. Total p97 and GST‐p47 served as loading controls for the GST‐pulldown assay.

Figure 5

p97 is indispensable for cytokinesis. (a) Knockdown of p97 resulted in cytokinetic defects in T47D cells. Forty‐eight hours after transfection, the p97 protein was significantly depleted in si‐p97‐treated T47D cells compared with si‐enhanced green fluorescent protein (EGFP)‐treated cells. β‐actin served as a loading control for immunoblotting analysis. By observation of cell morphology using a phase contrast microscopy, the p97‐depleted cells showed intercellular bridges as indicated by yellow arrows. The percentage of cells with cytokinetic defects were graphed after counting more than 1000 cells (P < 0.0001, Student’s t‐test). (b) Immunocytochemical staining of the T47D cells. To clarify a shape of cell, the actin structure was stained with Alexa Fluor 488 phalloidin (green), and nuclei were counter‐stained with DAPI (blue). The p97‐depleted cells failed in the cytokinesis and showed elongated intercellular bridges (yellow arrows). The white arrow indicates midbody formation of si‐EGFP‐treated cells at the cytokinesis. (c,d) Knockdown of p97 resulted in cytokinetic defects in ZR‐75‐1 cells. The depletion of p97 protein, cell shape, and percentages of the cells with cytokinetic defects were validated as mentioned above.

Figure 6

Schema of the roles of T‐LAK cell‐originated protein kinase (TOPK) in cancer cell mitosis. At an early stage of mitosis, TOPK autophosphorylation is induced though the inactivation of protein phosphatase 1 alpha (PP1α) by cyclin‐dependent kinase (CDK1)/cyclin B1 complex. Then, the activated TOPK phosphorylates p97 through its interaction with p47. Finally, the restored PP1α dephosphorylates and inactivates TOPK to the steady level at the exit of mitosis.