Present address: Division of Genome Medicine, Institute for Genome Research, The University of Tokushima, Tokushima, Japan.
Critical roles of T-LAK cell-originated protein kinase in cytokinesis
Article first published online: 12 OCT 2009
© 2009 Japanese Cancer Association
Volume 101, Issue 2, pages 403–411, February 2010
How to Cite
Park, J.-H., Nishidate, T., Nakamura, Y. and Katagiri, T. (2010), Critical roles of T-LAK cell-originated protein kinase in cytokinesis. Cancer Science, 101: 403–411. doi: 10.1111/j.1349-7006.2009.01400.x
- Issue published online: 29 JAN 2010
- Article first published online: 12 OCT 2009
- (Received August 29, 2009/Revised September 29, 2009/Accepted September 29, 2009/Online publication November 8, 2009)
Fig. S1. Cyclin-dependent (CDK1) phosphorylates T-LAK cell-originated protein kinase (TOPK) at Thr9 in vitro. (a) Co-localization of TOPK, CDK1, and cyclin B1 at the M-phase. T47D cells were treated with nocodazole (0.3 μg/mL for 18 h) and immunostained individually with monoclonal antibodies of TOPK (red), CDK1 (green), and cyclin B1 (green). Nuclei were counterstained with DAPI (blue) and arrows indicate the cells at mitosis. (b) In vitro kinase assay using recombinant proteins of CDK1/cyclin B1 and TOPK (Escherichia coli). Each of 5 μg of inactive TOPK-recombinant protein, wild-type (WT), and an alanine-substituted mutant (T9A) generated by E. coli was incubated with five units of CDK1/cyclin B1 complex (New England Biolabs, Ipswitch, MA, USA). After reactions for 1 h at 30°C, the mixtures were loaded on SDS-PAGE and autoradiographed (upper panel). The phosphorylated band of TOPK was diminished by competitive incubation with a peptide against the N-terminus of TOPK protein (TOPK1-18 peptide: MEGISNFKTPSKLSEKKK).
Fig. S2. (a) Cytokinetic failure in T47D cells after T-LAK cell-originated protein kinase (TOPK) -depletion. TOPK expression was drastically suppressed in si-TOPK-treated T47D cells compared with si-EGFP-treated cells. β-Actin served as a control for immunoblotting analysis. Cellular morphology was observed by phase-contrast microscopy at 2 days after transfection with si-EGFP or si-TOPK (upper panels), and the percentage of cells with cytokinetic defects was graphed after counting more than 1000 cells (middle panels). To clarify a shape of cell, the actin structure was stained with Alexa Fluor 594 phalloidin (red), and nuclei were counterstained with DAPI (blue) by immunocytochemistry (lower panels). The arrows indicate intercellular bridges. (b) Immunocytochemical staining with a protein marker for the midbody structure. The T47D cells transfected with si-EGFP or si-TOPK were immunostained with anti-PRC1 polyclonal antibody (green) to validate formation of the midbody structure during cytokinesis. (c) Cytokinetic failure in ZR-75-1 cells after TOPK-depletion. Similar to the results of T47D cells (a), many more cells of cytokinetic defects with the intercellular bridges (arrows) were observed in the TOPK-depleted ZR-75-1 cells.
Fig. S3. Identification of a T-LAK cell-originated protein kinase (TOPK)-interacting protein, p47. (a) T47D cells were transfected with plasmids encoding GST-only, GST-tagged TOPK protein of full-length (1-322), and GST-tagged TOPK protein depleted with the C-terminal ETDV motif (1-318). After nocodazole-treatment (0.3 μg/mL for 18 h) and GST pulldown, the co-precipitates with GST-tagged proteins were loaded on SDS-PAGE followed by immunoblotting and silver staining. A differentially appeared protein band in the lanes of GST-TOPKs (red arrow) was collected and analyzed by mass spectrometry. (b) Results from mass spectrometry. Mascot analysis (http://www.matrixscience.com) of peptide mass fingerprinting predicted the isolated sample as human p47 protein.
Fig. S4. T-LAK cell-originated protein kinase (TOPK) interacts with p47/p97. (a) TOPK directly interacts with p47, but not with p97. COS-7 cells were co-transfected with HA-TOPK, GST-p47, and p97-myc. Subsequently, co-precipitants with GST-p47 (left panels) or p97-myc (right panels) proteins were collected by GST-pulldown assay or immunoprecipitation, respectively (see the Materials and Methods). The arrow indicates HA-TOPK co-precipitated with GST-p47. The asterisk indicates a heavy chain of immunoglobulin from anti-myc monoclonal antibody used for immunoprecipitation. (b) TOPK binds to p97 via p47 as an adaptor. COS-7 cells were tri-transfected with GST-p47, myc-p97, or HA-TOPK constructs. The complex among those proteins were immunoprecipitated using anti-myc monoclonal antibody, and immunoblotted with anti-myc, -GST, -HA, and -TOPK monoclonal antibodies, respectively. The asterisk indicates a heavy chain of immunoglobulin from anti-myc monoclonal antibody. (c) Equal amounts of total protein were prepared from breast cancer cell lines (BT-549, HBC5, HCC1937, MCF-7, MDA-MB-231, MDA-MB-435S, SKBR3, T47D, and ZR-75-1), HBL100, and a human mammalian epithelial cell line (HMEC). After SDS-PAGE and membrane transfer, the proteins were immunoblotted with an anti-TOPK monoclonal antibody and anti-p47 or -p97 polyclonal antibodies. β-Actin served as a control for immunoblotting analysis. (d,e) Subcellular localizations of p47, p97, and TOPK in T47D were observed by immunocytochemistry using an anti-p47 polyclonal antibody or Alexa Fluor 488 phalloidin (green) and anti-p97 or -TOPK monoclonal antibodies (red). Nuclei were counter-stained with DAPI (blue). White and yellow arrows indicate mitotic cells (d) and the midbody structure of the cells at cytokinesis (e), respectively.
Fig. S5.In vitro kinase assay with T-LAK cell-originated protein kinase (TOPK) and immunoprecipitated proteins. (a) The candidate proteins to interact with TOPK were expressed in COS-7 cells and immunoprecipitated using an anti-Flag antibody. The endogenous p97 protein was co-precipitated with Flag-p47 and appeared in the SDS-PAGE gel of CBB staining (right panel). (b) In vitro kinase assay of TOPK and the precipitated proteins using radioisotope-labelled ATP. Each protein pool was reacted with 0.5 μg of active TOPK for 30 min at 30°C and autoradiographed. P1 is another candidate protein that may interact with TOPK, but not phosphorylated by TOPK. The asterisk indicates autophosphorylated TOPK. (c) In vitro kinase assay with recombinant proteins of TOPK, p47 and p97. The myc-tagged p97 recombinant protein (1 μg) was incubated with the active TOPK (0.5 μg) with/without GST-tagged p47 (0.5 μg). After reactions for 2 h at 30°C, the proteins were loaded on SDS-PAGE and analyzed by CBB staining and immunoblotting with anti-myc or -GST monoclonal antibodies and anti-phospho-Thr or -Ser specific polyclonal antibodies.
Fig. S6.In vivo phosphorylation of p97 by exogenously expressed T-LAK cell-originated protein kinase (TOPK). HEK293T cells were cotransfected with p97-myc and HA-tagged wild-type (WT) or kinase-dead (KD) of TOPK. Before cell collection, the cells were treated with 0.1 μg/mL of nocodazole for 18 h. After immunoprecipitation with myc-tag antibody, equal amounts of p97-myc protein were precipitated and then immunoblotted with anti-phospho-Thr polyclonal antibody. The arrow indicates the phosphorylated TOPK protein.
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