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First published online 15 June 2004
doi: 10.1242/jcs.01173

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Molecular interactions of Polo-like-kinase 1 with the mitotic kinesin-like protein CHO1/MKLP-1

¹ Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
² Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church Street, Minneapolis, MN 55455, USA

View larger version (26K): [in a new window]   Fig. 1. The C-terminal Polo-box domain of Plk1 is responsible for its binding to endogenous CHO1/MKLP-1. (A) FLAG-tagged Plk1 constructs used in the transfection experiment. The hatched and filled bars indicate the positions of kinase domain (residues 53-305) and Polo box (residues 410-440), respectively. Stars indicate the positions of three point mutations within the polo box (W414F, V415A, L427A). (B,C) Cos-7 cells were transiently transfected with 10 µg of the indicated Plk1 constructs per 10-cm Petri dish. 30 hours after transfection, cells were treated with 100 ng ml–1 nocodazole for 16 hours and harvested. Cell lysates were subjected to anti-CHO1/MKLP-1 antibody immunoprecipitation, and anti-FLAG western blotting. As a control, the same quantities of cell lysates were incubated with Protein-A beads only. About 5% of the total cell lysates was used to assess expression. (D) Different amounts of Plk1 constructs (2.5 µg, 5 µg or 10 µg of DNA per 10-cm dish) were used for transfection as described above. Cell lysates were immunoprecipitated with anti-CHO1/MKLP-1 antibody, followed by anti-FLAG immunoblotting.

View larger version (43K): [in a new window]   Fig. 2. The stalk domain of CHO1/MKLP-1 is responsible for its binding to endogenous Plk1. (A) HA-tagged CHO1/MKLP-1 constructs used in the transfection experiment. (B) Cos-7 cells were transiently transfected with CHO1/MKLP-1 constructs as indicated. 30 hours after transfection, cells were treated with 100 ng ml–1 nocodazole for 16 hours and harvested. About 5% of total soluble cell lysates (leftmost six lanes) or immunoprecipitates with anti-Plk1 rabbit polyclonal antibody from the transfected cells (rightmost six lanes) were subjected to western blotting with anti-HA antibody. The positions of molecular size markers are indicated at the left. Stars indicate non-specific proteins that cross-react with the anti-HA antibody. (C) Colocalization of the stalk domain of CHO1/MKLP-1 with Plk1 at the mid-body. GFP-fused CHO1/MKLP-1 domains were transfected into HeLa cells. 30 hours after transfection, cells were treated with 50 ng ml–1 nocodazole for 8 hours. Mitotic cells were mechanically shaken off and collected. After nocodazole was washed away over a 1 hour period with ice-cold DMEM, the cells were mixed with prewarmed medium and replated onto polylysine-coated coverslips for 75 minutes. Plk1 was stained with anti-Plk1 mouse monoclonal antibody, followed by Cy3-conjugated secondary antibody. DNA was stained with DAPI. Scale bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 3. Mislocalization of Plk1 in CHO1/MKLP-1-depleted cells during late mitosis. (A,B) HeLa cells on coverslips were first synchronized at late G1 phase with double-thymidine block. Upon release from the second thymidine block, cells were directly transfected with synthetic 21-nucleotide double-stranded RNA targeting CHO1/MKLP-1. Cells were fixed 10 hours later and subjected to double staining with anti-CHO1 and anti-Plk1 antibodies. DNA was stained with DAPI. (C,D) HeLa cells were transfected withpBS/U6-GFP-Plk1. 48 hours after transfection, cells were harvested and subjected to anti-Plk1 western blot (C) or anti-CHO1 staining (D). Scale bar, 10 µm.

View larger version (59K): [in a new window]   Fig. 4. Identification of Ser-904 and Ser-905 of CHO1/MKLP-1 as two major Plk1 phosphorylation sites. (A) GST-fused CHO1/MKLP-1 domains were purified (left) and subjected to in vitro kinase reactions with purified Plk1 (right). Only the tail domain (residues 643-953) was strongly phosphorylated by Plk1, indicated by the arrowhead on the right. (B,C) Different regions of the tail domain fused with GST were purified and subjected to in vitro kinase reactions with Plk1. The stars indicate the positions of the proteins based on Coomassie Blue staining. The size markers are indicated on the left. The region 901-953 contains Plk1 phosphorylation sites. (D) Wild-type or mutant GST fusion proteins of the 901-953 region were subjected to kinase assays with recombinant Plk1. (E) Wild-type or mutant GST fusion proteins of the 801-900 region (left) or the 643-953 region (right) were subjected to kinase assays with recombinant Plk1. The 2A mutant contains the mutations Ser805Ala and Ser807Ala; the 4A mutant contains the mutations Ser805Ala, Ser807Ala, Ser904Ala and Ser905Ala. The arrow on the left indicates the position of GST-Plk1, and the stars indicate the positions of the proteins based on Coomassie Blue staining. (F) Purified GST-fused CHO1 C-terminus (residues 406953) was subjected to kinase assay with either kinase defective (KM) or wild-type (WT) GST-Plk1. (G) CHO1 is highly phosphorylated during mitosis. Asynchronous or mitotic (treated with 200 ng ml–1 nocodazole for 10 hours) HeLa cells were labeled with [32P]orthophosphate for 4 hours. Cell lysates were either directly analysed on an anti-Plk1 western blot or immunoprecipitated with anti-CHO1 antibody first, then detected with anti-CHO1 western blot. Phosphorylated CHO1 was visualized by autoradiography and quantified using a phosphorimager. (H) HeLa cells were transfected with pBS/U6-Plk1 to deplete Plk1 first, then treated with nocodazole for 10 hours and labeled in vivo as in (G). (I) Cells were transfected with GFP-CHO1 wild-type or 4A mutant as indicated. 30 hours after transfection, cells were incubated with nocodazole for 10 hours and labeled with [32P]-orthophosphate for additional 4 hours. Cell lysates were immunoprecipitated with anti-GFP antibody and phosphorylated GFP-CHO1 was quantified using a phosphorimager.

View larger version (42K): [in a new window]   Fig. 5. Depletion of CHO1/MKLP-1 causes multinucleation. (A) Efficient depletion of CHO1/MKLP-1 with vector-based RNAi. HeLa cells were transfected with either control vector or pBS/U6-CHO1. Two days after transfection, cells were harvested and cell lysates were subjected to direct western blot using antibodies indicated on the left. (B) HeLa cells were cotransfected with pBS/U6-CHO1-1st half, GFP and pBabe-Puro (control), with pBS/U6-CHO1, GFP and pBabe-Puro (CHO1 depletion) or with pBS/U6-CHO1, GFP-hamster-CHO1 and pBabePuro (CHO1 depletion, then rescue) at the ratio of 5:5:1. After 2 days of puromycin selection of transfection-positive cells, cells were further incubated up to day 6 in the presence of puromycin. Cell numbers were counted with a cytometer. (C) FACS profiles of CHO1-depleted cells at 3 days and 4 days after transfection. (D) Representative confocal images of CHO1-depleted cells. Blue indicates DNA staining with DAPI and green indicates -tubulin staining. (E) Quantification of results of three independent experiments (400 cells each); bars indicate standard deviations. (F-H) CHO1-depleted cells have multiple centrosomes. (F) HeLa cells were transfected as described in (B). After 2 days of puromycin selection, cells were stained with antibodies against -tubulin (green) and -tubulin (red), and DNA was stained with DAPI (blue). (G) Quantification of results indicates that almost every multinucleate cell has multiple centrosomes. (H) CHO1/MKLP-1-depleted cells form multiple spindle poles during mitosis. Scale bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 6. Cytokinesis defect in CHO1-depleted cells. (A,B) HeLa cells were transfected with control vector (A) or pBS/U6-GFP-CHO1 (B), in which GFP was independently expressed as a marker for transfected cells. Only the GFP-positive cells produced hairpin RNA to induce CHO1 depletion. 30 hours after transfection, cells were fixed and subjected to anti--tubulin staining. Arrows indicated the regions of mid-zone/mid-body structure. Scale bar, 10 µm. (C) The protocol used to deplete CHO1 in well-synchronized cells. (D) Histogram quantifying the results.

View larger version (61K): [in a new window]   Fig. 7. Multinucleation induced by overexpression of unphosphorylatable CHO1 mutant. HeLa cells were transfected with GFP-CHO1-WT or -4A constructs using PolyFect reagents. 48 hours after transfection, cells were fixed and subjected to anti-CHO1 or anti-Plk1 staining as indicated. (A) GFP-CHO1 expression patterns during interphase. (B) Nuclear localization of GFP-CHO1 constructs with high resolution. (C,D) Intercellular bridge localization of GFP-CHO1-WT, but not the 4A mutant. (E) GFP-CHO1 expression patterns during mitosis. (F) Plk1 localization in GFP-CHO1-overexpressing cells. (G) Representative cells with overexpressed GFP-CHO1-4A. (H) Histogram quantifying the results. Scale bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 8. Dominant negative effect of Plk1-unphosphorylatable mutant CHO1 on cytokinesis. (A) The protocol used to overexpress CHO1 constructs in well-synchronized cells. (B) Representative images to show cells with overexpressed GFP-CHO1 using the protocol in (A). Arrows indicate the positions of mid-bodies. (C) Representative image to show cells with overexpressed GFP-CHO1-4A using the protocol in (A). (D) Histogram quantifying the results. Scale bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 9. Effect of Plk1-unphosphorylatable mutations on the rescue ability of hamster CHO1/MKLP-1 toward CHO1-depletion induced multinucleation in HeLa cells. (A) Sequence alignment of RNAi targeting site between human and hamster MKLP-1. The nonconserved sites are indicated in bold. (B) HeLa cells were cotransfected with hamster vectors that express CHO1 as indicated on the left. Three days after transfection, cells were harvested and subjected to FACS analysis. GFP-positive cells were gated and the proportion of cells with 8N DNA content was calculated. (C) Histogram showing results from six independent experiments; bars indicate standard deviations. (D) Representative cells with overexpressed GFP-CHO1 constructs in the absence of endogenous CHO1. (E) Histogram quantifying the results.

View larger version (24K): [in a new window]   Fig. 10. Failure of hamster GFP-CHO1-4A to rescue the CHO1-depletion-induced cytokinesis defect. (A,B) HeLa cells were first transfected with pBS/U6-CHO1 to deplete the endogenous CHO1. After overnight incubation, cells were transfected with either hamster GFP-CHO1-WT (A) or GFP-CHO1-4A (B) for 24 hours. Cells were fixed and subjected to anti-Plk1 staining. (C) The protocol used to rescue the CHO1-depletion-induced cytokinesis defect by hamster GFP-CHO1 in well-synchronized cells. (D,E) Histograms quantifying the results. Scale bar, 10 µm.

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