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A stringent requirement for Plk1 T210 phosphorylation during K-fiber assembly and chromosome congression

Abstract

Polo-like kinase 1 (Plk1) is an essential mitotic regulator and undergoes periodic phosphorylation on threonine 210, a conserved residue in the kinase’s activation loop. While phosphate-mimicking alterations of T210 stimulate Plk1’s kinase activity in vitro, their effects on cell cycle regulation in vivo remain controversial. Using gene targeting, we replaced the native PLK1 locus in human cells with either PLK1 T210A or PLK1 T210D in both dominant and recessive settings. In contrast to previous reports, PLK1 T210D did not accelerate cells prematurely into mitosis, nor could it fulfill the kinase’s essential role in chromosome congression. The latter was traced to an unexpected defect in Plk1-dependent phosphorylation of BubR1, a key mediator of stable kinetochore–microtubule attachment. Using chemical genetics to bypass this defect, we found that Plk1T210D is nonetheless able to induce equatorial RhoA zones and cleavage furrows during mitotic exit. Collectively, our data indicate that K-fibers are sensitive to even subtle perturbations in T210 phosphorylation and caution against relying on Plk1T210D as an in vivo surrogate for the natively activated kinase.

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Acknowledgments

We thank Sabine Elowe and Erich Nigg for kindly providing the pS676-BubR1 antibody. This study was supported by grants from the American Cancer Society (114520-RSG-08-093-01-CCG) and National Institutes of Health (R01GM094972) to P.V.J.

Author information

Correspondence to Prasad V. Jallepalli.

Additional information

Communicated by Erich Nigg

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure 1
figure5

Verification of gene targeting. a Southern blot analysis confirmed recovery of clones with each of the desired genotypes. b RT-PCR analysis confirmed monoallelic expression of PLK1T210A and PLK1T210D after AdCre infection. (JPEG 41 kb)

Supplementary Figure 2
figure6

Time course of mitotic arrest and slippage in cells hemizygous for PLK1 T210A or PLK1 T210D. a Cells of the indicated genotypes were infected with AdCre and harvested 3 days laster. Nocodazole was added to the media 12 h prior to cell harvest. Lysates were probed with an antibody against Plk1. b Cells were collected 0, 1, 2, or 3 days after AdCre infection and scored based on nuclear morphology. At least 3 sets of 100 cells were counted for each timepoint. Error bars represent SEM. (JPEG 39 kb)

Supplementary Figure 3
figure7

Chemical genetics confirms that Plk1T210D is unable to support chromosome congression and satisfy the spindle checkpoint. a Chromosome alignment assay. Cells were released from a nocodazole block into medium containing 3-MB-PP1 and MG132. Two hours later, cells were fixed and stained with DAPI (blue), CREST antisera (red) and α-tubulin antibodies (green). At least 50 cells were scored per sample. Scale bar represents 10 μm. b Cells were treated with 3-MB-PP1 and nocodazole, then stained for S676-phosphorylated and total BubR1 as in Fig. 3a. c Cold stability of K-fibers in 3-MB-PP1 treated Plk1as/wt and Plk1as/T210D cells was determined as in Fig. 2e. d Time course of mitotic arrest and slippage after allelic-specific inhibition in Plk1as/T210D cells. Percentages of mitotic and micronucleated cells were determined from three sets of 100 cells each. Error bars represent SEM. e Wild type, T210D, or T210A Plk1 were immunoprecipitated from Plk1as/wt, Plk1as/T210D, and Plk1as/T210A cells using mCherry-specific antibodies, then incubated with casein and [γ-32P]ATP for the times indicated. Samples were resolved by SDS-PAGE and either processed for Coomassie staining and phosphorimager detection (top and middle rows) or blotted to detect Plk1 (bottom row). (JPEG 99 kb)

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Paschal, C.R., Maciejowski, J. & Jallepalli, P.V. A stringent requirement for Plk1 T210 phosphorylation during K-fiber assembly and chromosome congression. Chromosoma 121, 565–572 (2012) doi:10.1007/s00412-012-0375-8

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Keywords

  • Spindle Checkpoint
  • Human Retinal Pigment Epithelial Cell
  • Mitotic Entry
  • Microtubule Attachment
  • Monastrol