A stringent requirement for Plk1 T210 phosphorylation during K-fiber assembly and chromosome congression
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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.
KeywordsSpindle Checkpoint Human Retinal Pigment Epithelial Cell Mitotic Entry Microtubule Attachment Monastrol
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.
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