Abstract
Plk1, an evolutionarily conserved M phase kinase, associates with not only spindle poles but also kinetochores during prometaphase. However, the role of Plk1 at kinetochores has been poorly understood. Here we show that BubR1 mediates the action of Plk1 at kinetochores for proper chromosome alignment. Our results show that BubR1 colocalizes with Plk1 at kinetochores of unaligned chromosomes and physically interacts with Plk1 in prometaphase cells. Down-regulation of Plk1 by small interfering RNA abolished the mobility-shifted, hyperphosphorylated form of BubR1 in the prometaphase-arrested cells. In addition, BubR1 was phosphorylated by Plk1 in vitro at two Plk1 consensus sites in the kinase domain of BubR1. The add-back of either wild-type BubR1 or BubR1 2E, in which the two Plk1 phosphorylation sites were replaced by glutamic acids, but not that of BubR1 2A, an unphosphorylatable mutant, rescued the chromosome alignment defects in BubR1-deficient cells. Moreover, when both Plk1 and BubR1 were down-regulated, the add-back of BubR1 2E, but not that of wild-type BubR1, rescued the chromosome alignment defects. These results taken together suggest that Plk1 facilitates chromosome alignment during prometaphase through BubR1.
Highlights
Plk1, a mammalian ortholog of Drosophila polo, plays a crucial role in multiple stages of mitosis (6 – 8)
When the kinetochores of a sisterchromatid pair are attached by microtubules from opposite spindle poles, tension develops across the sister kinetochores
BubR1 is able to inhibit APC/C by binding to Cdc20 [14] and associates with the kinetochore motor protein CENP-E (16 –18), which is shown to be essential for both chromosome alignment and spindle checkpoint (19 –21)
Summary
Cell Culture and Drug Treatment—HeLaS3 cells, HeLa cells expressing a GFP-histone H2B protein, and COS7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. When transfection was performed before the first thymidine block, HeLa cells were transfected in Opti-MEM with the use of Lipofectamine Plus (Invitrogen) for 2 h and washed into fresh medium containing thymidine for the first block. For transfection with DNA vectors and RNA oligonucleotides during synchronization, cells were transfected in Opti-MEM with the use of Lipofectamine Plus (Invitrogen) for 2 h and washed into fresh medium containing thymidine for the first block. Mitotic cells were treated with a calcium-containing buffer for 40 s during permeabilization and fixed for 10 min with 4% paraformaldehyde in the same buffer as described by Mitchison [25] Primary antibodies included those against BubR1 (mouse; Chemicon), Aurora-B (mouse; Transduction Laboratories), Myc (9E10, mouse; Santa Cruz Biotechnology), ␣-tubulin (DM1A, mouse; Sigma), and -tubulin (TUB2.1, mouse; Sigma). Image analysis was performed using either SoftWoRx or Adobe Photoshop (Adobe Systems)
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