POLO-LIKE KINASE-1 RECRUITS SPINDLE CHECKPOINT PROTEINS TO CENTROMERES AND CREATES THE TENSION-SENSING 3F3/2 PHOSPHOEPITOPE ON MITOTIC KINETOCHORES AND APC/C

Marko J. Kallio^{1, 2}, Leena J. Ahonen³, John R. Daum³, Margaret Bolton⁴, P. Todd Stukenberg⁴, and Gary J. Gorbsky³

¹University of Oklahoma Health Sciences Center, Department of Cell Biology, Oklahoma City, OK 73104, USA, ²Medical Biotechnology Group, Technical Research Centre of Finland and University of Turku, Finland, ³Molecular and Cell Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104,  ⁴Department of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, VA 22908, USA. *gary-gorbsky@omrf.ouhsc.edu

INTRODUCTION. The fidelity of mitosis is ensured by a mechano-chemical system, termed the spindle checkpoint or metaphase checkpoint (1). This checkpoint system monitors microtubule occupancy and/or tension at kinetochores and generates a “wait anaphase” signal if unaligned chromosomes are present. We investigated role of Polo-like kinase-1 (Plk1) in the spindle checkpoint signaling using siRNA-mediated gene-knock down. In addition, we tested various kinetochore-associated kinases for their ability to create 3F3/2 epitope at kinetochores using in vitro phosphorylation of kinetochores in detergent-lysed mitotic cells.

RESULTS. HeLa cells treated with siRNA against Plk1 showed several mitotic phenotypes. First, the spindle poles were unable to separate at the G2/M-phase transition resulting in formation of mono-polar spindles. Secondly, the cells arrested at prometaphase-like stage for several hours before they underwent apoptosis. The amount of Cdc20, a substrate specific activator of APC/C, was significantly reduced at the kinetochores of Plk1-depleted cells; three hours after entry into M-phase the intensity of Cdc20 fluorescence at the kinetochores had decreased by 80 ± 13% compared to the control cells. In addition, Mad2 and Cenp-E levels were significantly reduced at the kinetochores of Plk-depleted cells compared to controls while Ndc80-complex subunits were present at normal amounts.

The 3F3/2 monoclonal antibody identifies a kinetochore phosphoepitope that reversibly responds to the changes in the dynamics of microtubule-kinetochore interactions (2). The 3F3/2 phosphoepitope is expressed at kinetochores that lack mechanical tension and disappears when tension, induced by attachment of spindle microtubules, is applied.  Plk1 is sufficient and necessary for catalyzing the formation of the 3F3/2 phosphoepitope at the kinetochores of mitotic chromosomes. Xenopus egg extracts depleted of Plk1 fail to create the 3F3/2 phosphoepitope at kinetochores, and formation of the phosphoepitope is catalyzed by treatment of lysed mitotic cells with recombinant Plk1.

The 3F3/2 phosphoepitope is expressed in mitosis on certain components of the Anaphase-Promoting Complex/Cyclosome (APC/C), namely Apc1/BimE/Tsg24 and Apc3/Cdc27 (3). Now we show that the 3F3/2 phosphoepitope can be created on immunopurified APC/C with recombinant Plk-1.

DISCUSSION. Both Apc1 and Apc3 localize to mitotic kinetochores and/or chromosomes in living cells.  Moreover, the phosphorylation status of the chromosome-associated Apc1 and Apc3 differs from that of the same proteins in the bulk cytoplasm. We hypothesize that Plk1 phosphorylation of the APC/C at kinetochores may be regulated by tension and may contribute to signaling by the mitotic spindle checkpoint. In addition, Plk1 possibly regulates formation of spindle checkpoint protein complexes by facilitating their proper timely localization at the kinetochores.

ACKNOWLEDGEMENTS. This work was supported by National Institutes of Health
grants to P.T.S. and G.J.G.

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