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Molecular and Cellular Biology, June 2009, p. 3134-3150, Vol. 29, No. 11
0270-7306/09/$08.00+0 doi:10.1128/MCB.01663-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Functional Dynamics of Polo-Like Kinase 1 at the Centrosome
,
Kazuhiro Kishi,1
Marcel A. T. M. van Vugt,1
Ken-ichi Okamoto,2
Yasunori Hayashi,2 and
Michael B. Yaffe1*
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, E18-580, Cambridge, Massachusetts 02139,1 RIKEN-MIT Neuroscience Research Center, The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 46-4243A, 43 Vassar Street, Cambridge, Massachusetts 021392
Received 27 October 2008/ Returned for modification 20 November 2008/ Accepted 12 March 2009
Polo-like kinase 1 (Plk1) functions as a key regulator of mitotic events by phosphorylating substrate proteins on centrosomes, kinetochores, the mitotic spindle, and the midbody. Through mechanisms that are incompletely understood, Plk1 is released from and relocalizes to different mitotic structures as cells proceed through mitosis. We used fluorescence recovery after photobleaching to examine the kinetics of this process in more detail. We observed that Plk1 displayed a range of different recovery rates that differ at each mitotic substructure and depend on both the Polo-box domain and a functional kinase domain. Upon mitotic entry, centrosomal Plk1 becomes more dynamic, a process that is directly enhanced by Plk1 kinase activity. In contrast, Plk1 displays little dynamic exchange at the midbody, a process that again is modulated by the kinase activity of Plk1. Our findings suggest that the intrinsic kinase activity of Plk1 triggers its release from early mitotic structures and its relocalization to late mitotic structures. To assess the importance of Plk1 dynamic relocalization, Plk1 was persistently tethered to the centrosome. This resulted in a G2 delay, followed by a prominent prometaphase arrest, as a consequence of defective spindle formation and activation of the spindle checkpoint. The dynamic release of Plk1 from early mitotic structures is thus crucial for mid- to late-stage mitotic events and demonstrates the importance of a fully dynamic Plk1 at the centrosome for proper cell cycle progression. This dependence on dynamic Plk1 was further observed during the mitotic reentry of cells after a DNA damage G2 checkpoint, as this process was significantly delayed upon centrosomal tethering of Plk1. These results indicate that mitotic progression and control of mitotic reentry after DNA damage resides, at least in part, on the dynamic behavior of Plk1.
* Corresponding author. Mailing address: Department of Biology and Biological Engineering, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139. Phone: (617) 452-2103. Fax: (617) 452-4978. E-mail: myaffe{at}mit.edu
Published ahead of print on 23 March 2009.
Supplemental material for this article may be found at http://mcb.asm.org/.
Molecular and Cellular Biology, June 2009, p. 3134-3150, Vol. 29, No. 11
0270-7306/09/$08.00+0 doi:10.1128/MCB.01663-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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