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Vol. 16, Issue 4, 1711-1724, April 2005

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Dynamic Recruitment of Nek2 Kinase to the Centrosome Involves Microtubules, PCM-1, and Localized Proteasomal Degradation

Rebecca S. Hames ^*, Renarta E. Crookes ^* , Kees R. Straatman , Andreas Merdes , Michelle J. Hayes ^*, Alison J. Faragher ^* ||, and Andrew M. Fry ^*

^* Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom; Department of Genetics, University of Leicester, Leicester LE1 7RH, United Kingdom; and Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom

Submitted August 11, 2004; Revised December 3, 2004; Accepted January 6, 2005
Monitoring Editor: Tim Stearns

Centrosomes undergo dramatic changes in composition and activity during cell cycle progression. Yet mechanisms involved in recruiting centrosomal proteins are poorly understood. Nek2 is a cell cycle–regulated protein kinase required for regulation of centrosome structure at the G2/M transition. Here, we have addressed the processes involved in trafficking of Nek2 to the centrosome of human adult cells. We find that Nek2 exists in small, highly dynamic cytoplasmic particles that move to and from the centrosome. Many of these particles align along microtubules and a motif was identified in the Nek2 C-terminal noncatalytic domain that allows both microtubule binding and centrosome localization. FRAP experiments reveal that 70% of centrosomal Nek2 is rapidly turned over (t_1/2 3 s). Microtubules facilitate Nek2 trafficking to the centrosome but only over long distances. Cytoplasmic Nek2 particles colocalize in part with PCM-1 containing centriolar satellites and depletion of PCM-1 interferes with centrosomal recruitment of Nek2 and its substrate C-Nap1. Finally, we show that proteasomal degradation is necessary to allow rapid recruitment of new Nek2 molecules to the centrosome. Together, these data highlight multiple processes involved in regulating the abundance of Nek2 kinase at the centrosome including microtubule binding, the centriolar satellite component PCM-1, and localized protein degradation.

Abbreviations used: APC/C, anaphase promoting complex/cyclosome; C-Nap1, centrosomal Nek2-associated protein 1; EGFP, enhanced green fluorescent protein; FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; Nek2, NIMA-related kinase 2; PCM-1, pericentriolar material protein 1.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present address: Department of Cytogenetics, Sheffield Children's Hospital, Sheffield S10 2TH, United Kingdom

^|| Present address: MRC Toxicology Unit, Hodgkin Building, University of Leicester, Leicester LE1 7RH, United Kingdom.

Address correspondence to: Andrew M. Fry (amf5{at}le.ac.uk).

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