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Coordinate Regulation of the Mother Centriole Component Nlp by Nek2 and Plk1 Protein Kinases

Joseph Rapley ,^1,, Joanne E. Baxter,^1, Joelle Blot,¹ Samantha L. Wattam,¹ Martina Casenghi,² Patrick Meraldi,^2, Erich A. Nigg,² and Andrew M. Fry^1*

Department of Biochemistry, University of Leicester, Leicester, United Kingdom,¹ Department of Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany²

Received 16 July 2004/ Returned for modification 17 August 2004/ Accepted 12 November 2004

Mitotic entry requires a major reorganization of the microtubule cytoskeleton. Nlp, a centrosomal protein that binds -tubulin, is a G₂/M target of the Plk1 protein kinase. Here, we show that human Nlp and its Xenopus homologue, X-Nlp, are also phosphorylated by the cell cycle-regulated Nek2 kinase. X-Nlp is a 213-kDa mother centriole-specific protein, implicating it in microtubule anchoring. Although constant in abundance throughout the cell cycle, it is displaced from centrosomes upon mitotic entry. Overexpression of active Nek2 or Plk1 causes premature displacement of Nlp from interphase centrosomes. Active Nek2 is also capable of phosphorylating and displacing a mutant form of Nlp that lacks Plk1 phosphorylation sites. Importantly, kinase-inactive Nek2 interferes with Plk1-induced displacement of Nlp from interphase centrosomes and displacement of endogenous Nlp from mitotic spindle poles, while active Nek2 stimulates Plk1 phosphorylation of Nlp in vitro. Unlike Plk1, Nek2 does not prevent association of Nlp with -tubulin. Together, these results provide the first example of a protein involved in microtubule organization that is coordinately regulated at the G₂/M transition by two centrosomal kinases. We also propose that phosphorylation by Nek2 may prime Nlp for phosphorylation by Plk1.

J.R. and J.E.B. contributed equally to this work.

Present address: Department of Molecular Biology, Massachusetts General Hospital, Boston, Mass.

Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass.

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