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

Abstract

Mitotic entry requires a major reorganization of the microtubule cytoskeleton. Nlp, a centrosomal protein that binds γ-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 γ-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.

ACKNOWLEDGMENTS

We thank all members of the lab for useful discussion. We are grateful to the National Institute for Basic Biology (Okazaki, Japan) and the MRC Geneservice (Cambridge, United Kingdom) for providing ESTs, B. Edde (Montpellier, France) and H. Yamano (South Mimms, United Kingdom) for GT335 and Xenopus cyclin B2 antibodies, respectively, and D. Stott (Warwick, United Kingdom) for the pGAD-MakV plasmid.

J.R. and J.E.B. were supported by studentships from the BBSRC and Millennium Pharmaceuticals, respectively. This work was also supported by grants to A.M.F. from the BBSRC and the Wellcome Trust. A.M.F. is a Lister Institute Research Fellow.