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J. Biol. Chem., Vol. 282, Issue 36, 26431-26440, September 7, 2007

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Alternative Splicing Controls Nuclear Translocation of the Cell Cycle-regulated Nek2 Kinase^*

Wenjuan Wu¹, Joanne E. Baxter, Samantha L. Wattam, Daniel G. Hayward, Margarida Fardilha, Axel Knebel^¶, Eleanor M. Ford, Edgar F. da Cruz e Silva, and Andrew M. Fry²

From the Laboratório de Transdução de Sinais, Centro de Biologia Celular, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom, and ^¶Kinasource Ltd., Unit 9 South Dudhope Mill, 77 Douglas St., Dundee, DD1 5AN, United Kingdom

Nek2 is a cell cycle-regulated serine/threonine protein kinase that is up-regulated in human cancers. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells and chromatin condensation in meiotic cells. Two major splice variants have been described in vertebrates, Nek2A and Nek2B, that differ in their non-catalytic C termini. Recently, a third splice variant, Nek2C, was identified that lacks an eight-amino acid internal sequence within the C-terminal domain of Nek2A. This excision occurs at the same position as the Nek2A/Nek2B splice point. As predicted from their high degree of similarity, we show here that Nek2C shares many properties with Nek2A including kinase activity, dimerization, protein phosphatase 1 interaction, mitotic degradation, microtubule binding, and centrosome localization. Unexpectedly, though, the non-centrosomal pool of protein exhibits a marked difference in distribution for the three splice variants. Nek2C is mainly nuclear, Nek2B is mainly cytoplasmic, and Nek2A is evenly distributed within nuclei and cytoplasm. Mutagenesis experiments revealed a functional bipartite nuclear localization sequence (NLS) that spans the splice site leading to Nek2C having a strong NLS, Nek2A having a weak NLS, and Nek2B having no NLS. Finally, we identified a 28-kDa protein in nuclear extracts as a potential novel substrate of Nek2. Thus, alternative splicing provides an unusual mechanism for modulating Nek2 localization, enabling it to have both nuclear and cytoplasmic functions.

Received for publication, June 15, 2007 , and in revised form, July 11, 2007.

^* This work was supported by Fundação para a Ciência e Tecnologia of the Portuguese Ministry of Science and Higher Education Grants POCTI/CBO/39799/2001 and POCI/SAU-OBS/57394/2004 (to E. F. C. S.) and by The Wellcome Trust, the Association for International Cancer Research, Cancer Research UK, and the Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom (to A. M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of Ph.D. Fellowship SFRH/BD/6879/2001 from Fundação para a Ciência e Tecnologia.

² To whom correspondence should be addressed. Tel.: 44-116-229-7069; Fax: 44-116-229-7018; E-mail: amf5{at}le.ac.uk.

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