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J. Biol. Chem., Vol. 282, Issue 9, 6833-6842, March 2, 2007

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Structure and Regulation of the Human Nek2 Centrosomal Kinase^*

Peter Rellos¹, Frank J. Ivins¹, Joanne E. Baxter^¶12, Ashley Pike, Timothy J. Nott, Donna-Marie Parkinson^¶, Sanjan Das, Steven Howell, Oleg Fedorov, Qi Yu Shen^¶, Andrew M. Fry^¶3, Stefan Knapp⁴, and Stephen J. Smerdon⁵

From the Structural Genomics Consortium, Botnar Research Centre, University of Oxford, Oxford OX3 7LD, United Kingdom, Medical Research Council National Institute for Medical Research, Division of Molecular Structure, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom and ^¶Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom

The dimeric Ser/Thr kinase Nek2 regulates centrosome cohesion and separation through phosphorylation of structural components of the centrosome, and aberrant regulation of Nek2 activity can lead to aneuploid defects characteristic of cancer cells. Mutational analysis of autophosphorylation sites within the kinase domain identified by mass spectrometry shows a complex pattern of positive and negative regulatory effects on kinase activity that are correlated with effects on centrosomal splitting efficiency in vivo. The 2.2-Å resolution x-ray structure of the Nek2 kinase domain in complex with a pyrrole-indolinone inhibitor reveals an inhibitory helical motif within the activation loop. This helix presents a steric barrier to formation of the active enzyme and generates a surface that may be exploitable in the design of specific inhibitors that selectively target the inactive state. Comparison of this "auto-inhibitory" conformation with similar arrangements in cyclin-dependent kinase 2 and epidermal growth factor receptor kinase suggests a role for dimerization-dependent allosteric regulation that combines with autophosphorylation and protein phosphatase 1c phosphatase activity to generate the precise spatial and temporal control required for Nek2 function in centrosomal maturation.

Received for publication, October 16, 2006 , and in revised form, December 13, 2006.

The atomic coordinates and structure factors (code 2JAV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants (to A. M. F.) from The Wellcome Trust, the Association for International Cancer Research, and Cancer Research UK. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material, including supplemental Figs. S1-S3.

¹ These authors contributed equally to this work.

² Supported by a Ph.D. studentship from Millennium Pharmaceuticals Inc. (Boston).

³ To whom correspondence may be addressed. Tel.: 44-116-229-7069; E-mail: amf5{at}leicester.ac.uk.

⁴ To whom correspondence may be addressed. Tel.: 44-1865-227978; E-mail: Stefan.Knapp{at}sgc.ox.ac.uk.

⁵ To whom correspondence may be addressed. Tel.: 44-20-8816-2533; E-mail: stephen.smerdon{at}nimr.mrc.ac.uk.

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