Substrate Specificity and Cell Cycle Regulation of the Nek2 Protein Kinase, a Potential Human Homolog of the Mitotic Regulator NIMA of Aspergillus nidulans

doi:10.1074/jbc.270.21.12899

Substrate Specificity and Cell Cycle Regulation of the Nek2 Protein Kinase, a Potential Human Homolog of the Mitotic Regulator NIMA of Aspergillus nidulans(*)

From the (1) Swiss Institute for Experimental Cancer Research (ISREC), Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland and the
(2) Danish Cancer Society, Division for Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen, Denmark

** To whom correspondence should be addressed. Tel.: 41 21 692 5884; Fax: 41 21 652 6933.

↵^¶ Recipient of a postdoctoral fellowship from the European Molecular Biology Organization. Present address: Dept. of Microbiology, SC-42, University of Washington, Seattle, WA, 98195.

Abstract

The human Nek2 protein kinase is the closest known mammalian relative of the mitotic regulator NIMA of Aspergillus nidulans. The two kinases share 47% sequence identity over their catalytic domains and display a similar cell cycle-dependent expression peaking at the G² to M phase transition. Hence, it is attractive to speculate that human Nek2 and fungal NIMA may carry out similar functions at the onset of mitosis. To study the biochemical properties and substrate specificity of human Nek2 and compare them to those reported previously for other NIMA-related protein kinases, we have expressed Nek2 in insect cells. We show that recombinant Nek2 is active as a serine/threonine-specific protein kinase and may undergo autophosphorylation. Both human Nek2 and fungal NIMA phosphorylate a similar, albeit not identical, set of proteins and synthetic peptides, and β-casein was found to be a suitable substrate for assaying Nek2 in vitro. By exploiting these findings, we have studied the cell cycle regulation of Nek2 activity in HeLa cells. We show that Nek2 activity parallels its abundance, being low during M and G¹ but high during S and G² phase. Taken together, our results suggest that human Nek2 resembles fungal NIMA in its primary structure, cell cycle regulation of expression, and substrate specificity, but that Nek2 may function earlier in the cell cycle than NIMA.

Footnotes

↵^§ Recipient of a postdoctoral fellowhip from The Royal Society.
↵* This work was supported by grants from the Swiss National Science Foundation (31-33615.92), the Swiss Cancer League (FOR 205) (to E. A. N.), and the Danish Cancer League (to J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Nek

NIMA-related kinase

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

FACS

fluorescence-activated cell sorter

MAP

microtubule-associated protein

PLM

phospholemma-derived peptide.