SUBSTRATE-SPECIFICITY AND CELL-CYCLE REGULATION OF THE NEK2 PROTEIN-KINASE, A POTENTIAL HUMAN HOMOLOG OF THE MITOTIC REGULATOR NIMA OF ASPERGILLUS-NIDULANS

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 G(2) to M pha se transition. Hence, it is attractive to speculate that human Nek2 an d fungal NIMA may carry out similar functions at the onset of mitosis, To study the biochemical properties and substrate specificity of huma n Nek2 and compare them to those reported previously for other NIMA-re lated protein kinases, we have expressed Nek2 in insect cells. We show that recombinant Nek2 is active as a serine/threonine-specific protei n kinase and may undergo autophosphorylation. Both human Nek2 and fung al NIMA phosphorylate a similar, albeit not identical, set of proteins and synthetic peptides, and beta-casein was found to be a suitable su bstrate for assaying Nek2 in vitro. By exploiting these findings, we h ave studied the cell cycle regulation of Nek2 activity in HeLa cells. We show that Nek2 activity parallels its abundance, being low during M and G(1) but high during S and G(2) phase. Taken together, our result s suggest that human Nek2 resembles fungal NIMA in its primary structu re, cell cycle regulation of expression, and substrate specificity, bu t that Nek2 may function earlier in the cell cycle than NIMA.