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 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 beta-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.
Fry
A M
AM
Swiss Institute for Experimental Cancer Research (ISREC), Epalinges.
Schultz
S J
SJ
Bartek
J
J
Nigg
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D001233:Aspergillus nidulans / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity
D018797:Cell Cycle Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; C000606914:NEK1 protein, human; C089441:NEK2 protein, human; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; C442331:NIMA-related kinase 6; D017346:Protein Serine-Threonine Kinases