Phosphorylation of the mitotic regulator protein Hec1 by Nek2 kinase is essential for faithful chromosome segregation

Heel (highly expressed in cancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G(2)/M phase. Heel localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Heel is regulated, however, are not known. Here we show that human Heel is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G(2)/M. Nek2 phosphorylates Heel on serine residue 165, both in vitro and in vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Heel. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitive nima mutation in Aspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Heel but fails to phosphorylate it. Whereas wild-type human Heel rescues lethality resulting from deletion of Heel in Saccharomyces cerevesiae, a human Heel mutant or yeast Heel mutant changing Ser(165) to Ala or yeast Heel mutant changing Ser(201) to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Heel by Nek2 is essential for faithful chromosome segregation.