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

J Biol Chem. 2002 Dec 20;277(51):49408-16. doi: 10.1074/jbc.M207069200. Epub 2002 Oct 16.

Abstract

Hec1 (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. Hec1 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 Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G(2)/M. Nek2 phosphorylates Hec1 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 Hec1. 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 Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 in Saccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser(165) to Ala or yeast Hec1 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 Hec1 by Nek2 is essential for faithful chromosome segregation.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / chemistry
  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Blotting, Western
  • Cell Cycle
  • Chromosome Segregation
  • Cytoskeletal Proteins
  • DNA, Complementary / metabolism
  • Escherichia coli / metabolism
  • G2 Phase
  • Genotype
  • Glutathione Transferase / metabolism
  • Humans
  • Mitosis*
  • Molecular Sequence Data
  • Mutation
  • NIMA-Related Kinases
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Precipitin Tests
  • Protein Binding
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Serine / chemistry
  • Serine / metabolism
  • Temperature
  • Time Factors
  • Tumor Cells, Cultured

Substances

  • Amino Acids
  • Cytoskeletal Proteins
  • DNA, Complementary
  • NDC80 protein, human
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Serine
  • Glutathione Transferase
  • KIN3 protein, S cerevisiae
  • NEK2 protein, human
  • NIMA-Related Kinases
  • Protein-Serine-Threonine Kinases
  • Alanine