RRC ID 6683
Author Gräf R.
Title DdNek2, the first non-vertebrate homologue of human Nek2, is involved in the formation of microtubule-organizing centers.
Journal J. Cell. Sci.
Abstract Dictyostelium Nek2 (DdNek2) is the first structural and functional non-vertebrate homologue of human Nek2, a NIMA-related serine/threonine kinase required for centrosome splitting in early mitosis. DdNek2 shares 43% overall amino-acid identity with its human counterpart and 54% identity within the catalytic domain. Both proteins can be subdivided in an N-terminal catalytic domain, a leucine zipper and a C-terminal domain. Kinase assays with bacterially expressed DdNek2 and C-terminal deletion mutants revealed that catalytic activity requires the presence of the leucine zipper and that autophosphorylation occurs at the C-terminus. Microscopic analyses with DdNek2 antibodies and expression of a GFP-DdNek2 fusion protein in Dictyostelium showed that DdNek2 is a permanent centrosomal resident and suggested that it is a component of the centrosomal core. The GFP-DdNek2-overexpressing mutants frequently exhibit supernumerary microtubule-organizing centers (MTOCs). This phenotype did not require catalytic activity because it was also observed in cells expressing inactive GFP-K33R. However, it was shown to be caused by overexpression of the C-terminal domain since it also occurred in GFP-mutants expressing only the C-terminus or a leucine zipper/C-terminus construct but not in those mutants expressing only the catalytic domain or a catalytic domain/leucine zipper construct. These results suggest that DdNek2 is involved in the formation of MTOCs. Furthermore, the localization of the GFP-fusion proteins revealed two independent centrosomal targeting domains of DdNek2, one within the catalytic or leucine zipper domain and one in the C-terminal domain.
Volume 115(Pt 9)
Pages 1919-29
Published 2002-5-1
PMID 11956323
MeSH Animals Catalytic Domain / genetics Cells, Cultured Centrosome / metabolism* Centrosome / ultrastructure Dictyostelium / cytology Dictyostelium / genetics Dictyostelium / metabolism* Evolution, Molecular Gene Expression / physiology Green Fluorescent Proteins Humans Leucine Zippers / genetics Luminescent Proteins Microtubule-Associated Proteins / genetics Microtubule-Associated Proteins / isolation & purification* Microtubules / metabolism* Microtubules / ultrastructure Molecular Sequence Data NIMA-Related Kinases Phylogeny Protein-Serine-Threonine Kinases / genetics Protein-Serine-Threonine Kinases / isolation & purification* Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Sequence Homology, Amino Acid Sequence Homology, Nucleic Acid Xenopus Proteins / genetics Xenopus Proteins / metabolism
IF 4.431
Times Cited 26
Cellular slime molds G00317