DdNek2, the first non-vertebrate homologue of human Nek2, is involved in the formation of microtubule-organizing centers.
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.
Gräf
Ralph
R
Adolf-Butenandt-Institut/Zellbiologie, Universität München, Schillerstrasse 42, D-80336 Münich, Germany. ralph.graef@lrz.uni-muenchen.de
D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002478:Cells, Cultured; D018385:Centrosome / Q000378:metabolism* / Q000648:ultrastructure; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000302:isolation & purification*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D000072379:NIMA-Related Kinases; D010802:Phylogeny; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; C490316:NEK2 protein, Xenopus; C490315:Nek2B protein, Xenopus; C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases