The mechanism regulating the dissociation of the centrosomal protein C-Nap1 from mitotic spindle poles.
The centrosomal protein C-Nap1 is thought to play an important role in centrosome cohesion during interphase of the cell cycle. At the onset of mitosis, when centrosomes separate for bipolar spindle formation, C-Nap1 dissociates from centrosomes. Here we report the results of experiments aimed at determining whether the dissociation of C-Nap1 from mitotic centrosomes is triggered by proteolysis or phosphorylation. Specifically, we analyzed both the cell cycle regulation of endogenous C-Nap1 and the fate of exogenously expressed full-length C-Nap1. Western blot analyses suggested a reduction in the endogenous C-Nap1 level during M phase, but studies using proteasome inhibitors and destruction assays performed in Xenopus extracts argue against ubiquitin-dependent degradation of C-Nap1. Instead, our data indicate that the mitotic C-Nap1 signal is reduced as a consequence of M-phase-specific phosphorylation. Overexpression of full-length C-Nap1 in human U2OS cells caused the formation of large structures that embedded the centrosome and impaired its microtubule nucleation activity. Remarkably, however, these centrosome-associated structures did not interfere with cell division. Instead, centrosomes were found to separate from these structures at the onset of mitosis, indicating that a localized and cell-cycle-regulated activity can dissociate C-Nap1 from centrosomes. A prime candidate for this activity is the centrosomal protein kinase Nek2, as the formation of large C-Nap1 structures was substantially reduced upon co-expression of active Nek2. We conclude that the dissociation of C-Nap1 from mitotic centrosomes is regulated by localized phosphorylation rather than generalized proteolysis.
Mayor
Thibault
T
Department of Cell Biology, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany.
Hacker
Ulrike
U
Stierhof
York-Dieter
YD
Nigg
Erich A
EA
D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D018385:Centrosome / Q000378:metabolism*; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D000072379:NIMA-Related Kinases; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D029867:Xenopus Proteins
D001324:Autoantigens; D018797:Cell Cycle Proteins; C111329:CEP250 protein, human; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C490316:NEK2 protein, Xenopus; C490315:Nek2B protein, Xenopus; C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases