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Research Article |
1 Department of Cell Biology, Max-Planck-Institute for Biochemistry, Am
Klopferspitz 18a, D-82152 Martinsried, Germany
2 Department of Membrane Biology, Max-Planck-Institute for Biology,
Corrensstrasse 38, D-72076 Tübingen, Germany
* Present address: Division of Biology, California Institute of Technology, 1200
E. California Blvd, Pasadena, CA 91125, USA
Author for correspondence (e-mail:
nigg{at}biochem.mpg.de
)
Accepted 26 May 2002
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.
Key words: C-Nap1, Nek2, Centrosome, Mitotic spindle, Phosphorylation
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