Active Nercc1 Protein Kinase Concentrates at Centrosomes Early in Mitosis and Is Necessary for Proper Spindle Assembly
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Published:2005-10
Issue:10
Volume:16
Page:4827-4840
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ISSN:1059-1524
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Container-title:Molecular Biology of the Cell
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language:en
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Short-container-title:MBoC
Author:
Roig Joan1, Groen Aaron2, Caldwell Jennifer3, Avruch Joseph1
Affiliation:
1. Department of Molecular Biology and Medical Services, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02114 2. Department of Systems Biology, Harvard Medical School, Boston, MA 02115 3. MDS Proteomics, Charlottesville, VA 22911
Abstract
The Nercc1 protein kinase autoactivates in vitro and is activated in vivo during mitosis. Autoactivation in vitro requires phosphorylation of the activation loop at threonine 210. Mitotic activation of Nercc1 in mammalian cells is accompanied by Thr210 phosphorylation and involves a small fraction of total Nercc1. Mammalian Nercc1 coimmunoprecipitates γ-tubulin and the activated Nercc1 polypeptides localize to the centrosomes and spindle poles during early mitosis, suggesting that active Nercc has important functions at the microtubular organizing center during cell division. To test this hypothesis, we characterized the Xenopus Nercc1 orthologue (XNercc). XNercc endogenous to meiotic egg extracts coprecipitates a multiprotein complex that contains γ-tubulin and several components of the γ-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the γ-tubulin ring complex polypeptide Xgrip109 contain XNercc. Immunodepletion of XNercc from egg extracts results in delayed spindle assembly, fewer bipolar spindles, and the appearance of aberrant microtubule structures, aberrations corrected by addition of purified recombinant XNercc. XNercc immunodepletion also slows aster assembly induced by Ran-GTP, producing Ran-asters of abnormal size and morphology. Thus, Nercc1 contributes to both the centrosomal and the chromatin/Ran pathways that collaborate in the organization of a bipolar spindle.
Publisher
American Society for Cell Biology (ASCB)
Subject
Cell Biology,Molecular Biology
Reference28 articles.
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