Active Nercc1 protein kinase concentrates at centrosomes early in mitosis and is necessary for proper spindle assembly.
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 gamma-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 gamma-tubulin and several components of the gamma-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the gamma-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.
Roig
Joan
J
Department of Molecular Biology and Medical Services, Massachusetts General Hospital Harvard Medical School, Boston, MA 02114, USA. roig@molbio.mgh.harvard.edu
Groen
Aaron
A
Caldwell
Jennifer
J
Avruch
Joseph
J
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D018385:Centrosome / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000502:physiology*; D014404:Tubulin / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D020931:ran GTP-Binding Protein / Q000378:metabolism
D014404:Tubulin; D029867:Xenopus Proteins; D011494:Protein Kinases; C507760:NEK9 protein, Xenopus; D020931:ran GTP-Binding Protein