{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,4]],"date-time":"2026-10-04T11:53:21Z","timestamp":1791114801899,"version":"4.1.0"},"reference-count":37,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5554","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2002,1,18]]},"abstract":"<jats:p>\n                    Aberrant centrosome duplication is observed in many tumor cells and may contribute to genomic instability through the formation of multipolar mitotic spindles. Cyclin-dependent kinase 2 (Cdk2) is required for multiple rounds of centrosome duplication in\n                    <jats:italic>Xenopus<\/jats:italic>\n                    egg extracts but not for the initial round of replication. Egg extracts undergo periodic oscillations in the level of free calcium. We show here that chelation of calcium in egg extracts or specific inactivation of calcium\/calmodulin\u2013dependent protein kinase II (CaMKII) blocks even initial centrosome duplication, whereas inactivation of Cdk2 does not. Duplication can be restored to inhibited extracts by addition of CaMKII and calmodulin. These results indicate that calcium, calmodulin, and CaMKII are required for an essential step in initiation of centrosome duplication. Our data suggest that calcium oscillations in the cell cycle may be linked to centrosome duplication.\n                  <\/jats:p>","DOI":"10.1126\/science.1065693","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:47:15Z","timestamp":1027748835000},"page":"499-502","source":"Crossref","is-referenced-by-count":88,"title":["Calcium, Calmodulin, and CaMKII Requirement for Initiation of Centrosome Duplication in\n                    <i>Xenopus<\/i>\n                    Egg Extracts"],"prefix":"10.1126","volume":"295","author":[{"given":"Yutaka","family":"Matsumoto","sequence":"first","affiliation":[{"name":"Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine, Denver, CO 80262, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James L.","family":"Maller","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine, Denver, CO 80262, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.292.5516.426"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.271.5256.1744"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1101\/gad.894001"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.283.5403.851"},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.6.2817"},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(99)80191-2"},{"key":"e_1_3_1_8_2","doi-asserted-by":"publisher","DOI":"10.1038\/10054"},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)00093-3"},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(01)00411-1"},{"key":"e_1_3_1_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/0014-5793(94)00492-7"},{"key":"e_1_3_1_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/0955-0674(93)90111-3"},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.94.4.1194"},{"key":"e_1_3_1_14_2","doi-asserted-by":"publisher","DOI":"10.1002\/jcb.1140"},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.124.5.795"},{"key":"e_1_3_1_16_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.87.14.5341"},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1006\/bcmd.1995.0006"},{"key":"e_1_3_1_18_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00552a018"},{"key":"e_1_3_1_19_2","unstructured":"Y. Matsumoto J. L. Maller unpublished data."},{"key":"e_1_3_1_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0955-0674(99)00064-2"},{"key":"e_1_3_1_21_2","unstructured":"The S phase\u2013arrested extracts were prepared as described (4). Briefly freshly made cycling Xenopus egg extracts were aliquoted into 25 \u03bcl per tube. Aphidicolin (10 \u03bcg\/ml CalBiochem) and 400 demembranated sperm nuclei per microliter of extract were added before S phase started and the extracts were incubated for 1 or 6 hours at 2\u00b0C. For analysis the S phase\u2013arrested extracts were treated with nocodazole (30 \u03bcg\/ml CalBiochem) for 15 min at 20\u00b0C and kept on ice for 15 min. Centrosomes were then centrifuged onto a cover slip as described (21) and fixed with 1:1 methanol\/acetone for 10 min at \u201320\u00b0C. For centrosome duplication assays \u03b3-tubulin monoclonal antibody (mAb) (Sigma) was used. For double immunostaining of centrioles we used a rabbit polyclonal antibody to \u03b3-tubulin (Sigma) \u03b1-tubulin mAb (Sigma) and centrin mAb 20H5 (a gift of M. Winey) (14)."},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.100.4.1185"},{"key":"e_1_3_1_23_2","doi-asserted-by":"publisher","DOI":"10.1038\/341197a0"},{"key":"e_1_3_1_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)37923-7"},{"key":"e_1_3_1_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/366270a0"},{"key":"e_1_3_1_26_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)81337-6"},{"key":"e_1_3_1_27_2","unstructured":"CaMKII(281\u2013309) (CalBiochem) was added to CSF extracts on ice. After addition of calcium to 0.6 mM the extracts were incubated at room temperature for the indicated time. For cyclin B immunoblots a sheep affinity-purified polyclonal antibody to cyclin B2 was used. For immunoblots of phospho-Thr 286 in CaMKII\u03b1 the membrane was incubated in 2% (v\/v) goat serum (Sigma) containing rabbit anti-ACTIVE CaMKII polyclonal antibody (Promega). After washing in 0.5 M NaCl 20 mM tris-Cl (pH 7.5) and 0.05% (v\/v) Tween-20 the immunoblot was developed with anti-ACTIVE qualified horseradish peroxidase\u2013conjugated donkey antibody to rabbit immunoglobulin G (Promega)."},{"key":"e_1_3_1_28_2","doi-asserted-by":"publisher","DOI":"10.1016\/0896-6273(88)90109-2"},{"key":"e_1_3_1_29_2","unstructured":"The activity of the holoenzyme of CaM kinase II purified from rat brain (a kind gift of M. D. Browning) was 2.14 \u03bcmol min \u22121 mg \u22121 with synapsin I as substrate (29)."},{"key":"e_1_3_1_30_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.93.26.15451"},{"key":"e_1_3_1_31_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.92.22.10187"},{"key":"e_1_3_1_32_2","unstructured":"GST-\u039434Xic1 (30) and GST-Cdc25A(C432A) (32) were prepared from recombinant baculovirus-infected Sf9 cells as described."},{"key":"e_1_3_1_33_2","doi-asserted-by":"publisher","DOI":"10.1006\/dbio.1999.9361"},{"key":"e_1_3_1_34_2","unstructured":"Y. Matsumoto J. L. Maller unpublished data. Cdk2 phosphorylated on Tyr 15 was detected as a band with increased electrophoretic mobility relative to Cdc2 in immunoblots of egg extracts using an antibody to phospho-Tyr 15 of Cdc2 or Cdk2."},{"key":"e_1_3_1_35_2","unstructured":"After incubation of the S phase\u2013arrested extracts (4) for 6 hours centrosomes and microtubules were centrifuged onto cover slips at room temperature without depolymerization of microtubules. The cover slip was fixed with 99% methanol at \u201320\u00b0C for 5 min and then stained with a rabbit polyclonal antibody to \u03b3-tubulin (Sigma) and \u03b1-tubulin mAb (Sigma)."},{"key":"e_1_3_1_36_2","unstructured":"Y. Matsumoto J. L. Maller unpublished data. Immunofluorescence staining was performed using mAbs (No-185 and No-63) to NO38 a Xenopus homolog of nucleophosmin (B23) (36). The antibodies were a kind gift of M. S. Schmidt-Zachmann. The extracts were fixed with methanol methanol\/acetone (1:1) or 10% methanol\/10% formaldehyde as described (8)."},{"key":"e_1_3_1_37_2","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1987.tb02447.x"},{"key":"e_1_3_1_38_2","unstructured":"We thank G. Sluder for providing adaptors for spinning down centrosomes from extracts and for help in setting up polarization microscopy in this laboratory; M. Winey for the antibody to centrin; M. D. Browning for the CaM kinase II purified from rat brain; M. S. Schmidt-Zachmann for the antibodies to NO38; members of this laboratory for helpful discussions and support particularly F. Taieb for advice on the CaMKII experiments; and P. Eyers and E. Erikson for critical reading of the manuscript. Recombinant baculovirus-infected Sf9 cells were produced in the Tissue Culture\/Monoclonal Antibody Core Facility at the University of Colorado Cancer Center (CA46934). Y.M. is an Associate and J.L.M. is an Investigator of the Howard Hughes Medical Institute."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.1065693","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,9]],"date-time":"2024-01-09T16:09:53Z","timestamp":1704816593000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.1065693"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2002,1,18]]},"references-count":37,"journal-issue":{"issue":"5554","published-print":{"date-parts":[[2002,1,18]]}},"alternative-id":["10.1126\/science.1065693"],"URL":"https:\/\/doi.org\/10.1126\/science.1065693","relation":{"has-review":[{"id-type":"doi","id":"10.3410\/f.1003884.52404","asserted-by":"object"},{"id-type":"doi","id":"10.3410\/f.1003884.41606","asserted-by":"object"}]},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2002,1,18]]}}}