{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T08:57:06Z","timestamp":1789981026015,"version":"4.0.1"},"reference-count":37,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5533","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2001,8,17]]},"abstract":"<jats:p>\n                    Organelle transport by myosin-V is down-regulated during mitosis, presumably by myosin-V phosphorylation. We used mass spectrometry phosphopeptide mapping to show that the tail of myosin-V was phosphorylated in mitotic\n                    <jats:italic>Xenopus<\/jats:italic>\n                    egg extract on a single serine residue localized in the carboxyl-terminal organelle-binding domain. Phosphorylation resulted in the release of the motor from the organelle. The phosphorylation site matched the consensus sequence of calcium\/calmodulin\u2013dependent protein kinase II (CaMKII), and inhibitors of CaMKII prevented myosin-V release. The modulation of cargo binding by phosphorylation is likely to represent a general mechanism regulating organelle transport by myosin-V.\n                  <\/jats:p>","DOI":"10.1126\/science.1061086","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:39:52Z","timestamp":1027748392000},"page":"1317-1320","source":"Crossref","is-referenced-by-count":128,"title":["Cell Cycle Regulation of Myosin-V by Calcium\/Calmodulin-Dependent Protein Kinase II"],"prefix":"10.1126","volume":"293","author":[{"given":"Ryan L.","family":"Karcher","sequence":"first","affiliation":[{"name":"Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph T.","family":"Roland","sequence":"additional","affiliation":[{"name":"Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Zappacosta","sequence":"additional","affiliation":[{"name":"Department of Physical and Structural Chemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"Huddleston","sequence":"additional","affiliation":[{"name":"Department of Physical and Structural Chemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roland S.","family":"Annan","sequence":"additional","affiliation":[{"name":"Department of Physical and Structural Chemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven A.","family":"Carr","sequence":"additional","affiliation":[{"name":"Department of Physical and Structural Chemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vladimir I.","family":"Gelfand","sequence":"additional","affiliation":[{"name":"Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","unstructured":"G. Warren W. Wickner Cell 84 395 (1996).","DOI":"10.1016\/S0092-8674(00)81284-2"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.143.6.1547"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0169(1997)38:4<397::AID-CM9>3.0.CO;2-0"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(98)70063-6"},{"key":"e_1_3_1_6_2","first-page":"279","volume":"19","author":"Starodubov S. M.","year":"1988","unstructured":"Starodubov S. M., Golichenkov V. A., Ontogenez 19, 279 (1988).","journal-title":"Ontogenez"},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.113.2.347"},{"key":"e_1_3_1_8_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.133.3.585"},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.146.6.1265"},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.25.14799"},{"key":"e_1_3_1_11_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.143.7.1899"},{"key":"e_1_3_1_12_2","unstructured":"DNA encoding the globular tail of mouse myosin-Va (MGT) was amplified from the construct pCMV2-FLAG-MC-ST (10) by the overlap extension polymerase chain reaction (PCR) technique (29 30). Primers for the PCR reaction included the sequence encoding the myc-epitope tag. The PCR product was introduced into the multiple cloning site of the vector pGEX-4T-2 between the Bam HI and Xho I restriction sites. The plasmid pcDNA3-myc-MGT which allows for eukaryotic cell expression was created by ligation of the myc-MGT PCR product between the Bam HI and Xho I restriction sites of pcDNA3 (Invitrogen Carlsbad CA). The sequences were confirmed by direct DNA sequencing."},{"key":"e_1_3_1_13_2","unstructured":"The plasmid pGEX-4T-2-myc-MGT encodes the myc-epitope tag and the last 410 amino acids of mouse myosin-Va (dilute) fused to the COOH-terminus of GST. It was constructed from the vector pGEX-4T-2 (Pharmacia Piscataway NJ). Wild-type and mutant myc-MGT in pGEX-4T-2 were expressed in E. coli BL21(DE3) (Life Technologies Grand Island NY) and purified by the batch procedure on glutathone-agarose beads (Sigma). Bacterial pellets were resuspended in GST buffer [phosphate-buffered saline (PBS) 5 mM dithiothreitol pH 7.0] and supplemented with 1% Triton X-100 and protease inhibitors (10 \u03bcg\/ml each chymostatin leupeptin and pepstatin) before lysis in a French press at 7600 kPa. Lysates were clarified by centrifugation at 16 000 g for 10 min. Supernatants were applied to 1 ml of preswelled glutathione-agarose beads for 1 hour at 4\u00b0C while rotating. Beads were washed first in 10 resin volumes of lysis buffer followed by 10 volumes of GST buffer plus 300 mM NaCl and finally in 10 volumes of GST buffer."},{"key":"e_1_3_1_14_2","unstructured":"Half a milligram of GST-myc-MGT bound to glutathione-agarose beads was labeled in 100 \u03bcl of either mitotic or interphase Xenopus egg extracts supplemented with 20 \u03bcCi of [\u03b3- 32 P]ATP (ICN Costa Mesa CA) for 30 min. Beads were then washed in GST buffer GST buffer plus 300 mM NaCl and then with GST buffer again. Beads were resuspended in 100 \u03bcl of PBS and 40 \u03bcg of thrombin was allowed to cleave MGT from the GST for 1 hour at 37\u00b0C. After digestion beads were pelleted in a table-top centrifuge and discarded. Then 20 \u03bcl of 5\u00d7 Laemmli sample buffer was added to the supernatant boiled for 3 min and analyzed by SDS-PAGE and autoradiography."},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1021\/ac001130t"},{"key":"e_1_3_1_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/1044-0305(93)80049-5"},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1021\/ac960221g"},{"key":"e_1_3_1_18_2","unstructured":"Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr."},{"key":"e_1_3_1_19_2","doi-asserted-by":"publisher","DOI":"10.1006\/abio.1996.0313"},{"key":"e_1_3_1_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0955-0674(98)80121-X"},{"key":"e_1_3_1_21_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0955-0674(99)80036-2"},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)98436-X"},{"key":"e_1_3_1_23_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.274.22.15811"},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","unstructured":"A. Ishida et al. Biochem. Biophys. Res. Commun. 212 806 (1995).","DOI":"10.1006\/bbrc.1995.2040"},{"key":"e_1_3_1_25_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.274.12.7958"},{"key":"e_1_3_1_26_2","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.108.11.3557"},{"key":"e_1_3_1_27_2","doi-asserted-by":"crossref","unstructured":"R. S. Walikonis et al. J. Neurosci. 20 4069 (2000).","DOI":"10.1523\/JNEUROSCI.20-11-04069.2000"},{"key":"e_1_3_1_28_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.137.7.1589"},{"key":"e_1_3_1_29_2","doi-asserted-by":"publisher","DOI":"10.1038\/359417a0"},{"key":"e_1_3_1_30_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/16.15.7351"},{"key":"e_1_3_1_31_2","doi-asserted-by":"crossref","unstructured":"S. N. Ho et al. Gene 77 51 (1989).","DOI":"10.1016\/0378-1119(89)90358-2"},{"key":"e_1_3_1_32_2","unstructured":"Coomassie-blue\u2013stained SDS-PAGE bands from mitotic and interphase MGT were excised reduced alkylated and digested with trypsin in situ as described (32). Each sample was analyzed for phosphopeptide content by a modification of the multidimensional MS-based phosphopeptide mapping procedure reported in (14 15 33). To enhance the sensitivity of the previously reported method we used a microcapillary PepMap C18 high-performance liquid chromatography (HPLC) column (with a 180-\u03bcm inside diameter) flowing at 4 \u03bcl\/min and a modified PerkinElmer Sciex API III+ triple quadrupole mass spectrometer equipped with a micro-ionspray source. The HPLC flow was split post column with 0.4 \u03bcl\/min directed into the mass spectrometer and the remaining 3.6 \u03bcl\/min manually collected into chilled PCR tubes for further analysis. The MS was operated in a single ion monitoring mode for optimal sensitivity. Phosphopeptides were identified in selected fractions by MALDI-PSD analysis (16) on a Micromass TofSpec SE mass spectrometer. Single-segment PSD spectra were recorded on isolated peptide [M+H] + ions. Phosphopeptides were sequenced (18) by nanoelectrospray MS\/MS on a Micromass Q-ToF. Precursor ion spectra for m \/ z 79 (14 18) were recorded on a modified Sciex API III+ triple quadrupole mass spectrometer equipped with a nanospray source."},{"key":"e_1_3_1_33_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.24.15419"},{"key":"e_1_3_1_34_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.278.5337.455"},{"key":"e_1_3_1_35_2","doi-asserted-by":"publisher","DOI":"10.1016\/0076-6879(90)93460-3"},{"key":"e_1_3_1_36_2","unstructured":"Immortalized Xenopus melanophores were cultured as described (36). For in vivo binding and release experiments eight 100-mm plates of melanophores per treatment were each transfected by electroporation with 10 \u03bcg of pcDNA3-myc-MGT or Ser 1650 mutants of this construct. Transiently transfected cells were allowed to express protein for 48 hours before harvest."},{"key":"e_1_3_1_37_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.94.8.3720"},{"key":"e_1_3_1_38_2","unstructured":"This work was supported by NIH grant GM-52111 and NSF grant MCB95-13388. We thank R. Larson and A. Murray for discussions; R. Deshaies for Plx1; and J. Hammer (NHLB NIH) for pCMV2-FLAG-MC-ST."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.1061086","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,9]],"date-time":"2024-01-09T16:28:06Z","timestamp":1704817686000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.1061086"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2001,8,17]]},"references-count":37,"journal-issue":{"issue":"5533","published-print":{"date-parts":[[2001,8,17]]}},"alternative-id":["10.1126\/science.1061086"],"URL":"https:\/\/doi.org\/10.1126\/science.1061086","relation":{"has-review":[{"id-type":"doi","id":"10.3410\/f.1000762.11808","asserted-by":"object"}]},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2001,8,17]]}}}