Active MAP Kinase in Mitosis: Localization at Kinetochores and Association with the Motor Protein CENP-E

doi:10.1083/jcb.142.6.1547

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© The Rockefeller University Press, 0021-9525/1998//1547 $5.00
The Journal of Cell Biology, Volume 142, Number 6, , 1998 1547-1558

Regular Articles

Active MAP Kinase in Mitosis: Localization at Kinetochores and Association with the Motor Protein CENP-E

Maja Zecevic^*, Andrew D. Catling^*, Scott T. Eblen^*, Luigina Renzi, James C. Hittle, Tim J. Yen, Gary J. Gorbsky, and Michael J. Weber^*

^* Department of Microbiology and Cancer Center and Department of Cell Biology, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908; and The Fox Chase Cancer Institute, Philadelphia, Pennsylvania 19111

To investigate possible involvement of the mitogen-activatedprotein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulatedkinases) in somatic cell mitosis, we have used indirect immunofluorescencewith a highly specific phospho-MAP kinase antibody and foundthat a portion of the active MAP kinase is localized at kinetochores,asters, and the midbody during mitosis. Although the aster labelingwas constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase,started to fade during chromosome congression, and then disappearedat midanaphase. At telophase, active MAP kinase localized atthe midbody. Based on colocalization and the presence of aMAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substratefor MAP kinase. CENP-E was phosphorylated in vitro by MAP kinaseon sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with theactive MAP kinase during mitosis. Therefore, the presence ofactive MAP kinase at specific mitotic structures and its interactionwith CENP-E suggest that MAP kinase could play a role in mitosisat least in part by altering the ability of CENP-E to mediateinteractions between chromosomes and microtubules.

Key Words: MAP kinase • CENP-E • kinetochore • mitosis • phosphorylation

Abbreviations used in this paper: cdc2, cyclin-dependent kinase 2; CENP-E, centromere-binding protein E; CREST, calcinosis/Raynaud's phenomenon/esophageal dysmotility/sclerodactyly/telangiectasia variant of scleroderma; CSF, cytostatic factor; DAPI, 4',6-diamidino-2-phenylindole; ERK, extracellular signal–regulated kinase; FITC, fluorescein isothiocyanate; HA, hemagglutinin; KLH, keyhole limpet hemocyanin; MAP kinase, mitogen-activated protein kinase; MEK, MAPK/ERK kinase.

Address all correspondence to Michael J. Weber, Department ofMicrobiology and Cancer Center, University of Virginia HealthSciences Center, Charlottesville, VA 22908. Tel.: (804) 924-5022.Fax: (804) 982-0689. E-mail: mjw{at}virginia.edu

L. Renzi's current address is Center For Evolutionary Genetics-GNR, c/o Dept. of Genetics and Molecular Biology, University ofRome, Via degli Apuli 4, 00185 Rome, Italy.

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