J. Cell Biol., Volume 142, Number 6, September 21, 1998 1547-1558

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-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.

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