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© The Rockefeller University Press, 0021-9525/1998//1533 $5.00
The Journal of Cell Biology, Volume 142, Number 6, , 1998 1533-1545

Regular Articles

Activation of the MKK/ERK Pathway during Somatic Cell Mitosis: Direct Interactions of Active ERK with Kinetochores and Regulation of the Mitotic 3F3/2 Phosphoantigen



Paul S. Shapiro*, Eugeni Vaisberg§, Alan J. Hunt§, Nicholas S. Tolwinski*,{ddagger}, Anne M. Whalen*, J. Richard McIntosh§, and Natalie G. Ahn*,{ddagger}

* Department of Chemistry and Biochemistry, {ddagger} Howard Hughes Medical Institute, and § Molecular Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal–regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311–1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.

Key Words: MAP kinase • mitosis • kinetochore • cell cycle • phosphorylation

Abbreviations used in this paper: APC, anaphase promoting complex; DAPI, 4',6-diamidino-2-phenylindole; ERK, extracellular signal–regulated kinase; MAP, mitogen-activated protein; MKK, MAP kinase kinase; MEKK, MAP/ERK kinase kinase; PP2A, protein phosphatase type 2A; MPF, maturation promoting factor; NBF, neutral buffered formalin; NTA, Ni+2-nitrilotriacetic acid.

This research was supported by the Searle Scholars Program (N.G. Ahn), National Institutes of Health grants RO1 GM48521 (N.G. Ahn), F32 GM18151 (P.S. Shapiro), and RO1 GM33787 (J.R. McIntosh), and a Burroughs Wellcome career award (A.J. Hunt).

Address all correspondence to Paul S. Shapiro or Natalie G. Ahn, Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, CO 80309. Tel.: 303-492-4799. Fax: 303-492-2439.


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