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/

The mitogen-activated protein (MAP) kinase pathway, which includes ext racellular signal-regulated protein kinases 1 and 2 (ERK1, ERK2) and M AP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required f or cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established, We have examined the re gulation of ERK and MKK in mammalian cells during mitosis using antibo dies selective for active phosphorylated forms of these enzymes, In NI H 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and an aphase, and to the midbody during cytokinesis, During metaphase, activ e ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and s pindle pole localization of active ERK and MKK are also observed in Pt K1 cells. Discrete localization of active ERK at kinetochores is appar ent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate, The kinetochores of chr omosomes displaced from the metaphase plate, or in microtubule-disrupt ed cells, still react strongly with the active ERK antibody, This patt ern resembles that reported for the 3F3/2 monoclonal antibody, which r ecognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint fo r anaphase onset (Gorbsky and Ricketts, 1993, J, Cell Biol. 122:1311-1 321). The 3F3/2 reactivity of kinetochores on isolated chromosomes dec reases after dephosphorylation with protein phosphatase, and then incr eases after subsequent phosphorylation by purified active ERK or activ e 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 kin etochore attachment.