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

Abstract

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.

Role of MEK/ERK pathway in the MAD2-mediated cisplatin sensitivity in testicular germ cell tumour cells

“…For example, activation of the MEK/ERK pathway is required for exit from DNA damage-induced G2 cell cycle arrest (Abbott and Holt, 1999) and the transition from G2 into M (Lavoie et al, 1996;Wright et al, 1999). In addition, the localisation of active ERK to the mitotic kinetochore is also suggested to regulate proteins involved in chromosome segregation during metaphase to anaphase transition (Shapiro et al, 1998;Zecevic et al, 1998). Furthermore, ERK is also described to associate with kinetochores during early prophase, but this association is not apparent at later stages of mitosis (Knauf et al, 2005).…”

CENP-meta, an Essential Kinetochore Kinesin Required for the Maintenance of Metaphase Chromosome Alignment in Drosophila

“…Besides motor enzymes, a number of other proteins are at least transiently associated with kinetochores during mitosis. These include rod (Karess and Glover 1989; Starr et al, 1998), the MAP kinases ppERK (Shapiro et al 1998; Zecevic et al 1998) and ppMEK (Shapiro et al 1998), and an APC activator protein fizzy (called cdc20/cdh1 in other organisms; Kallio et al 1998). Likewise, the protein kinase polo (Logarinho and Sunkel 1998) and a number of components of the highly conserved spindle assembly checkpoint, including Bub1 (Taylor and McKeon 1997; Taylor et al 1998; Basu et al 1999), Bub3 (Taylor et al 1998; Basu et al 1998; Martinez-Exposito et al 1999), Mad1 (Jin et al 1998; Chen et al 1998), and Mad2 (Waters et al 1998; Chen et al 1996, Chen et al 1998), are transiently kinetochore-bound.…”

“…Wood, and D.W. Cleveland, manuscript submitted for publication). CENP-E is also a target of phosphorylation by the map kinase ppERK, an active kinase found at the kinetochore (Shapiro et al 1998; Zecevic et al 1998). CENP-E has also been implicated in the dynamic attachment of kinetochores to disassembling microtubules in vitro (Lombillo et al 1995).…”

Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2Phase and Promotes the G2/M Cell Cycle Transition

“…MEK inhibition causes defects in both spindle formation (Horne and Guadagno, 2003) and spindle checkpoint control (Minshull et al, 1994;Takenaka et al, 1997;Chung and Chen, 2003). MEK1 is also active during mammalian somatic cell mitosis (Shapiro et al, 1998;Colanzi et al, 2000;Hayne et al, 2000;Roberts et al, 2002). Activation is independent of extracellular growth factor input (Dangi and Shapiro, 2005), and inhibition causes spindle defects (Horne and Guadagno, 2003).…”