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Active MAP kinase in mitosis: localization at kinetochores and association with the motor protein CENP-E
M Zecevic1, A D Catling, S T Eblen
1Department of Microbiology and Cancer Center, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908, USA.
Abstract:
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.
Insights
Mitogen-activated protein (MAP) kinases ERK1 and ERK2 are active during cell division. Active MAP kinase interacts with CENP-E, a motor protein, suggesting a role in mitosis by regulating chromosome-microtubule interactions.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitogen-activated protein (MAP) kinases, including ERK1 and ERK2, are crucial signaling molecules.
- Their precise roles in somatic cell mitosis are not fully elucidated.
- Understanding MAP kinase involvement in mitosis is key to comprehending cell division regulation.
Purpose of the Study:
- To investigate the involvement of MAP kinases ERK1 and ERK2 in somatic cell mitosis.
- To identify potential mitotic substrates of MAP kinase.
- To explore the functional consequences of MAP kinase activity during cell division.
Main Methods:
- Indirect immunofluorescence using a phospho-MAP kinase antibody to detect active MAP kinase localization during mitosis.
- Identification of candidate substrates through colocalization and analysis of phosphorylation consensus sites.
- In vitro phosphorylation assays and in vivo association studies with CENP-E.
Main Results:
- Active MAP kinase was localized at kinetochores, asters, and the midbody during mitosis.
- Kinetochore localization peaked during prometaphase and decreased by mid-anaphase.
- The kinetochore motor protein CENP-E was identified as a mitotic substrate, phosphorylated by MAP kinase, and associated with active MAP kinase in vivo.
Conclusions:
- MAP kinase is present at key mitotic structures, including kinetochores, during specific stages of mitosis.
- MAP kinase directly phosphorylates CENP-E, potentially regulating its interaction with microtubules.
- These findings suggest MAP kinase plays a role in mitosis by modulating CENP-E-mediated chromosome-microtubule interactions.