MAP kinase regulation of the mitotic spindle checkpoint

Methods Mol Biol. 2010;661:497-505. doi: 10.1007/978-1-60761-795-2_31.

Abstract

Maintaining the integrity of the cell cycle is critical for ensuring that cells only undergo DNA replication and proliferation under controlled conditions in response to discrete stimuli. One mechanism by which the fidelity of this process is guaranteed is through the activation of cell cycle checkpoints. The mitotic spindle checkpoint, which is regulated by Aurora B kinase, ensures proper kinetochore attachment to chromosomes leading to equal distribution of chromosomes to daughter cells. We demonstrated that the mitogen-activated protein kinase (MAPK) cascade regulates mitotic progression and the spindle checkpoint. As demonstrated by immunofluorescence at kinetochores, depletion of Raf Kinase Inhibitory Protein (RKIP), an inhibitor of Raf/MEK/ERK signaling, causes an increase in MAPK activity that inhibits Aurora B kinase activity. By monitoring mitotic index and transit time from nuclear envelope breakdown to anaphase, we demonstrated that RKIP depletion leads to a defective spindle checkpoint and genomic instability, particularly in response to drugs that disrupt microtubule function.

MeSH terms

  • Fluorescent Antibody Technique / methods*
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Mitogen-Activated Protein Kinases / metabolism*
  • Mitosis*
  • Staining and Labeling
  • Thymidine / metabolism

Substances

  • Mitogen-Activated Protein Kinases
  • Thymidine