A New Methodology for the Quantification of In Vivo Cdc14 Phosphatase Activity

Methods Mol Biol. 2017:1505:89-96. doi: 10.1007/978-1-4939-6502-1_8.

Abstract

The phosphatase Cdc14 has a pivotal function in the mitotic exit of Saccharomyces cerevisiae. During interphase, Cdc14 remains inactive in the nucleolus bound to the inhibitor Net1. Cdc14 activation occurs in the metaphase to anaphase transition and it is promoted by at least two signaling pathways called FEAR (CdcFourteen Early Anaphase Release) and MEN (Mitotic Exit Network). These two pathways act in parallel and target the phosphorylation of Net1, thus decreasing Net1 affinity for Cdc14. The activity of Cdc14 can be used as a readout to assay functional interactions of different components of the mitotic exit signaling pathways.

Keywords: Assay; Cdc14; DiFMUP; Fluorescent; Immunoprecipitation; Mitotic exit; Net1; Phosphatase.

MeSH terms

  • Cell Culture Techniques / methods
  • Cell Cycle Proteins / analysis
  • Cell Cycle Proteins / metabolism*
  • Enzyme Assays / methods*
  • Immunoprecipitation / methods*
  • Mitosis
  • Phosphorylation
  • Protein Tyrosine Phosphatases / analysis
  • Protein Tyrosine Phosphatases / metabolism*
  • Saccharomyces cerevisiae / cytology*
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / analysis
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Signal Transduction

Substances

  • CDC14 protein, S cerevisiae
  • Cell Cycle Proteins
  • Saccharomyces cerevisiae Proteins
  • Protein Tyrosine Phosphatases