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Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
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Published on: September 17, 2012

Activation of the cdc25C phosphatase in mitotic HeLa cells

E Villa-Moruzzi1

  • 1Department of Biomedicine, University of Pisa, Italy.

Biochemical and Biophysical Research Communications
|November 15, 1993
PubMed

Insights

The cell division cycle regulator cdc25C shows significantly higher tyrosine phosphatase (PTP) activity during mitosis. This activation is linked to specific molecular weight changes and dephosphorylation by Phosphatase-2A.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The cdc25C protein is a crucial activator of the cdc2/cyclin B complex, regulating cell cycle progression.
  • Understanding the regulation of cdc25C activity is vital for comprehending cell cycle control and potential disruptions in diseases like cancer.

Purpose of the Study:

  • To investigate the activity and molecular state of cdc25C during different phases of the cell cycle.
  • To determine the factors contributing to the activation of cdc25C during mitosis.

Main Methods:

  • Immunoprecipitation of cdc25C from HeLa cell extracts.
  • Assay of tyrosine phosphatase (PTP) activity using tyrosine-phosphorylated myelin basic protein.
  • Western blot analysis to detect cdc25C protein levels and molecular weight.
  • Treatment with specific phosphatase catalytic subunits (Phosphatase-1 and Phosphatase-2A).
  • Analysis of cdc25C association with cdc2-p13-Sepharose and gel filtration.

Main Results:

  • Tyrosine phosphatase activity of cdc25C was 12-fold higher in mitotic cells compared to asynchronous cells.
  • Mitotic cdc25C exhibited a distinct molecular weight (M(r) 59,000) compared to asynchronous cells (M(r) 56,000-59,000 doublet).
  • Treatment with Phosphatase-2A catalytic subunit decreased the PTP activity of mitotic cdc25C and restored the 56,000 polypeptide.
  • cdc25C associated with cdc2-p13-Sepharose showed 7-fold higher PTP activity in mitotic samples.
  • Higher activity of mitotic cdc25C was retained through gel filtration and co-migrated with cdc2.

Conclusions:

  • Mitotic activation of cdc25C involves a significant increase in its tyrosine phosphatase activity.
  • The observed molecular weight shift and sensitivity to Phosphatase-2A suggest a role for phosphorylation in cdc25C regulation during mitosis.
  • The association with cdc2 and co-migration indicate that cdc25C functions within a complex during cell division.

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