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Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK

B Sarcevic1, R Lilischkis, R L Sutherland

  • 1Cancer Research Program, Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, New South Wales 2010, Australia. b.sarcevic@garvan.unsw.edu.au

The Journal of Biological Chemistry
|January 31, 1998
PubMed

Insights

This study identifies specific protein substrates for cyclin-dependent kinase (CDK) complexes involved in cell cycle progression. Findings reveal how cyclin and CDK subunits dictate substrate specificity, offering insights into G1 and S phase regulation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Cyclin-dependent kinases (CDKs) are crucial regulators of cell cycle transitions in mammalian cells through substrate phosphorylation.
  • The specific substrates for G1 and S phase cyclin-CDK complexes, such as cyclin D-CDK and cyclin E/A-CDK2, remain largely undefined.
  • Understanding these substrates is key to elucidating the precise mechanisms governing cell cycle progression.

Purpose of the Study:

  • To characterize the nuclear protein substrates phosphorylated by various G1 and S phase cyclin-CDK complexes.
  • To investigate the role of both cyclin and CDK subunits in determining substrate specificity.
  • To identify potential targets critical for cell cycle regulation during G1 and S phases.

Main Methods:

  • Phosphorylation of nuclear lysates from T-47D breast cancer cells using purified baculovirus-expressed cyclin-CDK complexes (cyclin D1/D3-CDK4/CDK6, cyclin E/A-CDK2).
  • Partial purification of nuclear lysates via ion-exchange chromatography.
  • Comparison of substrate phosphorylation patterns across different cyclin-CDK complexes.
  • Protein sequencing to identify specific substrates, exemplified by nucleolin.

Main Results:

  • Distinct substrate specificities were observed for different cyclin D-CDK complexes, with some common and some unique phosphorylated proteins.
  • Cyclin D1-CDK4 and cyclin D3-CDK4/CDK6 exhibited specific substrate preferences, indicating subunit-dependent specificity.
  • Cyclin E-CDK2 and cyclin A-CDK2 phosphorylated a broader range of substrates compared to cyclin D-CDKs, with significant overlap and some unique targets.
  • Nucleolin was identified as a substrate, validating the approach for identifying cyclin-CDK targets.

Conclusions:

  • Both cyclin and CDK subunits contribute to the substrate specificity of the overall cyclin-CDK complex.
  • Numerous substrates for D-, E-, and A-type cyclin-CDK complexes were identified, potentially playing roles in G1 and S phase transit.
  • Cyclins E and A modulate the substrate specificity of CDK2, highlighting their distinct functions in cell cycle regulation.

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