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Interaction studies between the p21Cip1/Waf1 cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen

M Knibiehler1, F Goubin, N Escalas

  • 1Institut de Pharmacologie et de Biologie Structurale, Université, P. Sabatier, Toulouse, France.

FEBS Letters
|August 5, 1996
PubMed

Insights

The cyclin-dependent kinase inhibitor p21Cip1 interacts with proliferating cell nuclear antigen (PCNA). Surface plasmon resonance (SPR) quantified this interaction, revealing its dependence on PCNA

Area of Science:

  • Molecular biology
  • Biochemistry
  • Cell cycle regulation

Background:

  • p21Cip1 is a cyclin-dependent kinase (CDK) inhibitor with distinct domains for CDK and proliferating cell nuclear antigen (PCNA) interaction.
  • Understanding the p21Cip1-PCNA interaction is crucial for comprehending cell cycle control and DNA replication.

Purpose of the Study:

  • To quantitatively investigate the kinetic parameters of the p21Cip1-PCNA interaction using Surface Plasmon Resonance (SPR).
  • To compare SPR findings with other interaction detection methods like the yeast two-hybrid system.
  • To explore the structural requirements of PCNA for binding to p21Cip1.

Main Methods:

  • Surface Plasmon Resonance (SPR) technology was employed to measure real-time binding kinetics.
  • Yeast two-hybrid system was used for comparative analysis.
  • Site-directed mutagenesis was utilized to generate PCNA mutants.

Main Results:

  • SPR enabled precise determination of kinetic parameters, yielding an apparent equilibrium constant (KD) of 3.2 x 10(-7) M for p21Cip1-PCNA interaction.
  • The interaction is dependent on the trimeric conformation of PCNA; a mutant disrupting PCNA-PCNA interaction also abolished p21Cip1 binding.
  • SPR successfully characterized interactions involving mutant proteins and in the presence of competitive peptides.

Conclusions:

  • SPR provides a robust and quantitative method for studying protein-protein interactions like that between p21Cip1 and PCNA.
  • The p21Cip1-PCNA interaction necessitates the trimeric structure of PCNA.
  • SPR is versatile for analyzing mutant interactions and the effects of competitive molecules.

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