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Mutation of Cys105 inhibits dimerization of p12CDK2-AP1 and its growth suppressor effect

Yong Kim1, Hiroe Ohyama, Vipel Patel

  • 1School of Dentistry and Dental Research Institute, UCLA, Los Angeles, California 90095, USA.

The Journal of Biological Chemistry
|April 21, 2005
PubMed

Insights

p12 (CDK2-associated protein 1) forms nuclear dimers essential for growth inhibition. This dimerization, dependent on cysteine 105, is crucial for p12

Area of Science:

  • Cell cycle regulation
  • Protein dimerization
  • Molecular biology

Background:

  • p12 (CDK2-associated protein 1) negatively regulates CDK2 kinase activity.
  • Contact inhibition in normal diploid cells leads to p27(kip1) induction and reduced CDK2 levels.
  • A nuclear 25-kDa molecule (p25), recognized by anti-p12 antibody, appears in growth-arrested cells.

Purpose of the Study:

  • To investigate the nature of the nuclear p25 molecule and its relationship to p12.
  • To determine the role of p12 dimerization in its growth-inhibitory function.
  • To elucidate the mechanism by which p12 affects CDK2 kinase activity.

Main Methods:

  • Biochemical analysis of His-tagged p12 and its conversion to p25.
  • Site-directed mutagenesis of the p12 cysteine residue (Cys105).
  • Transient transfection studies in U2OS and CCD18LU cells.
  • Short interfering RNA (siRNA) for p12 knockdown.

Main Results:

  • p25 is a homodimeric form of p12, dependent on a reducing agent and the Cys105 residue.
  • Mutation of Cys105 (C105A) abolished p12 dimerization and its growth-inhibitory effect.
  • The C105A mutation prevented p12 association with CDK2 without affecting nuclear localization.
  • siRNA-mediated reduction of p12 expression reduced p25 levels.

Conclusions:

  • p12 forms nuclear homodimers in contact-inhibited normal diploid cells.
  • Dimerization of p12, mediated by Cys105, is essential for its growth inhibition.
  • p12 dimerization is required for its inhibitory effect on CDK2 kinase activity.

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