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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
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Substrate recognition and ubiquitination of SCFSkp2/Cks1 ubiquitin-protein isopeptide ligase

Shuichan Xu1, Mahan Abbasian, Palka Patel

  • 1Department of Biochemistry and Biomarker Development, Signal Pharmaceuticals, LLC, San Diego, California 92121, USA. sxu@celgene.com

The Journal of Biological Chemistry
|April 6, 2007
PubMed

Insights

The cell cycle regulator p27 is targeted for degradation by the SCF(Skp2/Cks1) complex. Both Cdk2/E binding and Thr187 phosphorylation are crucial for p27 recognition and ubiquitination.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • p27 is a key regulator of the G1/S cell cycle transition.
  • Ubiquitination and degradation control p27 levels during cell cycle progression.

Purpose of the Study:

  • To investigate the mechanism of p27 recognition and ubiquitination by the SCF(Skp2/Cks1) complex.
  • To elucidate the roles of Cdk2/E and p27 Thr187 phosphorylation in this process.

Main Methods:

  • Biochemical assays to study protein-protein interactions.
  • Analysis of ubiquitination and phosphorylation events.
  • Investigation of cooperativity in binding and ubiquitination.

Main Results:

  • Cdk2/E binding and Thr187 phosphorylation are essential for p27 recognition by SCF(Skp2/Cks1).
  • Cdk2/E provides high-affinity binding, while phosphorylated Thr187 provides low-affinity binding.
  • Binding and ubiquitination of p27/Cdk2/E to the E3 complex exhibit positive cooperativity.
  • p27 inhibits Skp2 phosphorylation and auto-ubiquitination, preventing Skp2-mediated degradation.

Conclusions:

  • The study reveals a cooperative mechanism for p27 ubiquitination mediated by Cdk2/E and Thr187 phosphorylation.
  • p27 actively regulates its own degradation pathway by inhibiting Skp2 activity.

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