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Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase
Kui Xu1, Charles Belunis, Wei Chu
1Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA.
Abstract:
The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2.
Insights
The SCF(Skp2) complex targets the cell cycle inhibitor p27(Kip1) for degradation in cancer. Cks1 enhances Skp2 binding to phosphorylated p27, revealing its role as an allosteric effector.
Area of Science:
- Molecular biology
- Biochemistry
- Cell cycle regulation
Background:
- p27(Kip1) is a cyclin-dependent kinase inhibitor crucial for cell cycle control.
- Reduced p27(Kip1) levels are observed in many human cancers due to increased degradation.
- The SCF(Skp2) complex is the ubiquitin ligase responsible for p27(Kip1) degradation, with Skp2 binding phosphorylated p27.
Purpose of the Study:
- To characterize the molecular interactions within the SCF(Skp2) complex.
- To investigate the role of Cks1 in p27(Kip1) ubiquitination and degradation.
- To quantify the binding affinities between Skp2, Cks1, and phosphorylated p27.
Main Methods:
- Development of a homogeneous time-resolved fluorescence (HTRF) assay.
- Quantification of molecular interactions using recombinant human Skp2, Cks1, and a phosphorylated p27-derived peptide.
- Determination of dissociation constants (Kd) for Skp2-Cks1 and Skp2-p27 peptide complexes.
Main Results:
- The dissociation constant for the Skp2-Cks1 complex was determined to be 140 +/- 14 nM.
- Skp2 exhibited high-affinity binding to the phosphorylated p27 peptide only in the presence of Cks1 (Kd = 37 +/- 2 nM).
- Cks1 did not directly bind to the p27 phosphopeptide or Skp1, supporting its allosteric role.
Conclusions:
- Cks1 acts as an allosteric effector, enhancing the binding of Skp2 to phosphorylated p27.
- This interaction is critical for the ubiquitination and subsequent degradation of p27(Kip1).
- Understanding these interactions provides insights into cancer progression and potential therapeutic targets.