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Three different binding sites of Cks1 are required for p27-ubiquitin ligation
Danielle Sitry1, Markus A Seeliger, Tun K Ko
1Unit of Biochemistry, the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Abstract:
Previous studies have shown that the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) is targeted for degradation by an SCF(Skp2) ubiquitin ligase complex and that this process requires Cks1, a member of the highly conserved Suc1/Cks family of cell cycle regulatory proteins. All proteins of this family have Cdk-binding and anion-binding sites, but only mammalian Cks1 binds to Skp2 and promotes the association of Skp2 with p27 phosphorylated on Thr-187. The molecular mechanisms by which Cks1 promotes the interaction of the Skp2 ubiquitin ligase subunit to p27 remained obscure. Here we show that the Skp2-binding site of Cks1 is located on a region including the alpha2- and alpha1-helices and their immediate vicinity, well separated from the other two binding sites. All three binding sites of Cks1 are required for p27-ubiquitin ligation and for the association of Skp2 with Cdk-bound, Thr-187-phosphorylated p27. Cks1 and Skp2 mutually promote the binding of each other to a peptide similar to the 19 C-terminal amino acids of p27 containing phosphorylated Thr-187. This latter process requires the Skp2- and anion-binding sites of Cks1, but not its Cdk-binding site. It is proposed that the Skp2-Cks1 complex binds initially to the C-terminal region of phosphorylated p27 in a process promoted by the anion-binding site of Cks1. The interaction of Skp2 with the substrate is further strengthened by the association of the Cdk-binding site of Cks1 with Cdk2/cyclin E, to which phosphorylated p27 is bound.
Insights
The cell cycle regulator Cks1 (Suc1/Cks family) is crucial for targeting the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) for degradation by the SCF(Skp2) ubiquitin ligase complex. This study elucidates Cks1
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Protein Degradation
Background:
- The cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) is degraded by the SCF(Skp2) ubiquitin ligase complex.
- This degradation process requires Cks1, a conserved cell cycle regulatory protein.
- The precise molecular mechanism by which Cks1 facilitates Skp2-p27 interaction was previously unclear.
Purpose of the Study:
- To elucidate the molecular mechanisms by which Cks1 promotes the interaction between the Skp2 ubiquitin ligase and p27.
- To identify the specific binding sites on Cks1 involved in this interaction and subsequent p27 degradation.
Main Methods:
- Analysis of Cks1 binding sites for Skp2 and p27.
- Investigation of the role of Cks1's Cdk-binding, anion-binding, and Skp2-binding sites in p27 ubiquitination.
- Biochemical assays to assess the mutual promotion of binding between Cks1, Skp2, and a p27 peptide.
Main Results:
- The Skp2-binding site of Cks1 is distinct from its Cdk- and anion-binding sites.
- All three binding sites of Cks1 are essential for p27 ubiquitination and the association of Skp2 with Cdk-bound, phosphorylated p27.
- Cks1 and Skp2 mutually enhance their binding to a phosphorylated p27 peptide, requiring Cks1's Skp2- and anion-binding sites.
Conclusions:
- The Skp2-Cks1 complex initially binds to phosphorylated p27's C-terminus, facilitated by Cks1's anion-binding site.
- The interaction is further stabilized by Cks1's Cdk-binding site engaging with Cdk2/cyclin E, which is bound to p27.
- This detailed mechanism clarifies how Cks1 acts as a critical scaffold for SCF(Skp2)-mediated p27 degradation.