Three different binding sites of Cks1 are required for p27-ubiquitin ligation.
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.[1]References
- Three different binding sites of Cks1 are required for p27-ubiquitin ligation. Sitry, D., Seeliger, M.A., Ko, T.K., Ganoth, D., Breward, S.E., Itzhaki, L.S., Pagano, M., Hershko, A. J. Biol. Chem. (2002) [Pubmed]
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