Sitry Danielle, Seeliger Markus A, Ko Tun K, Ganoth Dvora, Breward Sadie E, Itzhaki Laura S, Pagano Michele, Hershko Avram
Unit of Biochemistry, the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
J Biol Chem. 2002 Nov 1;277(44):42233-40. doi: 10.1074/jbc.M205254200. Epub 2002 Jul 24.
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.
先前的研究表明，细胞周期蛋白依赖性激酶（Cdk）抑制剂p27（Kip1）被SCF（Skp2）泛素连接酶复合物靶向降解，并且该过程需要Cks1，它是细胞周期调节蛋白的高度保守的Suc1/Cks家族的成员。该家族的所有蛋白质都具有Cdk结合位点和阴离子结合位点，但只有哺乳动物的Cks1与Skp2结合并促进Skp2与苏氨酸-187磷酸化的p27的结合。Cks1促进Skp2泛素连接酶亚基与p27相互作用的分子机制仍不清楚。在这里，我们表明Cks1的Skp2结合位点位于一个包括α2-和α1-螺旋及其紧邻区域的区域，与其他两个结合位点相距甚远。Cks1的所有三个结合位点对于p27-泛素连接以及Skp2与Cdk结合的、苏氨酸-187磷酸化的p27的结合都是必需的。Cks1和Skp2相互促进彼此与类似于含有磷酸化苏氨酸-187的p27的19个C末端氨基酸的肽的结合。后一过程需要Cks1的Skp2和阴离子结合位点，但不需要其Cdk结合位点。有人提出，Skp2-Cks1复合物最初在Cks1的阴离子结合位点促进的过程中与磷酸化p27的C末端区域结合。Cks1的Cdk结合位点与Cdk2/细胞周期蛋白E的结合进一步加强了Skp2与底物的相互作用，磷酸化的p27与Cdk2/细胞周期蛋白E结合。
