Center for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, ON, Canada M5G 1X5.
Proc Natl Acad Sci U S A. 2012 Feb 28;109(9):3287-92. doi: 10.1073/pnas.1116455109. Epub 2012 Feb 10.
The ubiquitin ligase SCF(Cdc4) (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.
泛素连接酶 SCF(Cdc4)(Skp1/Cul1/F-box 蛋白)以多位点磷酸化依赖的方式识别其底物细胞周期蛋白依赖性激酶抑制剂 Sic1。虽然源自 Sic1 的短双磷酸化肽可以与 Cdc4 高亲和力结合，但通过系统的诱变和定量生物物理分析，我们表明单个较弱、分散的 Sic1 磷酸化位点使 Cdc4 处于动态平衡中。单个磷酸化表位的亲和力有助于调节有效识别所需的整体磷酸化位点阈值。值得注意的是，在全长 Sic1 的情况下，磷酸化表位与 Cdc4 的亲和力显着减弱，这表明结合相互作用的区域环境的重要性。Sic1-Cdc4 相互作用的多位点性质赋予了在平衡反应条件下对激酶活性的协同依赖性，以用于 Sic1 的识别和泛素化。低亲和力位点的复合动态相互作用可能是在生物反应中建立磷酸化阈值的一般机制。
