Structural Determinants in Cell Growth Control by p21 and p27

描述(申请人提供)：两种人类蛋白质，p21和p27，结合并调节真核细胞分裂的主要计时器--细胞周期蛋白依赖性蛋白激酶(CDK)。P21和p27是固有的非结构蛋白。重要的是，p27固有的灵活性在与CDK/Cyclin复合体结合时调节顺序折叠。虽然缺乏三级结构，但p27在与CDK/Cyclin复合体结合之前表现出部分折叠的局部结构元件，这影响了顺序结合机制。Pi‘s小组最近阐明了一种两步磷酸化机制，通过p27的酪氨酸(步骤1)和苏氨酸(步骤2)磷酸化来解除p27对CDK2的抑制。在酪氨酸88上磷酸化后，CDK2本身将苏氨酸187上的p27磷酸化，目标是泛素化和降解。促进细胞增殖的酪氨酸激酶被证明启动了这一两步机制，消除了p27介导的细胞周期停滞在G1/S检查点。此外，研究表明，bcr-Abl是与某些类型的白血病相关的具有结构性活性的致癌基因，它通过过度激活p27的两步p27磷酸化来驱动细胞增殖。结合CDK2/Cyclin A的磷酸化p27的核磁共振研究揭示了p27的柔韧性在这两步磷酸化机制中所起的关键作用，强调了内在紊乱在不同调节功能中的作用。我们建议整合我们在p27和CDK2/Cyclin A方面的专业知识，将我们的研究扩展到其他CDK/Cyclin复合体，以全面了解p21和p27如何调控细胞分裂。这些研究不仅将提供对控制正常细胞分裂的基本机制的洞察，还将提供有关这些控制机制如何在癌细胞中被破坏的知识。我们将利用生化、生物物理和结构方法，确定p27单独磷酸化以及与Ab1的酪氨酸88(Y88)磷酸化一起，如何改变p27对CDK2/Cyclin A的活性；2)确定已发现的CDK2/Cyclin A的两步p27磷酸化/调控机制与其他CDK/Cyclin复合体，包括CDK4/Cyclin D1和CDK1/Cyclin E2的调控的相关性；3)确定我们关于p27磷酸化的发现与相关的细胞周期调节因子p21的相关性；以及4)研究p27和p21不同亚域的内在灵活性与功能之间的关系。

DESCRIPTION (provided by applicant): Two human proteins, p21 and p27, bind to and regulate cyclin-dependent kinases (Cdks), the master time- keepers of eukaryotic cell division. p21 and p27 are intrinsically unstructured proteins. Importantly, the intrinsic flexibility of p27 mediates sequential folding upon binding to Cdk/cyclin complexes. While lacking tertiary structure, p27 exhibits partially folded local structural elements prior to binding Cdk/cyclin complexes, which influences the sequential binding mechanism. The PI's group recently elucidated a two-step phosphorylation mechanism that relieves p27-mediated inhibition of Cdk2 through tyrosine (step 1) and threonine (step 2) phosphorylation of p27. After phosphorylation on tyrosine 88, Cdk2 itself phosphorylates p27 on threonine 187, targeting it for ubiquitination and degradation. Tyrosine kinases that promote cell proliferation were shown to initiate this two-step mechanism that eliminates p27-mediated cell cycle arrest at the G1/S checkpoint. In addition, it was shown that Bcr-Abl, the constitutively active, causative oncogene associated with some types of leukemia, drives cell proliferation by over-activating the two-step p27 phosphorylation of p27. NMR studies of phosphorylated p27 bound to Cdk2/cyclin A revealed the critical role that p27 flexibility plays in this two step phosphorylation mechanism, highlighting the role of intrinsic disorder in diverse regulatory function. We propose to marshal our significant expertise with p27 and Cdk2/cyclin A to extend our studies to other Cdk/cyclin complexes to comprehensively understand how p21 and p27 regulate cell division. These studies will not only provide insights into the fundamental mechanisms that control division in normal cells, but also will provide knowledge relevant to how these control mechanisms are corrupted in cancer cells. The four specific aims are: 1) To determine the effect of phosphorylation (of p27) on tyrosine 74 (Y74) by Src on the activity of p27 toward Cdk2/cyclin A. We will determine how Y74 phosphorylation, alone and in combination with tyrosine 88 (Y88) phosphorylation by Abl, alters the activity of p27 toward Cdk2/cyclin A using biochemical, biophysical and structural methods; 2) To determine the relevance of the two-step p27 phosphorylation/regulatory mechanism discovered for Cdk2/cyclin A to the regulation of other Cdk/cyclin complexes, including Cdk4/cyclin D1 and Cdk1/cyclin E2; 3) To determine the relevance of our discoveries concerning p27 phosphorylation to the related cell cycle regulator, p21; and 4) To investigate relationships between intrinsic flexibility and function for the different sub-domains of p27 and p21.