Structural Determinants in Cell Growth Control by p21 and p27

描述（由申请人提供）：两种人类蛋白，p21和p27，结合并调节细胞周期蛋白依赖性激酶（Cdks），真核细胞分裂的主要时间守护者。P21和p27本质上是非结构化蛋白。重要的是，p27固有的灵活性介导了与Cdk/cyclin复合物结合后的顺序折叠。由于缺乏三级结构，p27在结合Cdk/cyclin复合物之前显示出部分折叠的局部结构元件，这影响了序列结合机制。PI的团队最近阐明了一种两步磷酸化机制，通过酪氨酸（步骤1）和苏氨酸（步骤2）磷酸化p27来缓解p27介导的Cdk2抑制。在酪氨酸88磷酸化后，Cdk2自身磷酸化苏氨酸187上的p27，靶向其泛素化和降解。研究表明，促进细胞增殖的酪氨酸激酶启动了这种两步机制，消除了G1/S检查点p27介导的细胞周期阻滞。此外，研究表明，Bcr-Abl是与某些类型的白血病相关的组成型活性致癌基因，它通过过度激活p27的两步磷酸化来驱动细胞增殖。磷酸化p27与Cdk2/cyclin A结合的NMR研究揭示了p27柔韧性在这两步磷酸化机制中起关键作用，突出了内在失调在多种调控功能中的作用。我们建议利用我们在p27和Cdk2/cyclin A方面的重要专业知识，将我们的研究扩展到其他Cdk/cyclin复合物，以全面了解p21和p27如何调节细胞分裂。这些研究不仅将提供对控制正常细胞分裂的基本机制的见解，而且将提供有关这些控制机制如何在癌细胞中被破坏的相关知识。四个具体目标是：1)确定Src对酪氨酸74 （Y74）的磷酸化（p27）对p27对Cdk2/cyclin A活性的影响。我们将使用生化，生物物理和结构方法确定Y74磷酸化，单独或与Abl对酪氨酸88 （Y88）的磷酸化联合，如何改变p27对Cdk2/cyclin A的活性；2)确定Cdk2/cyclin A的两步p27磷酸化/调控机制与其他Cdk/cyclin复合物（包括Cdk4/cyclin D1和Cdk1/cyclin E2）调控的相关性；3)确定p27磷酸化与相关细胞周期调节因子p21的相关性；4)研究p27和p21不同子结构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.