Structural biololgy of tumor suppressor PP2A and its regulatory proteins

描述（由申请人提供）：可逆蛋白质磷酸化，即蛋白质磷酸化和去磷酸化，是生物学各个方面的基本调控机制。蛋白磷酸酶2A （PP2A）是哺乳动物细胞中主要的丝氨酸/苏氨酸蛋白磷酸酶，也是一种主要的肿瘤抑制蛋白。PP2A的核心成分由支架亚基（A亚基）和催化亚基（C亚基）组成。由PP2A甲基转移酶（PMT）和PP2A甲基酯酶（PME）控制的C亚基甲基化对PP2A的功能至关重要。PP2A a - c异二聚体和调控亚基（B亚基）组装成一个功能性全酶。DMA病毒SV40小肿瘤抗原（ST）拮抗PP2A的功能，至少部分是通过与B亚基竞争PP2A A-C异源二聚体的结合。Dlpha4蛋白通过与PP2A的C亚基形成复合物而拮抗PP2A的正常功能。尽管进行了大量的研究，但PP2A的结构和机制在很大程度上仍然未知。系统的x射线晶体学和生化分析的PP2A核心成分，其调控蛋白，其修饰酶已经启动。取得了重大进展；本文提出的工作将建立在初步结果的基础上，具有以下具体目标：(1)确定PME， PMT及其与底物/抑制剂的同源配合物的晶体结构。(2) PP2A核心组分A-C异质二聚体的结构测定。(3) a - c异二聚体和B亚基的PP2A全酶结构的测定。(4)小肿瘤抗原（Small Tumor Antigen， ST）结合PP2A a - c异二聚体的结构测定。(5)测定Dlpha4单独及与PP2A的C亚基复合物的结构。本拨款申请中提出的具体目标代表了阐明PP2A调控结构机制的重要和系统的努力。蛋白质及其同源复合物的一般优异的溶液性质使这项工作可行。

DESCRIPTION (provided by applicant): Reversible protein phosphorylation, namely protein phosphorylation and dephosphorylation, is a fundamental regulatory mechanism in all aspects of biology. Protein phosphatase 2A (PP2A) is a dominant Ser/Thr protein phosphatase in mammalian cells and a principal tumor suppressor protein against oncogenic transformation. The core component of PP2A consists of the scaffolding subunit (A subunit) and the catalytic ubunit (C subunit). The methylation of the C subunit, controlled by the PP2A methyl transferase (PMT) and the PP2A methyl esterase (PME), is essential to the function of PP2A. The PP2A A-C hetero-dimer and the regulatory subunit (B subunit) assemble into a functional holoenzyme. The DMA virus SV40 Small Tumor Antigen (ST) antagonizes the function of PP2A at least in part by competing with the B subunit for binding to the PP2A A-C hetero-dimer. The Dlpha4 protein antagonizes the normal function of PP2A by forming a complex with the C subunit of PP2A. Despite intense investigation, the structure and mechanisms of PP2A remain largely unknown. Systematic X-ray crystallographic and biochemical analyses of the PP2A core component, its regulatory proteins, and its modifying enzymes have been initiated. Significant progress has been achieved; the work proposed here will build on the preliminary results with the following specific aims: (1) Determination of the crystal structures of PME, PMT, and their cognate complexes with substrate/inhibitor. (2) Determination of the structure of the core component A-C hetero-dimer of PP2A. (3) Determination of the structure of a PP2A holoenzyme involving A-C hetero-dimer and a B subunit. (4) Determination of the structure of a PP2A A-C hetero-dimer bound to Small Tumor Antigen (ST). (5) Determination of the structure of Dlpha4 alone and in complex with the C subunit of PP2A. The proposed specific aims in this grant application represent an important and systematic effort to unravel the structural mechanisms of PP2A regulation. The generally excellent solution properties of the proteins and their cognate complexes make this undertaking feasible.