Structural and Biochemical Insights into PP2A Holoenzyme Biogenesis

描述(申请人提供)：蛋白磷酸酶2A(PP2A)是一种主要的丝氨酸/苏氨酸磷酸酶，调节不同的途径和细胞过程。PP2A的解除管制与许多类型的癌症和阿尔茨海默病有关。PP2A在两个主要水平上受到高度调控：三聚体全酶控制底物专一性；活性部位构象调节调节酶活性水平。我们最近在理解PP2A特异性甲基转移酶LCMT-1方面的进展表明，PP2A甲基化是一种增强全酶组装的修饰，受到PP2A磷酸酶活性的刺激。我们的研究进一步提示了PP2A抑制蛋白A4和PP2A磷酸酶激活剂(PTPA)的强制机制，并指出了全酶生物发生的层级控制和线性途径：部分折叠的PP2A由A4以非活性形式稳定，并被PTPA转化为活性形式；然后，激活的PP2A选择性甲基化并增强以形成底物特异性的全酶。我们最近在与A4和PTPA结合的PP2A的结晶方面取得了关键突破，这是一种高度动态的相互作用高度调节的络合物。本文提出的研究将结合X射线结晶学与生物化学、生物物理学、酵母遗传学和细胞生物学来确定A4、PTPA和PP2A甲基化如何控制PP2A的结构和功能，以精确驱动全酶的生物发生。我们将确定PP2A-A4复合体的高分辨率结构，并解决它们的相互作用如何控制PP2A稳定性和影响细胞生存(目标1)。我们将确定PTPA与PP2A活性位点的伴侣功能的结构基础，以深入了解PP2A的激活，催化金属负载和底物偏好的控制，并阐明PTPA在PP2A激活和细胞生存中的机制(目标2)。我们将确定A4和PTPA中的缺陷如何影响全酶生物发生的后续步骤，并破译甲基化在控制全酶结构、构象和稳定性中的作用(目标3)。这些研究将揭示全酶生物发生的引人注目的机制和层级控制，限制模糊的磷酸酶活性，并确保活性全酶的形成，这对细胞周期、生存和药物敏感性具有根本影响。

DESCRIPTION (provided by applicant): Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase that regulates diverse pathways and cellular processes. Deregulation of PP2A is associated with many types of cancers and Alzheimer's Disease. PP2A is highly regulated at two major levels: trimeric holoenzyme controls substrate specificity; modulation of active site conformation regulates the level of enzyme activity. Our recent advance in understanding PP2A-specific methyltransferase LCMT-1 shows that PP2A methylation, a modification that enhances holoenzyme assembly, is stimulated by PP2A's phosphatase activity. Our studies further suggest compelling mechanisms for PP2A inhibitory protein a4 and PP2A phosphatase activator (PTPA), and point to hierarchy controls and a linear pathway of holoenzyme biogenesis: partially-folded PP2A is stabilized by a4 in an inactive form, and converted to an active form by PTPA; activated PP2A is then selectively methylated and enhanced to form substrate-specific holoenzymes. We recently made key breakthrough in crystallization of PP2A bound to a4 and PTPA, the highly dynamic complexes with highly regulated interactions. The research proposed here will combine x-ray crystallography with biochemistry, biophysics, yeast genetics, and cell biology to determine how a4, PTPA and PP2A methylation control PP2A structure and function to precisely drive holoenzyme biogenesis. We will determine the high-resolution structure of the PP2A-a4 complex, and address how their interaction controls PP2A stability and affects cell survival (Aim 1). We will determine the structural basis for the chaperone function of PTPA to the PP2A active site to gain insight into PP2A activation, controls of catalytic metal loading and substrate preferences, and elucidate mechanisms of PTPA in PP2A activation and cell survival (Aim 2). We will determine how defects in a4 and PTPA affect subsequent steps of holoenzyme biogenesis, and decipher the role of methylation in controlling holoenzyme structure, conformation and stability (Aim 3). These studies will reveal compelling mechanisms and hierarchy controls of holoenzyme biogenesis that restrict ambiguous phosphatase activity and ensure formation of active holoenzymes, which has a fundamental impact on cell cycle, survival, and drug sensitivity.