Structural and Biochemical Insights into PP2A Holoenzyme Biogenesis

描述（由申请人提供）：蛋白磷酸酶2A （PP2A）是一种主要的丝氨酸/苏氨酸磷酸酶，调节多种途径和细胞过程。PP2A的失调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特异性；活性位点构象的调节调节酶的活性水平。我们最近在了解PP2A特异性甲基转移酶LCMT-1方面的进展表明，PP2A的磷酸酶活性刺激了PP2A甲基化，这是一种增强全酶组装的修饰。我们的研究进一步揭示了PP2A抑制蛋白a4和PP2A磷酸酶激活剂（PTPA）的机制，并指出了层次控制和全酶生物发生的线性途径：部分折叠的PP2A被a4稳定为非活性形式，并通过PTPA转化为活性形式；然后，活化的PP2A选择性地甲基化并增强形成底物特异性全酶。我们最近在PP2A结合a4和PTPA的结晶方面取得了关键突破，这两种高动态配合物具有高度调节的相互作用。本研究将x射线晶体学与生物化学、生物物理学、酵母遗传学和细胞生物学相结合，确定a4、PTPA和PP2A甲基化如何控制PP2A的结构和功能，从而精确地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的稳定性和影响细胞存活（目的1）。我们将确定PTPA对PP2A活性位点的伴侣功能的结构基础，以深入了解PP2A的活化、对催化金属负载和底物偏好的控制，并阐明PTPA在PP2A活化和细胞存活中的机制（Aim 2）。我们将确定a4和PTPA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（目的3）。这些研究将揭示全酶生物发生的令人信服的机制和层次控制，这些机制和层次控制限制了模糊的磷酸酶活性，并确保了活性全酶的形成，这对细胞周期、生存和药物敏感性具有根本性的影响。

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.