Unraveling the complexity of substrate specificity of PP2A/B55a, a major eukaryote Serine/Threonine Phosphatase

 描述（由申请人提供）：PP 2A是一种主要的Ser/Thr蛋白磷酸酶，其功能为三聚体全酶，由支架蛋白（A）组成，其桥接催化亚基（C）和调节B亚基。调节亚基被认为介导底物特异性并整合来自多种信号通路的调节输入。B55α是B/B55家族的一个广泛表达的调节亚基，据报道，它靶向大量在细胞分裂、分化、存活以及组织特异性特化过程中具有关键功能的关键底物。与PP 2A-AC核心二聚体复合的B55α的结构已得到解决。B55α是一个WD 40 β螺旋桨，其中心孔周围有一个酸性表面，在叶片3和4的顶部有一个明显的凹槽。虽然对其他β-螺旋桨蛋白的研究已经将中心孔附近的区域确定为它们的结合位点， B55α的更卷曲的表面表明底物/调节亚基接触的更复杂的集合。我们之前已经证明，视网膜母细胞瘤相关蛋白p107是PP 2A/B55α的底物，并且p107在软骨细胞中响应FGF信号转导而去磷酸化，其动力学与MAP激酶途径中其他无关的关键PP 2A/B55α底物RAF 1和KSR 1相当。此外，先前的研究已经确定了微管相关蛋白TAU中富含带正电荷的Lys残基的结构域，这些残基是B55α/PP 2A去磷酸化所需的。由此推测TAU的两个结合结构域中带正电荷的残基可能与B55α顶部带负电荷的残基相互作用。为了了解B55α如何结合底物，我们对p107和B55α进行了广泛的结构/功能突变分析。我们的研究结果表明，p107的内在无序间隔区（R1和R2）中的两个独立区域参与接触B55α，并且这两个结构域都含有对结合很重要的带正电荷的残基。我们还发现，B55α顶部的几个酸性残基被报道对TAU去磷酸化至关重要，但对p107结合至关重要。我们的初步数据表明，在接触TAU和p107的表面有限的重叠。虽然PP 2A/B55全酶的晶体结构有助于开发TAU识别的假设机制，但尚不清楚B55α的表面如何结合如此多种不相关的底物，并且表征这些底物的基序仍然难以捉摸。我们的假设是B55α/PP 2A底物特异性是由B55α的顶部凹槽和孔内的双金属表面介导的，所述双金属表面由底物特异性残基组合组成，所述底物特异性残基组合靶向底物中固有无序结构域内的带正电荷残基簇。这种机制允许B55α表面定向底物，使磷酸位点位于酶活性位点。因此，本提案的总体目标是确定B55α的表面如何结合不相关的底物，并开发与实验数据（现有的和根据本提案获得的）一致的详细模型，最初关注p107，随后扩展到其他底物。这将通过以下目标来实现：（1）。鉴定B55α中介导与p107中底物特异性基序结合的底物特异性决定簇，并建立可测试的 高分辨率对接模型。（二）、为了鉴定B55α β-螺旋桨顶部上的特异性结合表面，其区分不相关的底物（例如，RAF 1、KSR 1、TAU和模型有丝分裂底物PRC 1）构建高分辨率模型，解释磷酸化残基向PP 2A/C催化位点的呈递。（三）、使用基于溶液的蛋白质组学分析确定选定细胞类型中底物多样性的程度，并确定B55α的扰动如何影响底物结合和总体细胞信号传导。了解这种PP 2A全酶的底物特异性的决定因素是真核生物信号传导中的一个基本问题，具有重要的治疗意义，因为PP 2A活性的调节正在各种癌症中积极探索。此外，已发现PP 2A/B55α在阿尔茨海默病中下调，其中发现TAU异常过度磷酸化和聚集。

 DESCRIPTION (provided by applicant): PP2A is a major Ser/Thr Protein Phosphatase that functions as a trimeric holoenzyme consisting of a scaffold protein (A), which bridges a catalytic subunit (C) and a regulatory B subunit. The regulatory subunit is thought to mediate substrate specificity and integrate regulatory inputs from a variety of signaling pathways. B55α is a ubiquitously expressed regulatory subunit of the B/B55 family and has been reported to target a considerable number of key substrates with critical functions in cell division, differentiation, survival, as well as tissue-specific specialized processes. The structure of B55α complexed with the PP2A-AC core dimer has been solved. B55α is a WD40 β-propeller with an acidic surface surrounding the central hole and a marked groove on top of blades 3 and 4. While studies with other β-propeller proteins have identified the region near the central hole as their binding site, the more convoluted surface of B55α suggests a more complex set of substrate/regulatory subunit contacts. We have previously shown that the retinoblastoma related protein p107 is a substrate of PP2A/B55α and that p107 is dephosphorylated in response to FGF signaling in chondrocytes with kinetics comparable to other unrelated key PP2A/B55α substrates in the MAP kinase pathway, RAF1 and KSR1. Additionally, a previous study had identified domains in the microtubule-associated protein TAU rich in positively charged Lys residues that are required for dephosphorylation by B55α/PP2A. This led to speculation that the positively charged residues in the two binding domains of TAU may interact with the negatively charged residues on the top of B55α. With the aim of understanding how B55α binds substrates, we have performed extensive structure/function mutation analysis of both p107 and B55α. Our results show that two separate regions within the intrinsically disordered spacer of p107 (R1 and R2) are involved in contacting B55α, and both domains contain positively charged residues that are important for binding. We have also found that several of the acidic residues in the top of B55α reported to be critical for TAU dephosphorylation are dispensable for p107 binding. Our preliminary data suggest limited overlap in the surfaces contacting TAU and p107. While the crystal structure of the PP2A/B55 holoenzyme has been instrumental in developing a hypothetical mechanism for TAU recognition, it is not known how the surface of B55α can bind such a variety of unrelated substrates, and the motifs that characterize these substrates remain elusive. Our hypotheses is that B55α/PP2A substrate specificity is mediated by discriminator surfaces within the top groove and hole of B55α that consist of substrate specific combinations of residues targeting clusters of positively charged residues within intrinsically disordered domains in substrates. This mechanism allows the B55α surface to orient substrates such that the phosphosite is positioned in the enzyme active site. Therefore, the overall goal of this proposal is to determine how the surface of B55α can bind unrelated substrates and to develop a detailed model consistent with experimental data (both existing and to be obtained under this proposal), with an initial focus on p107 and subsequently extended to other substrates. This will be accomplished with the following aims: (1). To identify the determinants of substrate specificity in B55α that mediate binding to substrate specific motifs in p107 and build a testable high-resolution docking model. (2). To identify the specific binding surfaces on the B55α β-propeller top that discriminate unrelated substrates (e.g., RAF1, KSR1, TAU and a model mitotic substrate, PRC1) to build a high-resolution model that explains presentation of the phosphorylated residue to the PP2A/C catalytic site. (3). To identify the extent of substrate diversity in select cell types using solution based proteomics analysis and to establish how perturbation on B55α affects substrate binding and overall cellular signaling. Understanding the determinants of substrate specificity for this PP2A holoenzyme is a fundamental question in eukaryotic signaling with important therapeutic implications as modulation of PP2A activity is actively being explored in a variety of cancers. Moreover, PP2A/B55α has been found to be down regulated in Alzheimer disease, where TAU is found abnormally hyperphosphorylated and aggregates.