Conformational dynamics and focal adhesion kinase function

描述（由申请人提供）：粘着斑激酶（FAK）是一种125 kDa的蛋白质，在细胞粘附于细胞外基质时与整联蛋白共定位于粘着斑。FAK在控制细胞运动和存活的整合素介导的信号传导事件中提供催化和支架功能。多种证据表明FAK可能在人类癌症和血管疾病的病理学中起作用，因此被认为是药物开发的潜在靶点。FAK的C-末端粘着斑靶向（FAT）结构域介导FAK定位于细胞中称为粘着斑的离散区域，并且对于FAK信号传导是重要的，因为定位的破坏防止响应于整合素依赖性细胞粘附的FAK活化和下游底物的磷酸化。桩蛋白是一种与调节细胞运动性有关的粘着斑相关的衔接蛋白，它与FAT结构域结合并促进FAK定位于粘着斑。此外，在FAT结构域中严格保守的酪氨酸的磷酸化调节FAK定位、FAK信号传导和粘着斑周转。 我们以前已经解决了NMR解决方案的FAT结构域的存在和不存在的桩蛋白衍生肽。我们还开发了一种新的方法，将氢交换（HX）数据集成到离散分子动力学（DMD）模拟中。DMD/HX方法应用于FAK的FAT结构域，并揭示了FAT中间状态的存在。实验数据完全支持这种中间状态的存在，这使我们提出FAT结构域的构象动力学调节桩蛋白结合和磷酸化，从而调节FAK功能。这项建议的主要目标是调查的FAT结构域的结构和动力学特征，促进“切换”之间的磷酸化和桩蛋白结合状态的FAK使用核磁共振（NMR）实验和突变研究结合生物化学和生物物理学的方法。这些研究的结果可能揭示了FAT结构域的构象动力学如何调节FAK功能，并可能提供有助于通过改变FAT结构域配体结合和磷酸化来抑制FAK功能的信息。 粘着斑激酶（FAK）作为一种支架蛋白和激酶，调节大量的细胞过程，如细胞增殖，细胞死亡和运动。FAK的异常调节可导致癌症和血管疾病。黏着斑靶向结构域（FAT）位于蛋白质的C末端。由于FAT结构域调节FAK功能，因此所提出的研究应提供有助于通过改变FAT结构域配体结合-结合和磷酸化来抑制FAK功能的信息。

DESCRIPTION (provided by applicant): Focal adhesion kinase (FAK) is a 125 kDa protein that colocalizes with integrins at focal adhesions upon cell adhesion to the extracellular matrix. FAK provides catalytic and scaffolding functions in integrin-mediated signaling events that control cell motility and survival. Multiple lines of evidence suggest that FAK may function in the pathology of human cancer and vascular disease and is therefore considered to be a potential target for drug development. The C-terminal focal adhesion targeting (FAT) domain of FAK mediates localization of FAK to discrete regions in the cell called focal adhesions and is important for FAK signaling since disruption of localization prevents the activation of FAK and phosphorylation of downstream substrates in response to integrin-dependent cell adhesion. Paxillin, a focal adhesion-associated adaptor protein that has been implicated in regulating cell motility, binds to the FAT domain and promotes FAK localization to focal adhesions. Moreover, phosphorylation of a strictly conserved tyrosine in the FAT domain modulates FAK localization, FAK signaling and focal adhesion turnover. We have previously solved NMR solution structures of the FAT domain in the presence and absence of a paxillin-derived peptide. We have also developed a novel methodology that integrates hydrogen exchange (HX) data into discrete molecular dynamics (DMD) simulations. The DMD/HX methodology was applied to the FAT domain of FAK and revealed the presence of a FAT intermediate state. The presence of this intermediate state is fully supported by experimental data leading us to propose that conformational dynamics of the FAT domain modulates paxillin binding and phosphorylation and therefore FAK function. The primary goal of this proposal is to investigate structural and dynamic features of the FAT domain that facilitate `switching' between phosphorylated and paxillin bound states of FAK using nuclear magnetic resonance (NMR) experiments and mutation studies combined with biochemical and biophysical approaches. Results from these studies are likely to shed light on how conformational dynamics of the FAT domain regulates FAK function and may provide information helpful for inhibition of FAK function by altering FAT domain ligand-binding and phosphorylation. Focal adhesion kinase (FAK) functions as both a scaffold protein and kinase that regulates a plethora of cellular processes such as cell proliferation, cell death and motility. Aberrant regulation of FAK can result in cancer and vascular disease. The focal adhesion targeting domain (FAT) is located at the C-terminus of the protein. Since the FAT domain regulates FAK function, the proposed investigation should provide information helpful for inhibiting FAK function by altering FAT domain ligand binding- binding and phosphorylation.