Conformational dynamics and focal adhesion kinase function

描述(申请人提供)：粘着斑激酶(FAK)是一种125 kDa的蛋白质，当细胞与细胞外基质粘连时，它与整合素在焦点粘连处共定位。FAK在整合素介导的控制细胞运动和存活的信号事件中提供催化和支架功能。多条证据表明，FAK可能在人类癌症和血管疾病的病理过程中发挥作用，因此被认为是药物开发的潜在靶点。FAK的C端局灶性黏附靶向(FAT)结构域介导FAK定位于细胞内称为灶性黏附的离散区域，对FAK信号转导很重要，因为定位的中断阻止了FAK的激活和下游底物的磷酸化，以响应整合素依赖的细胞黏附。Paxlin是一种粘着斑联接适配子蛋白，参与调节细胞的运动，结合脂肪区域并促进FAK定位到粘着斑。此外，脂肪区域中一个严格保守的酪氨酸的磷酸化调节了FAK的定位、FAK信号和焦点粘连翻转。 我们以前已经解决了脂肪结构域在存在和不存在巴西林衍生多肽的情况下的核磁共振溶液结构。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。将DMD/HX方法应用于FAK的脂肪结构域，发现FAK中存在脂肪中间态。这一中间状态的存在得到了实验数据的充分支持，这导致我们提出，脂肪域的构象动力学调节了帕西林的结合和磷酸化，从而调节了FAK的功能。这项建议的主要目标是利用核磁共振实验和突变研究，结合生物化学和生物物理方法，研究FAK脂肪域的结构和动态特征，这些特征有助于FAK在磷酸化和帕西林结合状态之间的“切换”。这些研究结果可能有助于阐明脂肪区的构象动力学如何调控FAK功能，并可能为通过改变脂肪区的配体结合和磷酸化来抑制FAK功能提供帮助。 粘着斑激酶(FAK)既是一种支架蛋白，又是一种调节细胞增殖、细胞死亡和运动等多种细胞过程的蛋白。FAK的异常调节可能导致癌症和血管疾病。焦点粘附靶向结构域(FAT)位于蛋白质的C末端。由于脂肪结构域调控FAK功能，本研究为通过改变脂肪结构域的配体结合和磷酸化来抑制FAK功能提供了有用的信息。

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.