Conformational dynamics and focal adhesion kinase function

描述（由申请人提供）：局灶黏附激酶（FAK）是一种125 kDa的蛋白，在细胞黏附到细胞外基质的局灶黏附中与整合素共定位。FAK在整合素介导的信号事件中提供催化和支架功能，控制细胞运动和存活。多种证据表明，FAK可能在人类癌症和血管疾病的病理中起作用，因此被认为是药物开发的潜在靶点。FAK的c端局灶粘附靶向（FAT）结构域介导FAK定位到细胞中称为局灶粘附的离散区域，并且对FAK信号传导很重要，因为定位的破坏会阻止FAK的激活和下游底物的磷酸化，以响应整合素依赖性细胞粘附。Paxillin是一种局灶性粘连相关的连接蛋白，参与调节细胞运动，与FAT结构域结合并促进FAK定位为局灶性粘连。此外，FAT结构域中严格保守的酪氨酸的磷酸化调节FAK定位，FAK信号传导和局灶粘附转换。

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.