Regulation of cadherin-based cell adhesions by talin proteolysis and arginylation

描述（由申请人提供）：基于钙粘蛋白的粘附连接（AJ）是一种关键的细胞-细胞粘附装置，其失调会导致发育失败和癌症。已知AJs的形成依赖于局部肌动蛋白网络的组装，但其调控机制尚不清楚。我们的初步数据表明，talin（一种以调节与整合素为基础的细胞-基质粘附相关的肌动蛋白网络而知名的蛋白质）也通过一种新的talin蛋白水解片段参与AJ，该片段含有血管蛋白和肌动蛋白结合域以及二聚化区域（称为VAD片段）。该片段在体内也经历翻译后精氨酸化。我们假设蛋白水解改变了talin的结构域结构，允许产生的VAD片段定位到基于钙粘蛋白的AJ上。该片段通过其功能域增强了局部肌动蛋白的组装，并稳定了AJ。此外，精氨酸化改变了VAD片段与其结合伙伴的亲和力，增强了其在AJs中的生理功能。在本研究中，我们将从以下三个方面验证这一假设：(1)talin VAD片段如何被招募到钙粘蛋白介导的AJ中，以及它如何增强肌动蛋白组装？(2)基于钙粘蛋白的AJs的形成是否依赖于VAD片段？(3)精氨酸化如何增强VAD片段的功能？通过这项研究，我们将阐明一种新的由talin加工介导的基于钙粘蛋白的细胞-细胞粘附的调节机制，传统上认为talin只与基于整合素的粘附有关。此外，该项目将为保守但知之甚少的翻译后精氨酸化修饰如何影响细胞中的蛋白质功能提供新的见解。

DESCRIPTION (provided by applicant): The cadherin-based adherens junction (AJ) is a critical cell-cell adhesion apparatus, misregulation of which leads to development failures and cancers. The formation of AJs is known to be dependent on the assembly of a local actin network, but the regulation of this remains unclear. Our preliminary data suggest that talin, a protein known for its role in the regulation of the actin network associated with integrin-based cell-matrix adhesion, is also involved in the AJ through a novel talin proteolytic fragment containing the vinculin- and actin- binding domains and a dimerizing region (referred as the VAD fragment). This fragment also undergoes posttranslational arginylation in vivo. We hypothesize that the proteolysis alters the domain architecture of talin, allowing the resulting VAD fragment to localize to the cadherin- based AJ. There the fragment enhances local actin assembly with its functional domains and stabilizes the AJ. Furthermore, arginylation changes the affinities of the VAD fragment to its binding partners and enhances its physiological function in AJs. In this proposed study we will test this hypothesis in these three specific aims: (1) How is the talin VAD fragment recruited to the cadherin-mediated AJ, and how does it enhance actin assembly? (2) Is the formation of cadherin-based AJs dependent on the VAD fragment? (3) How does arginylation enhance the function of the VAD fragment? Through this study, we will elucidate a novel regulatory mechanism for cadherin-based cell-cell adhesions mediated by the novel processing of talin, which was traditionally considered only associated with integrin-based adhesions. Also this project will provide new insight into how the conserved but poorly understood posttranslational arginylation modification affects protein function in cells.