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

描述(申请人提供)：基于钙粘附素的黏附连接(AJ)是一种重要的细胞-细胞黏附装置，其调控不当会导致发育失败和癌症。已知AJ的形成依赖于局部肌动蛋白网络的组装，但对此的调控仍不清楚。我们的初步数据表明，talin是一种以调节与基于整合素的细胞-基质黏附相关的肌动蛋白网络而闻名的蛋白质，它也通过一个新的talin蛋白分解片段(称为VAD片段)参与AJ，该片段包含vinculin和肌动蛋白结合域以及二聚化区域。该片段在体内也经历翻译后精氨酸化。我们假设蛋白质降解改变了talin的结构域结构，允许产生的VAD片段定位于基于钙粘素的AJ。在那里，该片段通过其功能结构域增强局部肌动蛋白组装，并稳定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AJ中的生理功能。在这项拟议的研究中，我们将在以下三个特定目标中检验这一假说：(1)talin VAD片段如何招募到钙粘素介导的AJ，以及它如何增强肌动蛋白组装？(2)基于钙粘素的AJ的形成依赖于VAD片段吗？(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调控机制，通过Talin的新工艺介导基于钙粘附素的细胞-细胞黏附，传统上认为Talin只与基于整合素的黏附相关。此外，该项目还将提供新的见解，了解保守但鲜为人知的翻译后精氨酸化修饰如何影响细胞中的蛋白质功能。

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.