Functional analysis of the DLC tumor suppressor family in 3D cell culture models

肝癌中缺失3 （DLC3）是一种具有潜在肿瘤抑制功能的Rho gtpase激活蛋白（GAP）。我们实验室的工作表明，DLC3在维持细胞-细胞粘附和控制rhob依赖的内吞膜运输中起着重要的gap依赖作用。DLC3表达的缺失不仅破坏了3D上皮的形态发生，而且还促进了跨膜蛋白的异常循环，如表皮生长因子受体（EGFR）和金属蛋白酶MT1-MMP。我们发现DLC3在细胞接触和内体膜上的差异募集分别由PDZ适配器蛋白Scribble和SNX27介导。与我们的生物物理学合作伙伴一起，我们现在已经在DLC3的一个内在无序区域中发现了一个多碱基区域，该区域介导与膜的相互作用，其结构和膜结合特性似乎受到磷酸化的调节。这可能是DLC3调控靶膜识别和时空Rho信号传导的中枢机制。有趣的是，我们的数据库搜索显示，DLC3-PBR中存在几种与癌症相关的突变。在这个项目中，我们将利用先进的生物物理学、分子细胞生物学和成像技术的结合来揭示DLC3活性的分子机制，并询问DLC3的突变是否会导致其功能失活并与细胞转化有关。

Deleted in Liver Cancer 3 (DLC3) is a still poorly characterized Rho GTPase-activating protein (GAP) with potential tumor-suppressive function. Work from our lab has shown that DLC3 plays an important GAP-dependent role in maintaining cell-cell adhesions and in controlling RhoB-dependent endocytic membrane transport. The loss of DLC3 expression not only disrupts 3D epithelial morphogenesis, but also promotes the aberrant recycling of transmembrane proteins such as e.g. the epidermal growth factor receptor (EGFR) and the metalloproteinase MT1-MMP. We revealed that the differential recruitment of DLC3 to cell contacts and endosomal membranes is mediated by the PDZ adapter proteins Scribble and SNX27, respectively. Together with our biophysical partners, we have now identified within an intrinsically disordered region in DLC3 a polybasic region mediating the interaction with membranes and whose structural and membrane binding properties appear to be regulated by phosphorylation. This might constitute a central mechanism controlling target membrane recognition and spatiotemporal Rho signaling by DLC3. Interestingly, our database searches revealed the presence of several cancer-associated mutations within the DLC3-PBR. In this project, we will utilize a combination of advanced biophysical, molecular cell biological and imaging techniques to unravel the molecular mechanisms underlying DLC3 activity and interrogate whether mutations in DLC3 can lead to its functional inactivation and are associated with cellular transformation.