Wojtowicz Stephanie, Lee Sanghyun, Chan Eddie, Ng Evelyn, Campbell Craig I, Di Guglielmo Gianni M
Department of Physiology and Pharmacology, Western University, London, ON, Canada.
Department of Physiology and Pharmacology, Western University, London, ON, Canada.
Cell Signal. 2020 Aug;72:109627. doi: 10.1016/j.cellsig.2020.109627. Epub 2020 Apr 10.
TGFβ-dependent signal transduction is facilitated by Smad anchor for receptor activation (SARA) and inhibited by the inhibitory-Smad, Smad7, which recruits the E3 ubiquitin ligase, Smurf2, to catalyze the degradation of TGFβ receptors. Since the signalling and degradation pathways target active receptor complexes, we assessed if SARA and Smurf2/Smad7 interact and if Smad7/Smurf2 would affect SARA steady state levels. We observed that the Smurf2/Smad7 complex induces a decrease of SARA steady state levels in a process that is dependent on the HECT ubiquitin E3 ligase activity of Smurf2 but is independent of SARA associating with TGFβ receptors or Smad2. We observed that Smurf2/Smad7-dependent reduction of SARA levels is dependent on proteasome activity, as the pharmacological inhibition of the proteasome using MG132 blocked degradation of SARA. When we assessed the functional outcome of reducing endogenous SARA levels via siRNA-mediated silencing, we observed that siRNA directed at SARA decreased both TGFβ-dependent Smad2 membrane recruitment and phosphorylation, as assessed by subcellular fractionation and western blotting. Furthermore, siRNA targeting SARA decreased TGFβ-dependent epithelial to mesenchymal transition, as measured by cellular E- and N-Cadherin protein levels, and the reorganization of actin from cortical actin to stress fiber formation. These data describe a previously undescribed mechanism where the robustness of the TGFβ signalling is regulated by interplay between SARA and Smurf2/Smad7 complexes.
转化生长因子β（TGFβ）依赖性信号转导由受体激活的Smad锚定蛋白（SARA）促进，并受到抑制性Smad蛋白Smad7的抑制，Smad7招募E3泛素连接酶Smurf2来催化TGFβ受体的降解。由于信号传导和降解途径靶向活性受体复合物，我们评估了SARA与Smurf2之间是否相互作用，以及Smad7/Smurf2是否会影响SARA的稳态水平。我们观察到，Smurf2/Smad7复合物在一个依赖于Smurf2的HECT泛素E3连接酶活性，但独立于SARA与TGFβ受体或Smad2结合的过程中，诱导SARA稳态水平降低。我们观察到，Smurf2/Smad7依赖性的SARA水平降低依赖于蛋白酶体活性，因为使用MG132对蛋白酶体进行药理学抑制可阻断SARA的降解。当我们通过小干扰RNA（siRNA）介导的沉默来评估降低内源性SARA水平的功能结果时，我们观察到，针对SARA的siRNA降低了TGFβ依赖性的Smad2向膜的募集和磷酸化，这通过亚细胞分级分离和蛋白质印迹法进行评估。此外，靶向SARA的siRNA降低了TGFβ依赖性的上皮-间充质转化，这通过细胞中E-钙黏蛋白和N-钙黏蛋白的蛋白质水平以及肌动蛋白从皮质肌动蛋白向应力纤维形成的重组来衡量。这些数据描述了一种先前未描述的机制，即TGFβ信号传导的稳健性通过SARA与Smurf2/Smad7复合物之间的相互作用来调节。
