Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells.

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-09 DOI:10.1016/j.jbc.2023.105233
Kate Tarvestad-Laise, Brian P Ceresa
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引用次数: 1

Abstract

In many cell types, the E3 ubiquitin ligases c-Cbl and Cbl-b induce ligand-dependent ubiquitylation of the hepatocyte growth factor (HGF)-stimulated c-Met receptor and target it for lysosomal degradation. This study determines whether c-Cbl/Cbl-b are negative regulators of c-Met in the corneal epithelium (CE) and if their inhibition can augment c-Met-mediated CE homeostasis. Immortalized human corneal epithelial cells were transfected with Cas9 only (Cas9, control cells) or with Cas9 and c-Cbl/Cbl-b guide RNAs to knockout each gene singularly (-c-Cbl or -Cbl-b cells) or both genes (double KO [DKO] cells) and monitored for their responses to HGF. Cells were assessed for ligand-dependent c-Met ubiquitylation via immunoprecipitation, magnitude, and duration of c-Met receptor signaling via immunoblot and receptor trafficking by immunofluorescence. Single KO cells displayed a decrease in receptor ubiquitylation and an increase in phosphorylation compared to control. DKO cells had no detectable ubiquitylation, had delayed receptor trafficking, and a 2.3-fold increase in c-Met phosphorylation. Based on the observed changes in receptor trafficking and signaling, we examined HGF-dependent in vitro wound healing via live-cell time-lapse microscopy in control and DKO cells. HGF-treated DKO cells healed at approximately twice the rate of untreated cells. From these data, we have generated a model in which c-Cbl/Cbl-b mediate the ubiquitylation of c-Met, which targets the receptor through the endocytic pathway toward lysosomal degradation. In the absence of ubiquitylation, the stimulated receptor stays phosphorylated longer and enhances in vitro wound healing. We propose that c-Cbl and Cbl-b are promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.

c-Cbl/Cbl-b的敲除减缓了c-Met的运输,从而增强了角膜上皮细胞中的信号传导。
在许多细胞类型中,E3泛素连接酶c-Cbl和Cbl-b诱导肝细胞生长因子(HGF)刺激的c-Met受体的配体依赖性泛素化,并将其靶向溶酶体降解。本研究确定了c-Cbl/Cbl-b是否是角膜上皮(CE)中c-Met的负调节因子,以及它们的抑制是否可以增强c-Met介导的CE稳态。仅用Cas9(Cas9,对照细胞)或用Cas9和c-Cbl/Cbl-b引导RNA转染永生化的人角膜上皮细胞,以单独敲除每个基因(-c-Cbl或-Cbl-b细胞)或同时敲除两个基因(双KO[DKO]细胞),并监测其对HGF的反应。通过免疫沉淀评估细胞的配体依赖性c-Met泛素化,通过免疫印迹评估c-Met受体信号传导的大小和持续时间,并通过免疫荧光评估受体运输。与对照相比,单个KO细胞显示出受体泛素化减少和磷酸化增加。DKO细胞没有可检测的泛素化,受体运输延迟,c-Met磷酸化增加2.3倍。基于观察到的受体运输和信号传导的变化,我们通过活细胞延时显微镜检查了对照细胞和DKO细胞中HGF依赖性的体外伤口愈合。HGF处理的DKO细胞的愈合速率大约是未处理细胞的两倍。根据这些数据,我们生成了一个模型,其中c-Cbl/Cbl-b介导c-Met的泛素化,该泛素化通过溶酶体降解的内吞途径靶向受体。在缺乏泛素化的情况下,受刺激的受体保持磷酸化的时间更长,并增强体外伤口愈合。我们认为c-Cbl和Cbl-b是增强c-Met介导的CE再上皮化的有前景的药理学靶点。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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