Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells.
Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells.
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DOI：
10.1016/j.jbc.2023.105233
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发表时间：
2023-10
影响因子：
4.8
通讯作者：
Ceresa, Brian P.
中科院分区：
文献类型：
--
作者：
Tarvestad-Laise, Kate;Ceresa, Brian P.
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受体的配体依赖泛素化，并靶向于溶酶体的降解。本研究确定c-Cbl/Cbl-b是否是c-Met在角膜上皮细胞(CE)中的负调节因子，以及它们的抑制是否能增强c-Met介导的CE动态平衡。永生化的人角膜上皮细胞分别用Cas9(对照细胞)或Cas9和c-Cbl/Cbl-b引导RNA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)，并观察其对HGF的反应。通过免疫沉淀、通过免疫印迹传递c-Met受体信号的幅度和持续时间以及通过免疫荧光传递受体来评估细胞的配体依赖的c-Met泛素化。与对照组相比，单个KO细胞的受体泛素化水平降低，而磷酸化水平升高。DKO细胞没有检测到泛素化，受体转运延迟，c-Met磷酸化增加2.3倍。基于观察到的受体运输和信号转导的变化，我们通过活细胞时间推移显微镜在对照组和DKO细胞中检测了HGF依赖的细胞在体外伤口愈合过程中的作用。HGF处理的DKO细胞的愈合率大约是未处理细胞的两倍。根据这些数据，我们建立了一个模型，其中c-Cbl/Cbl-b介导c-Met的泛素化，c-Met通过内吞途径靶向溶酶体降解的受体。在没有泛素化的情况下，受刺激的受体保持磷酸化的时间更长，并在体外伤口愈合中增强能力。我们认为c-Cbl和Cbl-b是促进c-Met介导的CE再上皮化的有前景的药理靶点。
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.
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影响因子：
8
作者：
Hammond, DE;Urbé, S;Clague, MJ
通讯作者：
Clague, MJ
影响因子：
1.7
作者：
Hashimoto, N;Yamanaka, H;Noguchi, K
通讯作者：
Noguchi, K
影响因子：
2.8
作者：
Foerster, Claudia G.;Cursiefen, Claus;Kruse, Friedrich E.
通讯作者：
Kruse, Friedrich E.
影响因子：
2.6
作者：
Crotchett BLM;Ceresa BP
通讯作者：
Ceresa BP
DOI：
10.1073/pnas.0403412101
发表时间：
2004-07-20
影响因子：
11.1
作者：
Borowiak, M;Garratt, AN;Birchmeier, C
通讯作者：
Birchmeier, C