Knockout of c-Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation.
Knockout of c-Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation.
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DOI：
10.1002/prp2.756
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发表时间：
2021-04
影响因子：
2.6
通讯作者：
Ceresa BP
中科院分区：
文献类型：
--
作者：
Crotchett BLM;Ceresa BP
表皮生长因子受体（EGFR）活性是维持角膜上皮稳态的必要条件和充分条件。然而，外源性表皮生长因子（EGF）的加入并不能可靠地恢复角膜上皮损伤。这可能是由于泪液中高水平的内源性EGF以及配体刺激后EGFR的脱敏。我们假设，防止受体下调是增强EGFR信号传导和促进受损角膜修复的另一种机制。配体依赖性EGFR泛素化与受体的靶向降解有关。在这篇论文中，我们确定敲除c‐Cbl（一种E3泛素连接酶，使EGFR泛素化）是否足以延长EGFR磷酸化并维持信号传导。利用CRISPR/Cas9基因编辑，我们生成了缺乏c‐Cbl的永活人角膜上皮（hTCEpi）细胞。敲除（KO）细胞表达其他E3连接酶的水平与对照细胞相同，表明其他E3连接酶没有上调。与对照细胞相比，EGF刺激的EGFR泛素化在KO细胞中降低，但未完全消除。同样，EGF:EGFR的转运被减缓，内吞率下降35%，受体半衰期增加两倍。这导致EGFR磷酸化的幅度增加了两倍，而持续时间没有变化。相反，与对照细胞相比，丝裂原激活蛋白激酶（MAPK）的磷酸化程度没有增加，但持续了2-3小时。我们提出拮抗c - Cbl会部分改变受体泛素化和内吞运输，但这足以增强下游信号。敲除c - Cbl可减缓EGFR内吞作用并增强EGFR再循环
Epidermal growth factor receptor (EGFR) activity is necessary and sufficient for corneal epithelial homeostasis. However, the addition of exogenous Epidermal Growth Factor (EGF) does not reliably restore the corneal epithelium when wounded. This is likely due to high levels of endogenous EGF in tear fluid as well as desensitization of the EGFR following ligand stimulation. We hypothesize that preventing receptor downregulation is an alternative mechanism to enhance EGFR signaling and promote the restoration of compromised corneas. Ligand‐dependent EGFR ubiquitylation is associated with the targeted degradation of the receptor. In this manuscript, we determine whether knockout of c‐Cbl, an E3 ubiquitin ligase that ubiquitylates the EGFR, is sufficient to prolong EGFR phosphorylation and sustain signaling. Using CRISPR/Cas9 gene editing, we generated immortalized human corneal epithelial (hTCEpi) cells lacking c‐Cbl. Knockout (KO) cells expressed the other E3 ligases at the same levels as the control cells, indicating other E3 ligases were not up‐regulated. As compared to the control cells, EGF‐stimulated EGFR ubiquitylation was reduced in KO cells, but not completely abolished. Similarly, EGF:EGFR trafficking was slowed, with a 35% decrease in the rate of endocytosis and a twofold increase in the receptor half‐life. This resulted in a twofold increase in the magnitude of EGFR phosphorylation, with no change in duration. Conversely, Mitogen Activating Protein Kinase (MAPK) phosphorylation did not increase in magnitude but was sustained for 2–3 h as compared to control cells. We propose antagonizing c‐Cbl will partially alter receptor ubiquitylation and endocytic trafficking but this is sufficient to enhance downstream signaling. Knockout of c‐Cbl slows EGFR endocytosis and enhances EGFR recycling
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DOI：
10.1111/j.1600-0854.2011.01305.x
发表时间：
2012-02
期刊：
Traffic (Copenhagen, Denmark)
影响因子：
--
作者：
Eden ER;Huang F;Sorkin A;Futter CE
通讯作者：
Futter CE
DOI：
10.1007/bf00414564
发表时间：
1979-01-01
期刊：
ALBRECHT VON GRAEFES ARCHIV FUR KLINISCHE UND EXPERIMENTELLE OPHTHALMOLOGIE
影响因子：
--
作者：
DANIELE, S;FRATI, L;SANTONI, G
通讯作者：
SANTONI, G
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：
4.5
作者：
Katz, M;Shtiegman, K;Yarden, Y
通讯作者：
Yarden, Y
影响因子：
3.3
作者：
Huang, FT;Sorkin, A
通讯作者：
Sorkin, A