Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization.
Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization.
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DOI：
10.18632/aging.204820
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发表时间：
2023-07-03
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EGFR信号传导起始于配体结合，其导致受体-配体复合物的活化和内化。在这里，我们评估了BUB 1是否通过调节EGFR受体内化和激活来影响EGFR信号传导。BUB 1在细胞中被基因组学（siRNA）或生物化学（2 OH-BNPP 1）消融。EGF配体用于启动EGFR信号传导，而二琥珀酰亚胺辛二酸酯（DSS）用于交联细胞蛋白。通过蛋白质免疫印迹测量EGFR信号传导，并通过荧光显微镜（pEGFR（pY 1068）与早期内体标记物EEA 1共定位）评估受体内化。siRNA介导的BUB 1消耗导致总EGFR水平的总体增加和更多的磷酸化EGFR（Y845、Y1092和Y1173）二聚体，而总EGFR（非磷酸化）二聚体的量保持不变。BUB 1抑制剂（BUB 1 i）以时间依赖性的方式降低EGF介导的EGFR信号传导，包括pEGFR Y845、pAKT S473和pERK 1/2。此外，BUB 1 i还减少EGF介导的pEGFR（Y845）二聚体（不对称二聚体），而不影响总EGFR二聚体（对称二聚体），表明无活性EGFR的二聚化不受BUB 1影响。此外，BUB 1 i阻断EGF介导的EGFR降解（EGFR半衰期增加），而不影响HER 2或c-MET的半衰期。BUB 1 i还减少了pEGFR与EEA 1阳性内体的共定位，表明BUB 1可能调节EGFR内吞作用。我们的数据提供的证据表明，BUB 1蛋白及其激酶活性可以调节EGFR的激活，内吞作用，降解和下游信号，而不影响受体酪氨酸激酶家族的其他成员。
EGFR signaling initiates upon ligand binding which leads to activation and internalization of the receptor-ligand complex. Here, we evaluated if BUB1 impacted EGFR signaling by regulating EGFR receptor internalization and activation. BUB1 was ablated genomically (siRNA) or biochemically (2OH-BNPP1) in cells. EGF ligand was used to initiate EGFR signaling while disuccinimidyl suberate (DSS) was used for cross linking cellular proteins. EGFR signaling was measured by western immunoblotting and receptor internalization was evaluated by fluorescent microscopy (pEGFR (pY1068) colocalization with early endosome marker EEA1). siRNA mediated BUB1 depletion led to an overall increase in total EGFR levels and more phospho-EGFR (Y845, Y1092, and Y1173) dimers while the amount of total EGFR (non-phospho) dimers remained unchanged. BUB1 inhibitor (BUB1i) decreased EGF mediated EGFR signaling including pEGFR Y845, pAKT S473 and pERK1/2 in a time dependent manner. Additionally, BUB1i also reduced EGF mediated pEGFR (Y845) dimers (asymmetric dimers) without affecting total EGFR dimers (symmetric dimers) indicating that dimerization of inactive EGFR is not affected by BUB1. Furthermore, BUB1i blocked EGF mediated EGFR degradation (increase in EGFR half-life) without impacting half-lives of HER2 or c-MET. BUB1i also reduced co-localization of pEGFR with EEA1 positive endosomes suggesting that BUB1 might modulate EGFR endocytosis. Our data provide evidence that BUB1 protein and its kinase activity may regulate EGFR activation, endocytosis, degradation, and downstream signaling without affecting other members of the receptor tyrosine kinase family.
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影响因子：
9.9
作者：
Chia, Joanne;Goh, Germaine;Racine, Victor;Ng, Susanne;Kumar, Pankaj;Bard, Frederic
通讯作者：
Bard, Frederic
影响因子：
13.5
作者：
Cuneo KC;Nyati MK;Ray D;Lawrence TS
通讯作者：
Lawrence TS
影响因子：
4.8
作者：
Barcellos-Hoff, Mary Helen
通讯作者：
Barcellos-Hoff, Mary Helen
DOI：
10.1007/978-3-319-96704-2_9
发表时间：
2018-01-01
期刊：
ENDOCYTOSIS AND SIGNALING
影响因子：
--
作者：
Caldieri, Giusi;Malabarba, Maria Grazia;Sigismund, Sara
通讯作者：
Sigismund, Sara
影响因子：
64.5
作者：
Freed DM;Bessman NJ;Kiyatkin A;Salazar-Cavazos E;Byrne PO;Moore JO;Valley CC;Ferguson KM;Leahy DJ;Lidke DS;Lemmon MA
通讯作者：
Lemmon MA