Department of Physiology, School of Medicine and Center for Interdisciplinary Studies of the Nervous System (CISNe), Universidad Austral de Chile, Valdivia 5090000, Chile.
Center for Cell Biology and Biomedicine, School of Science and Medicine, Universidad San Sebastián, Santiago 7510235, Chile.
Int J Mol Sci. 2020 Nov 23;21(22):8880. doi: 10.3390/ijms21228880.
Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.
当正常细胞获得肿瘤表型时，蛋白质运输会发生改变。调节蛋白质运输的关键亚细胞区室是高尔基体，但它在癌变中的作用尚未明确界定。高尔基体磷蛋白 3（GOLPH3）是一种主要位于反式高尔基体网络的外周膜蛋白，在多种肿瘤类型中过表达，包括多形性胶质母细胞瘤（GBM），这是最致命的原发性脑肿瘤。此外，GOLPH3 目前被认为是一种癌蛋白，但它在 GBM 中的确切功能尚未完全了解。在这里，我们分析了表达高水平表皮生长因子受体（EGFR）的 GBM 细胞 T98G 中，稳定 RNAi 介导的 GOLPH3 敲低的影响。我们发现，沉默 GOLPH3 导致 T98G 细胞的增殖显著减少，而总 EGFR 水平意外增加，甚至在细胞表面，但其对配体诱导的自动磷酸化的倾向较小。此外，沉默 GOLPH3 降低了 EGFR 的唾液酸化和岩藻糖化，这与配体诱导的 EGFR 下调及其在内体溶酶体区室中的积累延迟相关。最后，我们发现当 GOLPH3 水平降低时，EGF 无法促进 EGFR 的泛素化。总之，我们的结果表明，GOLPH3 在 T98G 细胞中通过影响 EGFR 的糖基化和泛素化程度来调节 EGFR 的内吞运输和激活。
