Department of General, Visceral and Transplant Surgery, University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg, Germany.
Exp Cell Res. 2010 Jul 15;316(12):1914-24. doi: 10.1016/j.yexcr.2010.02.020. Epub 2010 Feb 23.
Eps8 controls actin dynamics directly through its barbed end capping and actin-bundling activity, and indirectly by regulating Rac-activation when engaged into a trimeric complex with Eps8-Abi1-Sos1. Recently, Eps8 has been associated with promotion of various solid malignancies, but neither its mechanisms of action nor its regulation in cancer cells have been elucidated. Here, we report a novel association of Eps8 with the late endosomal/lysosomal compartment, which is independent from actin polymerization and specifically occurs in cancer cells. Endogenous Eps8 localized to large vesicular lysosomal structures in metastatic pancreatic cancer cell lines, such as AsPC-1 and Capan-1 that display high Eps8 levels. Additionally, ectopic expression of Eps8 increased the size of lysosomes. Structure-function analysis revealed that the region encompassing the amino acids 184-535 of Eps8 was sufficient to mediate lysosomal recruitment. Notably, this fragment harbors two KFERQ-like motifs required for chaperone-mediated autophagy (CMA). Furthermore, Eps8 co-immunoprecipitated with Hsc70 and LAMP-2, which are key elements for the CMA degradative pathway. Consistently, in vitro, a significant fraction of Eps8 bound to (11.9+/-5.1%) and was incorporated into (5.3+/-6.5%) lysosomes. Additionally, Eps8 binding to lysosomes was competed by other known CMA-substrates. Fluorescence recovery after photobleaching revealed that Eps8 recruitment to the lysosomal membrane was highly dynamic. Collectively, these results indicate that Eps8 in certain human cancer cells specifically localizes to lysosomes, and is directed to CMA. These results open a new field for the investigation of how Eps8 is regulated and contributes to tumor promotion in human cancers.
Eps8 通过其加帽和肌动蛋白束状结构活性直接控制肌动蛋白动力学，并且通过与 Eps8-Abi1-Sos1 三聚体复合物结合时调节 Rac 激活间接控制。最近，Eps8 与各种实体恶性肿瘤的促进有关，但尚未阐明其作用机制及其在癌细胞中的调节作用。在这里，我们报告了 Eps8 与晚期内体/溶酶体隔室的新关联，该关联独立于肌动蛋白聚合，并且仅发生在癌细胞中。内源性 Eps8 定位于转移性胰腺癌细胞系（例如具有高 Eps8 水平的 AsPC-1 和 Capan-1）中的大囊泡溶酶体结构。此外，Eps8 的异位表达增加了溶酶体的大小。结构功能分析表明，包含 Eps8 的氨基酸 184-535 的区域足以介导溶酶体募集。值得注意的是，该片段包含两个 KFERQ 样基序，这些基序是伴侣介导的自噬（CMA）所必需的。此外，Eps8 与 Hsc70 和 LAMP-2 共免疫沉淀，Hsc70 和 LAMP-2 是 CMA 降解途径的关键要素。一致地，在体外，Eps8 与（11.9+/-5.1%）显著部分结合并被（5.3+/-6.5%）纳入溶酶体。此外，Eps8 与溶酶体的结合被其他已知的 CMA 底物竞争。光漂白后荧光恢复显示，Eps8 向溶酶体膜的募集具有高度动态性。总的来说，这些结果表明，在某些人类癌细胞中，Eps8 特异性定位于溶酶体，并被定向到 CMA。这些结果为研究 Eps8 如何在人类癌症中被调节以及如何促进肿瘤提供了一个新的领域。
