Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering.
Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering.
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DOI：
10.1038/srep24165
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发表时间：
2016-04-18
影响因子：
4.6
通讯作者：
Abankwa D
中科院分区：
文献类型：
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作者：
Blaževitš O;Mideksa YG;Šolman M;Ligabue A;Ariotti N;Nakhaeizadeh H;Fansa EK;Papageorgiou AC;Wittinghofer A;Ahmadian MR;Abankwa D
Galectin-1 (Gal-1) 二聚体可交联细胞表面受体上的碳水化合物。碳水化合物衍生的抑制剂已被开发用于癌症治疗。在细胞内，Gal-1 被认为与 Ras 的法尼基化 C 末端相互作用，从而特异性稳定膜中的 GTP-H-ras 纳米级信号中枢，称为纳米簇。后一种活性可能为过度表达的 Gal-1 如何刺激肿瘤发生提供了另一种机制。在这里，我们修改了 Gal-1 与 H-ras 相互作用的当前模型。我们发现它通过与 Ras 效应子的 Ras 结合域 (RBD) 的高亲和力相互作用，间接与 GTP-H-ras 形成复合物。通过突变分析验证了 Gal-1/C-Raf-RBD 复合物的计算生成模型。细胞 FRET 以及邻近连接测定实验均证实了哺乳动物细胞中 Gal-1 与 Raf 蛋白的相互作用。一致地，干扰 H-rasG12V-效应子相互作用基本上消除了 H-ras 纳米簇。此外，Gal-1 需要完整的二聚体界面才能正向调节 H-rasG12V 纳米簇，但负向调节 K-rasG12V 纳米簇。我们的研究结果表明，H-ras、Raf 和 Gal-1 的堆叠二聚体是高 Gal-1 水平的 GTP-H-ras-纳米簇的构建模块。根据我们的结果，Gal-1/效应器界面代表了 Ras 信号异常疾病中的潜在药物靶位点。
Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling.