Abstract:
The RhoGEF Trio is a large multi-domain protein and an activator of the small GTPases Rac1, RhoG, and RhoA. Although Trio has been implicated in many cellular mechanisms like leukocyte transendothelial migration, cell-cell junction stability, lamellipodia formation, axon outgrowth, and muscle fusion, it remains unclear how Trio is activated. Using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of endothelial cells, we identified two serine residues (S1785/S1786) located in between the two exchange domains of Trio that were highly phosphorylated upon short thrombin treatment. Using phosphomimetic Trio S1785D/S1786D double mutants, we did not find an increase in Rac1/RhoG activity, indicating that the phosphorylation events do not increase Trio exchange activity. However, we found that the Trio mutants localized more strongly at cell-cell junctions and prevented junction destabilization upon thrombin treatment, judged by junction linearity. Our data suggest that serine phosphorylation of Trio potentiates the localization of Trio to junctional regions, resulting in locally promoting the exchange for Rac1 at junction regions and increasing endothelial cell-cell junction stability upon permeability-inducing reagents such as thrombin.
摘要:
RhoGEFTrio是一种大型多结构域蛋白,是小型GTPasesRac1,RhoG,还有RhoA.尽管Trio参与了许多细胞机制,如白细胞跨内皮迁移,细胞-细胞连接稳定性,层状足形成,轴突生长,和肌肉融合,目前还不清楚Trio是如何激活的.在基于细胞培养(SILAC)的基于质谱的内皮细胞分析中使用氨基酸的稳定同位素标记,我们鉴定了位于Trio的两个交换域之间的两个丝氨酸残基(S1785/S1786),它们在短凝血酶处理后高度磷酸化.使用磷模拟三重奏S1785D/S1786D双突变体,我们没有发现Rac1/RhoG活性增加,表明磷酸化事件不增加三重奏交换活性。然而,我们发现Trio突变体更强烈地定位在细胞-细胞连接处,并防止凝血酶处理后的连接处不稳定,通过结线性度判断。我们的数据表明,Trio的丝氨酸磷酸化增强了Trio到连接区的定位,导致在连接区域局部促进Rac1的交换,并在渗透性诱导试剂如凝血酶时增加内皮细胞-细胞连接的稳定性。