A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway.
A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway.
复制标题
FRET 分析揭示了胆固醇在 InlB/Met 信号通路中 Rac1 和 PI 3 激酶激活中的作用。
DOI：
10.1111/j.1462-5822.2006.00832.x
复制
发表时间：
2007
影响因子：
3.4
通讯作者：
Cossart,Pascale
中科院分区：
文献类型：
--
作者：
Seveau,Stéphanie;Tham,ToN;Payrastre,Bernard;Hoppe,AdamD;Swanson,JoelA;Cossart,Pascale
肝细胞生长因子受体Met/HGF-R的信号通路被细菌表面蛋白InlB劫持，以诱导单核细胞增生李斯特菌进入非吞噬细胞。我们先前的研究表明李斯特菌通过与宿主细胞质膜上称为脂筏的特殊微区相互作用而侵入宿主细胞。在这项研究中，我们使用基于荧光共振能量转移的显微镜方法分析了活细胞中对李斯特菌进入至关重要的信号事件。监测磷酸肌醇（PI）3-激酶活性和李斯特菌与上皮细胞相互作用诱导的Rac 1信号传导，以及可溶性InIB和Met天然配体HGF诱导的信号传导。我们发现InlB和HGF诱导PI 3-激酶和Rac 1活化的相似动力学。PI 3-激酶激活位于上游，独立于Rac 1激活。进行了胆固醇耗竭实验，以阐明脂筏在Met信号传导中的作用。PI 3-激酶产生的3′-磷酸肌醇的量不受胆固醇消耗的影响，而它们的膜动力学是胆固醇依赖性的。PI 3-激酶下游的Rac 1活化是胆固醇依赖性的，这表明膜微区内3′-磷酸肌醇的空间分布对Rac 1活化至关重要，因此对细菌进入位点的F-肌动蛋白组装至关重要。
The signalling pathway for the hepatocyte growth factor receptor, Met/HGF‐R, is hijacked by the bacterial surface protein InlB to induceListeria monocytogenesentry into non‐phagocytic cells. We previously showed thatListeriainvades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial forListeriaentry using a fluorescence resonance energy transfer‐based microscopic method. Phosphoinositide (PI) 3‐kinase activity and Rac1 signalling induced byListeriainteracting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3‐kinase and Rac1 activation. PI 3‐kinase activation was upstream and independent of Rac1 activation. Cholesterol‐depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3′‐phosphoinositides produced by PI 3‐kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol‐dependent. Rac1 activation, downstream from PI 3‐kinase, was cholesterol‐dependent suggesting that the spatial distribution of 3′‐phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F‐actin assembly at bacterial entry site.