Regulation of HRAS reactive cysteines in endothelial cell migration - HRAS signaling from the endomembranes
Haeussler, Dagmar Johanna Franziska
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The small GTPase HRas is a major molecular switch, regulating various cellular responses including proliferation, survival, migration and apoptosis. After binding GTP, Ras undergoes a conformational change, and activates downstream signaling partners by recruiting them to distinct cellular membrane localizations. Thus activation through Ras occurs by interaction with effector Ras-binding-domains (RBD) such as that found in Raf and the catalytic subunit p110 of phosphatidylinositol 3-kinase (PI3K). HRas subcellular localization and therefore effector interaction is regulated by its state of S-palmitoylation. Biotin switch assays are a molecular tool for the detection of reversible protein oxidation and S-palmitoylation. To assess the state of H as cysteine modifications during VEGF signaling in EC in the second aim of this thesis, various adaptations were introduced to the protocol allowing accurate detection and differential assessment of reversible oxidative modifications from S-palmitoylation when using the biotin switch assay. By depleting HAECs of endogenous HRas, I provided evidence that HRas was essential in VEGF-mediated activation of the PI3K/Akt/eNOS (endothelial nitric oxide synthase) pathway and EC migration. However, using optimized thiol labeling strategies and immune-fluorescent cell staining demonstrated that only 31% of total HRas is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane localized HRas unexplained. Utilizing a knock-down/reconstitution approach, I showed for the first time that endomembrane-delimited HRas mediates VEGF-induced phosphorylation of Akt and activation of eNOS, leading subsequently to EC migration in response to VEGF. The loss of migratory response in cells lacking endogenous HRas was fully restored overexpression of an endomembrane-delimited HRas palmitoylation by modest mutant. These studies define a newly recognized role for endomembrane localized HRAs in mediating nitric oxide-dependent proangiogenic signaling.
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