Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA.
Cell Rep. 2018 Sep 18;24(12):3312-3323.e5. doi: 10.1016/j.celrep.2018.08.061.
Ubiquitination is essential for protein degradation and signaling and pivotal to many physiological processes. Ubiquitination of a subset of G-protein-coupled receptors (GPCRs) by the E3 ligase NEDD4-2 is required for p38 activation, but how GPCRs activate NEDD4-2 to promote ubiquitin-mediated signaling is not known. Here, we report that the GPCR protease-activated receptor-1 (PAR1) stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4-2 to promote p38 signaling and endothelial barrier disruption. Using mass spectrometry, we identified a unique phosphorylated tyrosine (Y)-485 within the 2,3-linker peptide between WW domain 2 and 3 of NEDD4-2 in agonist-stimulated cells. Mutation of NEDD4-2 Y485 impaired E3 ligase activity and failed to rescue PAR1-stimulated p38 activation and endothelial barrier permeability. The purinergic P2Y receptor also required c-Src and NEDD4-2 tyrosine phosphorylation for p38 activation. These studies reveal a novel role for c-Src in GPCR-induced NEDD4-2 activation, which is critical for driving ubiquitin-mediated p38 inflammatory signaling.
泛素化对于蛋白质降解和信号转导至关重要，是许多生理过程的关键。E3 连接酶 NEDD4-2 对一组 G 蛋白偶联受体（GPCR）的泛素化对于 p38 的激活是必需的，但是 GPCR 如何激活 NEDD4-2 以促进泛素介导的信号转导尚不清楚。在这里，我们报告 GPCR 蛋白酶激活受体 1（PAR1）刺激 c-Src 介导的 NEDD4-2 的酪氨酸磷酸化和激活，以促进 p38 信号转导和内皮屏障破坏。使用质谱法，我们在激动剂刺激的细胞中鉴定到 NEDD4-2 的 WW 结构域 2 和 3 之间的 2,3-连接肽内的一个独特的磷酸化酪氨酸（Y）-485。NEDD4-2 Y485 的突变会损害 E3 连接酶的活性，并且无法挽救 PAR1 刺激的 p38 激活和内皮屏障通透性。嘌呤能 P2Y 受体也需要 c-Src 和 NEDD4-2 酪氨酸磷酸化来激活 p38。这些研究揭示了 c-Src 在 GPCR 诱导的 NEDD4-2 激活中的新作用，这对于驱动泛素介导的 p38 炎症信号转导至关重要。
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