Wang Jia, Sinnett-Smith James, Stevens Jan V, Young Steven H, Rozengurt Enrique
From the Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine.
From the Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, CURE: Digestive Diseases Research Center, and the Veterans Affairs Greater Los Angeles Health Care System, Los Angeles, California 90073.
J Biol Chem. 2016 Aug 19;291(34):17988-8005. doi: 10.1074/jbc.M115.711275. Epub 2016 Jul 1.
We examined the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in intestinal epithelial cells. Our results show that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II, a potent mitogen for these cells, induced rapid translocation of YAP from the nucleus to the cytoplasm (within 15 min) and a concomitant increase in YAP phosphorylation at Ser(127) and Ser(397) Angiotensin II elicited YAP phosphorylation and cytoplasmic accumulation in a dose-dependent manner (ED50 = 0.3 nm). Similar YAP responses were provoked by stimulation with vasopressin or serum. Treatment of the cells with the protein kinase D (PKD) family inhibitors CRT0066101 and kb NB 142-70 prevented the increase in YAP phosphorylation on Ser(127) and Ser(397) via Lats2, YAP cytoplasmic accumulation, and increase in the mRNA levels of YAP/TEAD-regulated genes (Ctgf and Areg). Furthermore, siRNA-mediated knockdown of PKD1, PKD2, and PKD3 markedly attenuated YAP nuclear-cytoplasmic shuttling, phosphorylation at Ser(127), and induction of Ctgf and Areg expression in response to GPCR activation. These results identify a novel role for the PKD family in the control of biphasic localization, phosphorylation, and transcriptional activity of YAP in intestinal epithelial cells. In turn, YAP and TAZ are necessary for the stimulation of the proliferative response of intestinal epithelial cells to GPCR agonists that act via PKD. The discovery of interaction between YAP and PKD pathways identifies a novel cross-talk in signal transduction and demonstrates, for the first time, that the PKDs feed into the YAP pathway.
我们研究了肠道上皮细胞中Yes相关蛋白（YAP）的定位、磷酸化及转录活性的调控机制。我们的研究结果表明，用G蛋白偶联受体（GPCR）激动剂血管紧张素II刺激肠道上皮IEC-18细胞（一种对这些细胞有效的促有丝分裂原），可诱导YAP在15分钟内迅速从细胞核转移至细胞质，并伴随Ser(127)和Ser(397)位点YAP磷酸化水平的升高。血管紧张素II以剂量依赖的方式引发YAP磷酸化和细胞质积累（半数有效浓度ED50 = 0.3 nM）。加压素或血清刺激也能引发类似的YAP反应。用蛋白激酶D（PKD）家族抑制剂CRT0066101和kb NB 142-70处理细胞，可通过Lats2阻止Ser(127)和Ser(397)位点YAP磷酸化水平的升高、YAP的细胞质积累以及YAP/TEAD调控基因（Ctgf和Areg）mRNA水平的增加。此外，小干扰RNA（siRNA）介导的PKD1、PKD2和PKD3基因敲低显著减弱了YAP的核质穿梭、Ser(127)位点的磷酸化以及对GPCR激活的Ctgf和Areg表达的诱导。这些结果确定了PKD家族在控制肠道上皮细胞中YAP的双相定位、磷酸化和转录活性方面的新作用。反过来，YAP和TAZ对于刺激肠道上皮细胞对通过PKD起作用的GPCR激动剂的增殖反应是必需的。YAP和PKD途径之间相互作用的发现确定了信号转导中的一种新的相互作用，并首次证明PKD可作用于YAP途径。
