PDF(Pubmed)
Abstract：
The Hippo pathway plays a critical role in controlled cell proliferation. The tumor suppressor Merlin and large tumor suppressor kinase 1 (LATS1) mediate activation of Hippo pathway, consequently inhibiting the primary effectors, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid present in the plasma membrane (PM), binds to and activates Merlin. Phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) is an enzyme responsible for PIP2 production. However, the functional role of PIP5Kα in regulation of Merlin and LATS1 under Hippo signaling conditions remains unclear.
PIP5Kα, Merlin, or LATS1 knockout or knockdown cells and transfected cells with them were used. LATS1, YAP, and TAZ activities were measured using biochemical methods and PIP2 levels were evaluated using cell imaging. Low/high cell density and serum starvation/stimulation conditions were tested. Colocalization of PIP5Kα and PIP2 with Merlin and LATS1, and their protein interactions were examined using transfection, confocal imaging, immunoprecipitation, western blotting, and/or pull-down experiments. Colony formation and adipocyte differentiation assays were performed.
We found that PIP5Kα induced LATS1 activation and YAP/TAZ inhibition in a kinase activity-dependent manner. Consistent with these findings, PIP5Kα suppressed cell proliferation and enhanced adipocyte differentiation of mesenchymal stem cells. Moreover, PIP5Kα protein stability and PIP2 levels were elevated at high cell density compared with those at low cell density, and both PIP2 and YAP phosphorylation levels initially declined, then recovered upon serum stimulation. Under these conditions, YAP/TAZ activity was aberrantly regulated by PIP5Kα deficiency. Mechanistically, either Merlin deficiency or LATS1 deficiency abrogated PIP5Kα-mediated YAP/TAZ inactivation. Additionally, the catalytic domain of PIP5Kα directly interacted with the band 4.1/ezrin/radixin/moesin domain of Merlin, and this interaction reinforced interaction of Merlin with LATS1. In accordance with these findings, PIP5Kα and PIP2 colocalized with Merlin and LATS1 in the PM. In PIP5Kα-deficient cells, Merlin colocalization with PIP2 was reduced, and LATS1 solubility increased.
Collectively, our results support that PIP5Kα serves as an activator of the Hippo pathway through interaction and colocalization with Merlin, which promotes PIP2-dependent Merlin activation and induces local recruitment of LATS1 to the PIP2-rich PM and its activation, thereby negatively regulating YAP/TAZ activity. Video Abstract.
PIP5Kα, Merlin, or LATS1 knockout or knockdown cells and transfected cells with them were used. LATS1, YAP, and TAZ activities were measured using biochemical methods and PIP2 levels were evaluated using cell imaging. Low/high cell density and serum starvation/stimulation conditions were tested. Colocalization of PIP5Kα and PIP2 with Merlin and LATS1, and their protein interactions were examined using transfection, confocal imaging, immunoprecipitation, western blotting, and/or pull-down experiments. Colony formation and adipocyte differentiation assays were performed.
We found that PIP5Kα induced LATS1 activation and YAP/TAZ inhibition in a kinase activity-dependent manner. Consistent with these findings, PIP5Kα suppressed cell proliferation and enhanced adipocyte differentiation of mesenchymal stem cells. Moreover, PIP5Kα protein stability and PIP2 levels were elevated at high cell density compared with those at low cell density, and both PIP2 and YAP phosphorylation levels initially declined, then recovered upon serum stimulation. Under these conditions, YAP/TAZ activity was aberrantly regulated by PIP5Kα deficiency. Mechanistically, either Merlin deficiency or LATS1 deficiency abrogated PIP5Kα-mediated YAP/TAZ inactivation. Additionally, the catalytic domain of PIP5Kα directly interacted with the band 4.1/ezrin/radixin/moesin domain of Merlin, and this interaction reinforced interaction of Merlin with LATS1. In accordance with these findings, PIP5Kα and PIP2 colocalized with Merlin and LATS1 in the PM. In PIP5Kα-deficient cells, Merlin colocalization with PIP2 was reduced, and LATS1 solubility increased.
Collectively, our results support that PIP5Kα serves as an activator of the Hippo pathway through interaction and colocalization with Merlin, which promotes PIP2-dependent Merlin activation and induces local recruitment of LATS1 to the PIP2-rich PM and its activation, thereby negatively regulating YAP/TAZ activity. Video Abstract.
摘要：
背景：Hippo途径在受控细胞增殖中起关键作用。肿瘤抑制因子Merlin和大肿瘤抑制激酶1（LATS1）介导Hippo通路的激活，因此抑制了主要效应，Yes相关蛋白（YAP）和具有PDZ结合基序（TAZ）的转录共激活因子。磷脂酰肌醇4,5-双磷酸酯（PIP2），存在于质膜（PM）中的脂质，绑定并激活Merlin.磷脂酰肌醇4-磷酸5-激酶α(PIP5Kα)是负责PIP2产生的酶。然而,在Hippo信号传导条件下，PIP5Kα在Merlin和LATS1调节中的功能作用尚不清楚。
方法：PIP5Kα，梅林,或LATS1敲除或敲低细胞和用它们转染的细胞被使用。LATS1,YAP,使用生化方法测量和TAZ活性，并使用细胞成像评估PIP2水平。测试了低/高细胞密度和血清饥饿/刺激条件。PIP5Kα和PIP2与Merlin和LATS1的共定位，以及使用转染检查它们的蛋白质相互作用，共焦成像，免疫沉淀,西方印迹,和/或下拉实验。进行集落形成和脂肪细胞分化测定。
结果：我们发现PIP5Kα以激酶活性依赖性方式诱导LATS1激活和YAP/TAZ抑制。与这些发现一致，PIP5Kα抑制间充质干细胞的细胞增殖并增强脂肪细胞分化。此外，与低细胞密度相比，高细胞密度时PIP5Kα蛋白稳定性和PIP2水平升高，PIP2和YAP磷酸化水平最初下降，然后在血清刺激后恢复。在这些条件下,YAP/TAZ活性受PIP5Kα缺乏异常调控。机械上,Merlin缺陷或LATS1缺陷消除了PIP5Kα介导的YAP/TAZ失活。此外,PIP5Kα的催化结构域与Merlin的4.1/ezrin/radixin/moesin结构域直接相互作用，这种相互作用增强了Merlin与LATS1的相互作用。根据这些发现，PIP5Kα和PIP2在PM中与Merlin和LATS1共定位。在PIP5Kα缺陷细胞中，梅林与PIP2的共定位减少，和LATS1溶解度增加。
结论：总的来说，我们的结果支持PIP5Kα通过与Merlin的相互作用和共定位作为Hippo途径的激活剂，促进PIP2依赖的Merlin激活，并诱导LATS1局部募集到富含PIP2的PM及其激活，从而负调节YAP/TAZ活性。视频摘要。
方法：PIP5Kα，梅林,或LATS1敲除或敲低细胞和用它们转染的细胞被使用。LATS1,YAP,使用生化方法测量和TAZ活性，并使用细胞成像评估PIP2水平。测试了低/高细胞密度和血清饥饿/刺激条件。PIP5Kα和PIP2与Merlin和LATS1的共定位，以及使用转染检查它们的蛋白质相互作用，共焦成像，免疫沉淀,西方印迹,和/或下拉实验。进行集落形成和脂肪细胞分化测定。
结果：我们发现PIP5Kα以激酶活性依赖性方式诱导LATS1激活和YAP/TAZ抑制。与这些发现一致，PIP5Kα抑制间充质干细胞的细胞增殖并增强脂肪细胞分化。此外，与低细胞密度相比，高细胞密度时PIP5Kα蛋白稳定性和PIP2水平升高，PIP2和YAP磷酸化水平最初下降，然后在血清刺激后恢复。在这些条件下,YAP/TAZ活性受PIP5Kα缺乏异常调控。机械上,Merlin缺陷或LATS1缺陷消除了PIP5Kα介导的YAP/TAZ失活。此外,PIP5Kα的催化结构域与Merlin的4.1/ezrin/radixin/moesin结构域直接相互作用，这种相互作用增强了Merlin与LATS1的相互作用。根据这些发现，PIP5Kα和PIP2在PM中与Merlin和LATS1共定位。在PIP5Kα缺陷细胞中，梅林与PIP2的共定位减少，和LATS1溶解度增加。
结论：总的来说，我们的结果支持PIP5Kα通过与Merlin的相互作用和共定位作为Hippo途径的激活剂，促进PIP2依赖的Merlin激活，并诱导LATS1局部募集到富含PIP2的PM及其激活，从而负调节YAP/TAZ活性。视频摘要。
