Yasuda Takao, Homma Yuta, Fukuda Mitsunori
Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Biochem Biophys Res Commun. 2015 May 15;460(4):896-902. doi: 10.1016/j.bbrc.2015.03.099. Epub 2015 Mar 25.
Synaptotagmin-like protein 2-a (Slp2-a) was originally described as a membrane trafficking protein that consists of a Slp homology domain (SHD), a linker domain, and tandem C2 domains (named the C2A domain and C2B domain). Slp2-a mediates docking of Rab27-bearing vesicles to the plasma membrane through simultaneous interaction with Rab27 and phospholipids in the plasma membrane. We have recently reported that Slp2-a regulates renal epithelial cell size through interaction with Rap1GAP2 via the C2B domain independently of Rab27 and demonstrated the presence of excess activation of ezrin, a membrane-cytoskeleton linker and signal transducer, in Slp2-a-knockdown Madin-Darby canine kidney II (MDCK II) cells. However, the precise mechanism of ezrin inactivation by Slp2-a in cell size control has remained largely unknown. In this study, we investigated the functional relationship between Slp2-a and ezrin in MDCK II cells. The results showed that activation of ezrin in control MDCK II cells either pharmacologically or by overexpression of a constitutively active ezrin mutant caused an increase in cell size, whereas inactivation of ezrin in Slp2-a-knockdown cells by a specific ezrin inhibitor restored them to their normal cell size. We also found that Slp2-a interacts via its previously uncharacterized linker domain with protein phosphatase 1β (PP1β), which inactivates ezrin, and that the interaction is required for the plasma membrane localization of PP1β. These results indicate that Slp2-a inactivates ezrin by recruiting PP1 to the plasma membrane.
类突触结合蛋白2-a（Slp2-a）最初被描述为一种膜运输蛋白，它由一个Slp同源结构域（SHD）、一个连接结构域和串联的C2结构域（命名为C2A结构域和C2B结构域）组成。Slp2-a通过与Rab27和质膜中的磷脂同时相互作用，介导携带Rab27的囊泡与质膜的对接。我们最近报道，Slp2-a通过其C2B结构域与Rap1GAP2相互作用，独立于Rab27调节肾上皮细胞大小，并证明在Slp2-a敲低的Madin-Darby犬肾II型（MDCK II）细胞中，膜-细胞骨架连接蛋白和信号转导蛋白埃兹蛋白（ezrin）存在过度激活。然而，在细胞大小控制中，Slp2-a使ezrin失活的确切机制在很大程度上仍然未知。在本研究中，我们研究了MDCK II细胞中Slp2-a与ezrin之间的功能关系。结果表明，在对照MDCK II细胞中，通过药理学方法或组成型活性ezrin突变体的过表达激活ezrin会导致细胞大小增加，而在Slp2-a敲低的细胞中，通过特异性ezrin抑制剂使ezrin失活可使其恢复到正常细胞大小。我们还发现，Slp2-a通过其先前未被表征的连接结构域与使ezrin失活的蛋白磷酸酶1β（PP1β）相互作用，并且这种相互作用是PP1β定位于质膜所必需的。这些结果表明，Slp2-a通过将PP1募集到质膜来使ezrin失活。
