Nakahara Shinichiro, Tsutsumi Koji, Zuinen Takuya, Ohta Yasutaka
Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, Kanagawa 252-0373, Japan.
Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, Kanagawa 252-0373, Japan
J Cell Sci. 2015 Jun 1;128(11):2047-56. doi: 10.1242/jcs.160192. Epub 2015 Apr 23.
Rho family small GTPases are essential for the formation of adherens junctions in epithelial cells. Here, we found that FilGAP (also known as ARHGAP24), a Rac-specific Rho GTPase-activating protein, promoted the formation of adherens junctions in Madin-Darby canine kidney (MDCK) cells. Knockdown of FilGAP by siRNA stimulated the disassembly and migration of MDCK cells induced by hepatocyte growth factor (HGF). By contrast, forced expression of FilGAP induced accumulation of E-cadherin at adherens junctions. Endogenous FilGAP colocalized with E-cadherin at adherens junctions, and depletion of FilGAP reduced the amount of E-cadherin expressed at the surface. The Rac GAP domain of FilGAP was necessary for the suppression of cell scattering induced by HGF. In agreement with this, siRNA-mediated knockdown of both Rac1 and FilGAP suppressed cell scattering induced by HGF. Forced expression of Rho kinase (ROCK, of which there are two isoforms ROCK1 and ROCK2) induced the accumulation of E-cadherin at the adherens junction, and depletion of FilGAP prevented the accumulation of E-cadherin. Moreover, wild-type FilGAP but not a non-phosphorylatable FilGAP mutant rescued the accumulation of E-cadherin at adherens junctions. These results suggest that FilGAP might regulate cell-cell adhesion through inactivation of Rac downstream of Rho-ROCK-signaling in MDCK cells.
Rho家族小GTP酶对于上皮细胞中黏附连接的形成至关重要。在此，我们发现FilGAP（也称为ARHGAP24），一种Rac特异性的Rho GTP酶激活蛋白，促进了Madin-Darby犬肾（MDCK）细胞中黏附连接的形成。通过小干扰RNA敲低FilGAP会刺激肝细胞生长因子（HGF）诱导的MDCK细胞的解体和迁移。相比之下，强制表达FilGAP会诱导E-钙黏蛋白在黏附连接处积累。内源性FilGAP与E-钙黏蛋白在黏附连接处共定位，并且FilGAP的缺失会减少表面表达的E-钙黏蛋白的量。FilGAP的Rac GAP结构域对于抑制HGF诱导的细胞散射是必需的。与此一致的是，小干扰RNA介导的Rac1和FilGAP的敲低抑制了HGF诱导的细胞散射。强制表达Rho激酶（ROCK，有两种同工型ROCK1和ROCK2）会诱导E-钙黏蛋白在黏附连接处积累，并且FilGAP的缺失会阻止E-钙黏蛋白的积累。此外，野生型FilGAP而非不可磷酸化的FilGAP突变体挽救了E-钙黏蛋白在黏附连接处的积累。这些结果表明，FilGAP可能通过在MDCK细胞中Rho-ROCK信号下游使Rac失活来调节细胞间黏附。
