Department of Biochemistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, 770-8503, Japan.
Genes Cells. 2013 Sep;18(9):810-22. doi: 10.1111/gtc.12078. Epub 2013 Jul 25.
We previously showed that Rab13 and its effector protein, junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2), regulate junctional development by modulating cell adhesion molecule transport and actin cytoskeletal reorganization in epithelial cells. Here, we investigated how JRAB regulates reorganization of the actin cytoskeleton in NIH3T3 fibroblasts, in an attempt to obtain novel insights into the mechanism of JRAB action. To this end, we expressed mutant proteins that adopt a constitutively open or closed state and then examined effect on cellular morphology of the resulting actin cytoskeletal reorganization. Expression of the JRABΔCT mutant (constitutively 'closed' state) induced stress fibers, whereas expression of the JRABΔCC mutant (constitutively 'open' state) caused cell spreading with membrane ruffles. Next, we identified the proteins involved in JRAB-induced rearrangement of actin cytoskeleton leading to morphological changes. In NIH3T3 cells expressing HA-JRABΔCC, filamin, an actin cross-linking protein, coimmunoprecipitated with HA-JRABΔCC. Expression of ASB2 induced degradation of all three filamin isoforms and inhibited the JRABΔCC-induced cell spreading. Consistent with our previous results, actinin-1/-4 were also immunoprecipitated with HA-JRABΔCC. However, actinin-1/-4 have no effect on the cell spreading regulated by JRABΔCC. These data suggest that JRAB contributes to the rearrangement of the actin cytoskeleton during cell spreading via filamins.
我们之前曾表明 Rab13 及其效应蛋白，连接 Rab13 结合蛋白（JRAB）/与 CasL 样 2（MICAL-L2）相互作用的分子，通过调节细胞黏附分子运输和上皮细胞中的肌动蛋白细胞骨架重排来调节连接的发育。在这里，我们研究了 JRAB 如何调节 NIH3T3 成纤维细胞中肌动蛋白细胞骨架的重组，试图深入了解 JRAB 作用的机制。为此，我们表达了采用组成型开放或关闭状态的突变蛋白，然后检查了由此产生的肌动蛋白细胞骨架重排对细胞形态的影响。表达 JRABΔCT 突变体（组成型“关闭”状态）诱导应激纤维，而表达 JRABΔCC 突变体（组成型“打开”状态）导致细胞铺展并有膜皱襞。接下来，我们确定了参与 JRAB 诱导的肌动蛋白细胞骨架重排导致形态变化的蛋白。在表达 HA-JRABΔCC 的 NIH3T3 细胞中，肌联蛋白，一种肌动蛋白交联蛋白，与 HA-JRABΔCC 共免疫沉淀。表达 ASB2 诱导所有三种肌联蛋白同工型的降解，并抑制 JRABΔCC 诱导的细胞铺展。与我们之前的结果一致，肌动蛋白-1/-4 也与 HA-JRABΔCC 免疫沉淀。然而，肌动蛋白-1/-4 对 JRABΔCC 调节的细胞铺展没有影响。这些数据表明，JRAB 通过肌联蛋白有助于细胞铺展过程中肌动蛋白细胞骨架的重排。
