Yamanaka Daisuke, Akama Takeshi, Chida Kazuhiro, Minami Shiro, Ito Koichi, Hakuno Fumihiko, Takahashi Shin-Ichiro
Laboratory of Cell Regulation, Department of Animal Resource Sciences, Graduate School of Agriculture and Life Science, The University of Tokyo, Bunkyo-ku, Japan; Laboratory of Food and Physiological Models, Department of Veterinary Medical Sciences, Graduate School of Agriculture and Life Science, The University of Tokyo, Kasama, Japan; Department of Bioregulation, Nippon Medical School, Kawasaki, Japan.
Laboratory of Cell Regulation, Department of Animal Resource Sciences, Graduate School of Agriculture and Life Science, The University of Tokyo , Bunkyo-ku , Japan.
Front Endocrinol (Lausanne). 2016 Jul 11;7:89. doi: 10.3389/fendo.2016.00089. eCollection 2016.
Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830-840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250-1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and plays an important role in endocytosis.
肌动蛋白交联蛋白控制肌动蛋白丝网络和束，并参与多种细胞功能，包括调节细胞迁移、细胞形态和内吞作用。磷脂酰肌醇3激酶相关蛋白（PI3KAP）/XB130已被报道定位于肌动蛋白丝（F-肌动蛋白），并且在甲状腺癌细胞的细胞迁移中是必需的。在此，我们展示了PI3KAP/XB130作为一种肌动蛋白交联蛋白的作用。首先，我们发现PI3KAP/XB130的羧基末端区域（包含氨基酸残基830 - 840）对于在NIH3T3细胞中定位于F-肌动蛋白是必需且足够的，并且该区域在体外直接与F-肌动蛋白结合。此外，肌动蛋白交联分析表明重组PI3KAP/XB130交联F-肌动蛋白。一般来说，肌动蛋白交联蛋白常常多聚化以组装多个肌动蛋白结合位点。然后我们研究了PI3KAP/XB130是否能形成多聚体。蓝色非变性聚丙烯酰胺凝胶电泳分析表明，尽管重组PI3KAP/XB130的分子量约为125 kDa，但在250 - 1200 kDa处检测到它，这表明PI3KAP/XB130形成了多聚体。此外，通过在HEK293T细胞中的共免疫沉淀分析，我们发现多聚化需要氨基末端的40个氨基酸。缺乏肌动蛋白结合区域或多聚化区域的PI3KAP/XB130缺失突变体不能交联肌动蛋白丝，这表明PI3KAP/XB130的肌动蛋白结合和多聚化对于交联F-肌动蛋白是必需的。最后，我们研究了PI3KAP/XB130在内吞作用（一种与肌动蛋白相关的生物学过程）中的作用。PI3KAP/XB130的过表达增强了HEK 293细胞中葡聚糖的摄取。然而，大多数转染了缺乏肌动蛋白结合区域的缺失突变体的细胞摄取葡聚糖的程度与对照细胞相似。综上所述，这些结果表明PI3KAP/XB130通过其肌动蛋白结合区域和多聚化区域交联F-肌动蛋白，并在内吞作用中起重要作用。
