Ruiz-Lafuente Natalia, Alcaraz-García María-José, García-Serna Azahara-María, Sebastián-Ruiz Silvia, Moya-Quiles María-Rosa, García-Alonso Ana-María, Parrado Antonio
Immunology Service, Virgen de la Arrixaca Clinic University Hospital, Institute for Biohealth Research (IMIB-Arrixaca), Ctra. Madrid-Cartagena s/n, El Palmar, 30120, Murcia, Spain.
Immunology Service, Virgen de la Arrixaca Clinic University Hospital, Institute for Biohealth Research (IMIB-Arrixaca), Ctra. Madrid-Cartagena s/n, El Palmar, 30120, Murcia, Spain
Biol Open. 2015 Apr 10;4(5):627-35. doi: 10.1242/bio.20149050.
Dock10 is one of the three members of the Dock-D family of Dock proteins, a class of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Its homologs Dock9 and Dock11 are Cdc42 GEFs. Dock10 is required for maintenance of rounded morphology and amoeboid-type movement. Full-length isoforms of Dock10 have been recently cloned. Here, we address GTPase specificity and GEF activity of Dock10. In order of decreasing intensity, Dock10 interacted with nucleotide-free Rac1, Cdc42, and Rac3, and more weakly with Rac2, RhoF, and RhoG. Inducible expression of Dock10 in HeLa epithelial cells promoted GEF activity on Cdc42 and Rac1, and a morphologic change in two-dimensional culture consisting in loss of cell elongation, increase of filopodia, and ruffles. Area in contact with the substrate of cells that spread with non-elongated morphology was larger in cells expressing Dock10. Inducible expression of constitutively active mutants of Cdc42 and Rac1 in HeLa cells also induced loss of elongation. However, Cdc42 induced filopodia and contraction, and Rac1 induced membrane ruffles and flattening. When co-expressed with Dock10, Cdc42 potentiated filopodia, and Rac1 potentiated ruffles. These results suggest that Dock10 functions as a dual GEF for Cdc42 and Rac1, affecting cell morphology, spreading and actin cytoskeleton protrusions of adherent HeLa cells.
Dock10是Dock蛋白家族中Dock-D亚家族的三个成员之一，Dock蛋白是一类Rho GTP酶的鸟嘌呤核苷酸交换因子（GEF）。它的同源物Dock9和Dock11是Cdc42 GEF。维持圆形形态和阿米巴样运动需要Dock10。最近已克隆出Dock10的全长异构体。在此，我们研究了Dock10的GTP酶特异性和GEF活性。按相互作用强度递减顺序，Dock10与无核苷酸的Rac1、Cdc42和Rac3相互作用，与Rac2、RhoF和RhoG的相互作用较弱。在HeLa上皮细胞中诱导表达Dock10可促进对Cdc42和Rac是的GEF活性，并在二维培养中引起形态学变化，包括细胞伸长丧失、丝状伪足增加和褶皱增加。在表达Dock10的细胞中，以非伸长形态铺展的细胞与底物接触的面积更大。在HeLa细胞中诱导表达组成型活性的Cdc42和Rac1突变体也会导致伸长丧失。然而，Cdc42诱导丝状伪足和收缩，Rac1诱导膜褶皱和平坦化。当与Dock10共表达时，Cdc42增强丝状伪足，Rac1增强褶皱。这些结果表明，Dock10作为Cdc42和Rac1的双重GEF发挥作用，影响贴壁HeLa细胞的形态、铺展和肌动蛋白细胞骨架突起。
