Abstract
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.
Highlights
Rho GTPases are small proteins involved in actin cytoskeleton organization, cell shape, adhesion and movement (Wennerberg and Der, 2004; Aspenstrom et al, 2004; Heasman and Ridley, 2008)
Total protein extracts of 293T cells transfected with HA-Dock10.1, HA-Dock10.2, HADock11, and FLAG-Dock9 were assayed for precipitation by GSTbound Cdc42, Rac1, Rac2, Rac3, RhoA, RhoD, RhoF, RhoG, RhoJ (TCL), and RhoQ (TC10)
Using nucleotide-free forms of the GTPases, we found that both Dock10 isoforms interacted, in order of decreasing intensity, with Rac1, Cdc42, and Rac3, and weakly with Rac2, RhoF, and RhoG; Dock11 interacted with Cdc42, and weakly with Rac1, and RhoJ; and Dock9 interacted with Cdc42, and weakly with Rac1, and RhoD (Fig. 1A)
Summary
Rho GTPases are small proteins involved in actin cytoskeleton organization, cell shape, adhesion and movement (Wennerberg and Der, 2004; Aspenstrom et al, 2004; Heasman and Ridley, 2008). The ‘‘classic’’ Rho GTPases cycle between two forms, GDP- or GTP-bound. The GTP-bound is the ‘‘active’’ form, because it associates with downstream effectors. Received 5 June 2014; Accepted 2 March 2015 and RhoG), RhoA-related (RhoA, RhoB, and RhoC), RhoD, and RhoF/Rif. Additional 8 genes encode the ‘‘atypical’’ Rho GTPases, constitutively bound to GTP. The activity of Rho GTPases is regulated by 3 classes of proteins: 1) Guanosine nucleotide exchange factors (GEFs), which stimulate the weak intrinsic exchange activity of Rho GTPases to promote the formation of the GTP-bound form; 2) GTPase-activating proteins (GAPs), which stimulate GTPase activity and conversion to the GDP-bound form; and 3) Rho GDP dissociation inhibitors (GDIs), which retain the GDP-bound form in the cytoplasm
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