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doi: 10.1242/10.1242/jcs.00219

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Identification of an evolutionarily conserved superfamily of DOCK180-related proteins with guanine nucleotide exchange activity

Jean-François Côté and Kristiina Vuori*

The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA



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  		Fig. 1. Identification of the DOCK180 superfamily of proteins. (A) Phylogenetic tree of DOCK180-related proteins. Amino-acid sequences that cover the region of the DHR-2 domains (see below) of the indicated human, Drosophila and C. elegans family members were aligned with ClustalW. The tree was derived by neighbor-joining analysis applied to pairwise sequence distances calculated with the PHYLIP package using the Kimura two-parameter method to generate unrooted trees. The final output was generated with TREEVIEW. The number at each node represents the percentage of bootstrap replicates (out of 100). The four subfamilies of DOCK180-related proteins (DOCK-A, -B, -C and -D) are indicated. (B) Schematic diagram of the structure of representative members of the human DOCK180 superfamily. Percentage identity between the various SH3, DHR-1 and DHR-2 domains in amino-acid sequence to DOCK180 is indicated. Structures of DOCK180, DOCK2, DOCK3 and DOCK9 were derived from full-length sequences. The sequence for DOCK6 could be truncated at the N-terminus (indicated by a yellow dashed line).

 


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  		Fig. 1. Identification of the DOCK180 superfamily of proteins. (C,D) Multiple sequence alignment of the DHR-1 (C) and DHR-2 (D) domains. DHR-1 and DHR-2 sequences from representative members of the human DOCK180 superfamily were aligned with ClustalW and the final output was generated by BoxShade. Black and gray shading indicate identical and similar residues, respectively. Identical residues are indicated in capital letters and similar residues in lower-case letters in the consensus sequence.

 


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  		Fig. 2. The DHR-2 domain of DOCK180 binds to and activates Rac in vitro and is necessary and sufficient for DOCK180-mediated Rac activation in vivo. (A) Schematic representation of the DOCK180 DHR-2 domain. The boundaries of the fusion proteins that were used in GTPase binding and GEF assays are indicated. (B) Lysates from untransfected COS-1 cells (for RhoA detection) or cells transfected with Myc-Rac or Myc-Cdc42 were incubated with the indicated GST-fusion proteins bound to Glutathione beads. The precipitated proteins were detected by immunoblotting with anti-Myc or anti-RhoA antibodies. TCL, total cell lysate. (C) Bacterially produced, purified DHR-2 domain of DOCK180 and the DPC domain of Vav2 were used in an in vitro GEF assay toward [3H] GDP-loaded Rac, Cdc42 or RhoA. Reactions were stopped at 0, 15 and 30 minute time points. Radiolabelled GDP bound to the GTPases was measured by using a filter-binding assay, as described in Materials and Methods. (D) 293-T cells were transfected with a control vector or with vectors coding for Flag-DOCK180, DOCK180{Delta}DHR-2, Myc-DHR-2, Myc-DHR-2/DH domain or Myc-Docker (see text for details). Rac-GTP was pulled down from cell lysates using the p21-binding domain of PAK fused to GST (`PBD' assay). The amount of Rac in pull-downs and in total cell lysates (`TCL') was detected by anti-Rac immunoblotting. Expression levels of the various DOCK180 proteins were analyzed by anti-DOCK180 and anti-Myc immunoblotting in total cell lysates.

 


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  		Fig. 3. Coexpression of ELMO1 is not required for DOCK180-mediated Rac activation in LR73 cells. (A) LR73 cells were transfected with a control plasmid or with the indicated amounts of plasmids coding for Flag-DOCK180 and Myc-ELMO1. GTP loading of Rac was measured by the PBD pull-down assay, and the precipitated Rac was analyzed by immunoblotting with anti-Rac antibodies. Expression levels of Rac, Flag-DOCK180 and Myc-ELMO1 were analyzed by anti-Rac, anti-Flag and anti-Myc immunoblotting, respectively, in total cell lysates. (B) LR73 cells were transfected with a control plasmid or with the indicated amounts of plasmids coding for Flag-DOCK180 with or without cotransfection of 1 µg of ELMO1-GFP. GTP loading of Rac was measured by the PBD pull-down assay, and the precipitated Rac was analyzed by immunoblotting with anti-Rac antibodies. Expression levels of Rac, Flag-DOCK180 and ELMO1-GFP were analyzed by anti-Rac, anti-Flag and anti-GFP immunoblotting, respectively, in total cell lysates.

 


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  		Fig. 4. The DHR-2 domains of the various DOCK180-related proteins bind to nucleotide-free small GTPases. (A) The in vitro transcribed and translated DHR-2 domain of CED-5 was tested for its ability to interact with nucleotide-free Rac1, Cdc42 or RhoA, as described in Materials and Methods. The bound CED-5 DHR-2 domain was visualized by autoradiography. (B) COS-1 cell lysates were incubated with the indicated DHR-2 fusion proteins or with GST alone as described in Materials and Methods, and the precipitated Rac1 was visualized by anti-Rac immunoblotting. (C) The DHR-2 domains of DOCK7 and DOCK9 were in vitro transcribed and translated as in (A) and tested for their ability to interact with nucleotide-free Rac1, Cdc42 and RhoA. The DHR-2 domains were visualized by autoradiography.

 


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  		Fig. 5. The DHR-2 domains of the various DOCK180-related proteins have GEF activity toward small GTPases. (A,B) The indicated DHR-2 domains of various DOCK180-related proteins, GST alone or the DPC domain of Vav2 as a positive control were tested in an in vitro GEF assay toward [3H] GDP-loaded Rac1 (A) and Cdc42 (B). Reactions were stopped at 0, 15 and 30 minute time points. Radiolabelled GDP bound to the GTPases was measured by using a filter-binding assay. (C) Schematic representation of the various DOCK2 and DOCK9 constructs that were used to study the in vivo activation of Rac and Cdc42. (D) Various DOCK2 and DOCK9 constructs were expressed in 293-T cells as indicated, and Rac-GTP and Cdc42-GTP were pulled down from cell lysates using the p21-binding domain of PAK fused to GST (PBD pull-down assay). Precipitated Rac 1 and Cdc42 were detected by anti-Rac and anti-Cdc42 immunoblotting, respectively. Transfected proteins were detected by immunoblotting the lysates with anti-Flag or anti-Myc antibodies.

 

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