A novel and evolutionarily conserved PtdIns(3,4,5)P3-binding domain is necessary for DOCK180 signalling

Abstract

The evolutionarily conserved DOCK180 protein has an indispensable role in cell migration by functioning as an exchange factor for Rac GTPase via its DOCK homology region (DHR)-2 domain. We report here that the conserved DHR-1 domain also has an important signalling role. A form of DOCK180 that lacks DHR-1 fails to promote cell migration, although it is capable of inducing Rac GTP-loading. The DHR-1 domain interacts with PtdIns(3,4,5)P(3) in vitro and in vivo, and mediates the DOCK180 signalling complex localization at sites of PtdIns(3,4,5)P(3) accumulation in the cell's leading edge. A form of DOCK180 in which the DHR-1 domain has been replaced by a canonical PtdIns(3,4,5)P(3)-binding pleckstrin homology domain is fully functional at inducing cell elongation and migration, suggesting that the main function of DHR-1 is to bind PtdIns(3,4,5)P(3). These results demonstrate that DOCK180, via its DHR-1 and DHR-2 domains, couples PtdIns(3,4,5)P(3) signalling to Rac GTP-loading, which is essential for directional cell movement.

Fig. 1. The DHR-1 domain is required for DOCK180-mediated cell elongation and motility

a) Serum-starved LR73 cells that had been transfected with the indicated plasmids were detached and plated on fibronectin-coated cover slips for 2 h in the absence of serum prior to fixing. Cells in the left panels were stained with anti-DOCK180 (H-4), while the panels on the right represent an overlay of the anti-DOCK180, rhodamine-phalloidin and DAPI stains. Cells were photographed at a 60X magnification. b) Expression levels of the transfected proteins were analyzed by immunoblotting of cell lysates with anti-DOCK180 (C-19) and anti-Myc antibodies, as indicated. c, d) Serum-starved LR73 cells that had been transfected with a GFP plasmid together with the indicated plasmids were detached and placed in the upper compartment of a Boyden-chamber. In the haptotactic migration assay (c), the cells were allowed to migrate for 4 h towards fibronectin. In the chemotactic assay (d), the top and bottom sides of the membrane were precoated with fibronectin and the cells were allowed to migrate for 4 h towards 10% serum as a chemoattractant in the lower chamber. Cells were fixed and stained with DAPI. GFP/DAPI double positive cells that had migrated across the membrane were counted from photographs taken at a 40X magnification. The experiments described above were performed independently three times in duplicate. Data are shown as mean +/- SD. *, P < 0.001; one-way ANOVA. e) Expression levels of the transfected proteins were analyzed by immunoblotting of cell lysates with anti-DOCK180 (C-19) and anti-Myc antibodies, as indicated.

Fig. 2. The DHR-1 domain is required for cell spreading

a) Immunoblot analysis with an anti-DOCK180 antibody (C-19) of total LR73 cell lysates that had been treated with either control siRNA (ctrl) or siRNA against DOCK180 (siDOCK180), and simultaneously transfected with an empty vector (pcDNA), or a vector coding for the wild-type DOCK180 or the DOCK180 DHR-1 mutant (top panel). Anti-Tubulin blot was used as a loading control (bottom panel). b–c) siRNA-mediated downregulation of DOCK180 levels leads to an impaired cell spreading which can be rescued by re-expression of wild-type DOCK180 protein but not of DOCK180 DHR-1. Micrographs of cells from three independent experiments were pooled and used to score for two phenotypes: 1-Round and 2-Spread. Quantification of the results is presented in a table format in (b). Representative micrographs in (c) are an overlay of FITC-phalloidin and DAPI stains. The cells were photographed at a 60X magnification. d–e) Cells treated as in (b–c) were stained for DOCK180 (H-4) to allow for identification and specific analysis of those re-expressing the wild-type DOCK180 or the DOCK180 DHR-1 mutant proteins. Cells with equal green fluorescent intensity were scored for the round and spread phenotypes. Quantification of the results in a representative experiment is presented in a table format in (d), while representative micrographs of the same experiment are shown in (e). In (e), upper panels show anti-DOCK180 stains, while the bottom panels are overlays of DOCK180, rhodamine-phalloidin and DAPI stains.

Fig. 3. Intact GTP-loading of Rac and coupling to ELMO1 and CrkII by the DHR-1 mutant of DOCK180

a) LR73 cells were transfected with the indicated plasmids and the GTP-loading status of Rac was analyzed by affinity precipitation with the PBD domain of PAK immobilized to glutathione-sepharose beads (upper panel). As a loading control for Rac and to verify the expression of each protein, 20 μl of total cell lysates (TCL) were analyzed by immunoblotting with antibodies against Rac, DOCK180 and Myc (for ELMO1 and CrkII). The experiment was performed independently three times, and no statistical differences were observed between the capabilities of the wild-type DOCK180 and the DOCK180ΔDHR-1 mutant to induce GTP-loading of Rac (the DOCK180ΔDHR-1 mutant demonstrated, in arbitrary units, a 0.94±0.05-fold Rac activation when co-expressed with ELMO1 and CrkII, compared to 1.0-fold activation by wild-type DOCK180, Crk and ELMO). b) DOCK180 lacking the DHR-1 forms a trimolecular complex with ELMO1 and CrkII. LR73 cells were transfected with the indicated plasmids and Triton X-100 lysates were immunoprecipitated with an antibody against the Myc-epitope. The coprecipitation of the various DOCK180 proteins and ELMO1-GFP, in addition to the verification of equal Myc-tagged CrkII precipitation, was analyzed by immunoblotting with antibodies against DOCK180 (C19), GFP and Myc, respectively (left panels). The expression levels of each protein were analyzed by immunoblotting 10 μg of the total Triton X-100 lysates with antibodies against DOCK180 (C19), GFP and Myc (right panels).

Fig. 4. The DHR-1 domain of DOCK180 has lipid-binding activity toward phosphoinositides in vitro

a) The DHR-1 domain of DOCK180 binds to PtdIns(3,5)P₂ and PtdIns(3,4,5)P₃ in vitro. 5 μg of purified recombinant His-DHR-1 or GST-Akt-PH proteins were incubated with beads coated with the indicated phosphoinositides, or as a control, with beads alone. The bound proteins were detected by immunoblotting with anti-His or anti-GST antibodies. b) Mapping of the minimal PtdIns(3,4,5)P₃ binding site in the DHR-1 domain. A graphical representation of the various DHR-1 domain constructs is shown on the left. The carboxyl-terminal truncations of the DHR-1 domain were tested for their ability to interact with beads coated with PtdIns(3,4,5)P₃ and the bound material was detected as described in (a). Fractions of the purified proteins were analyzed on SDS-PAGE and stained with Coomassie Blue as a control. c) Liposome competition assay. The DHR-1 domain of DOCK180 and the PH domain of Akt were preincubated with various liposomes, or Ins(1,3,4,5)P₄, prior to a pull-down experiment with the indicated lipid-coated beads. The bound material was detected as described in (a). Relative binding is indicated. d) Mutation of six conserved lysines residues in the putative CBR1 and CBR3 loops of the DHR-1 domain abolishes the lipid binding activity. A graphical representation of the DHR-1 mutant is shown on the left. The indicated proteins were purified and subjected to binding to PtdIns(3,4,5)P₃-beads, as described in (a). In the lower panel, the purified proteins were analyzed on SDS-PAGE and stained with Coomassie Blue as a control. FAK-FAT, focal adhesion targeting motif of FAK. e) Protein-lipid overlay assay. Lipid-spotted membranes were overlayed with the indicated purified proteins. Bound proteins were detected by immunoblotting with anti-His and anti-GST antibodies, as indicated.

Fig. 5. The DHR-1 domain displays lipid binding activity in vivo

a) The DOCK180 DHR-1 mutant fails to interact with PtdIns(3,4,5)P₃. DOCK180 WT, DOCK180 DHR-1 or DOCK180 DHR-1+PH (see Fig. 7 and supplemental information, Fig. S1) were expressed in LR73 cells. Cell lysates were subjected to a pull-down by PtdIns(3,4,5)P₃-beads. Bound proteins were detected by immunoblotting with an anti-DOCK180 antibody (C19). The lower panels demonstrate the expression levels of the exogenous DOCK180 proteins. b) Endogenous DOCK180 localizes to the plasma membrane in response to PDGF stimulation in a PtdIns 3-kinase dependent manner. Serum-starved NIH3T3 cells were treated with either DMSO (top and middle panels) or 50 μM LY294002 for 30 min (bottom panels). Cells were subsequently left untreated (top panels) or stimulated with 10 ng/ml PDGF (middle and bottom panels) for 10 min prior to fixing. Cells in the left panels were stained with an anti-DOCK180 rabbit polyclonal antibody, while the panels on the right represent an overlay of the anti-DOCK180, rhodamine-phalloidin and DAPI stains. Cells were photographed at a 60X magnification. c) DOCK180 translocates to the membrane in response to PtdIns(3,4,5)P₃ production in a DHR-1-dependent manner. HEK293T cells were transfected with the indicated plasmids. After 24 h, the cytosolic and membrane fractions were biochemically purified and the distribution of DOCK180, DOCK180 DHR-1 and p110* was analyzed by immunoblotting with the indicated antibodies.

Fig 6. Generation of PtdIns(3,4,5)P³ is necessary for DOCK180-induced cell elongation and migration

a) CrkII-ELMO1-DOCK180-induced cell elongation is sensitive to the PtdIns 3-kinase inhibitor LY294002. Serum-starved LR73 cells expressing the indicated plasmids were detached and incubated with the LY294002 inhibitor (25 μM) for 30 min in suspension. The cells were then allowed to spread for 2 h on fibronectin-coated slides in the presence of LY294002, fixed and stained with anti-DOCK180 (H-4) antibody, Rhodamine-phalloidin and DAPI. Photographs were taken at a 60X magnification. (Compare to Fig. 1a). The expression levels of the transfected proteins were analyzed by immunoblotting of total cell lysates with anti-DOCK180 (C19) and anti-Myc antibodies, as indicated (right panel). b) CrkII-ELMO1-DOCK180-induced cell migration is sensitive to LY294002. Serum-starved LR73 cells expressing the indicated plasmids, together with a plasmid for GFP, were subjected to a haptotactic migration assay. For the sample treated with 25 μM of LY294002, the inhibitor was added to both the upper and the lower compartment of the modified Boyden chamber. Cell migration was analyzed as described in Fig. 1a. Data represent mean +/− SD of a representative experiment performed in duplicate. *, P<0.001; one-way ANOVA. In the lower panel, expression levels of the transfected proteins were analyzed by immunoblotting of cell lysates with anti-DOCK180 (C19) and anti-Myc antibodies, as indicated.

Fig. 7. The PH domain of BMX/Etk can functionally replace the DHR-1 domain in DOCK180

a) A chimeric DOCK180 protein (DOCK180 DHR-1+PH) in which the DHR-1 domain had been replaced with the PH domain of Bmx/Etk translocates to the membrane in response to PtdIns(3,4,5)P₃ production. HEK293T cells were transfected with the indicated plasmids. After 24 h, the cytosolic and membrane fractions were biochemically purified and the distribution of DOCK180 DHR-1+PH and p110* was analyzed by immunoblotting with the indicated antibodies. b) The introduction of the PH domain of Bmx/Etk in the DHR-1 mutant rescues the cell polarization defects. Serum-starved LR73 cells expressing the indicated plasmids were detached and allowed to spread on fibronectin for 2 h. Cells were then analyzed as described in Fig 1a. When treated with LY294002, the cells were preincubated with the inhibitor for 30 min prior to plating and the inhibitor was left on the cells throughout the experiment. In the right panel, expression levels of the proteins were analyzed by immunoblotting the cell lysates with anti-DOCK180 (C19) and anti-Myc antibodies. c) Quantification of the effect on cell elongation by the DOCK180 DHR-1+PH chimeric protein. Cells were processed exactly as in (b) and several independent fields were photographed. The cells were visually inspected and scored for three phenotypes: 1- Round (attached and minimally spread cells), 2- Spread (clearly spread and flat cells) and 3- Elongated (elongated cells with a polarity). This is a representative experiment of three independent assays. d–e) The introduction of the PH domain of Bmx/Etk in the DHR-1 mutant rescues the cell migration defects. Serum-starved LR73 expressing the indicated plasmids were detached and subjected to haptotactic and chemotactic migration assays, as described in Fig. 1. Data represent mean +/- SD of a representative experiment performed in duplicate. *, P<0.001; one-way ANOVA. f) Expression levels of the transfected proteins were analyzed by immunoblotting of cell lysates with anti-DOCK180 and anti-Myc antibodies, as indicated.