Figure 1.
The lobe A is required for dimerization, but not for the Rac GEF activity of DHR-2 domain.
(A) Ribbon diagram of the human DOCK2DHR-2-Rac1 complex. Overall structure (left) and dimerization interface (right) are shown. The coordinates and structure factors have been deposited in the Protein Data Bank (www.pdb.org) under accession code 3B13. (B) Size-exclusion chromatography analysis for DHR-2WT and DHR-2Δlobe A. For protein size estimation, their elution volumes were compared with those of protein standards, thyroglobulin (670 kDa), immunoglobulin G (158 kDa), ovalbumin (43 kDa), and myoglobulin (17 kDa). (C) The Rac GEF activity was compared among DHR-2WT, DHR-2Δlobe A, and DHR-2V1538A using BODIPY-FL-GTP.
Figure 2.
DOCK2 forms homodimer via lobe A of DHR-2 domain in cells.
(A) Following expression of FLAG-tagged WT or mutant DOCK2 (Δlobe A or 3A) in HEK-293T cells in combination with GFP-tagged WT or mutant DOCK2 (Δlobe A or 3A) or GFP alone, cell extracts were immunoprecipitated with anti-FLAG M2 antibody or anti-GFP antibody. Immunoblotting was carried out to detect homodimerization using the relevant antibodies. (B) Extracts from HEK-293T cells expressing FLAG-tagged WT DOCK2 or Δlobe A were pulled down with GST-fusion Rac1. Assays were done in Tris-buffered saline-Tween-20 supplemented with 10 mM EDTA (E) or 10 mM MgCl2 plus 30 µM GTPγS (M). (C) Following expression of FLAG-tagged WT DOCK2 or Δlobe A in HEK-293T cells, Rac activation was analyzed. In (A-C), data are representative of, at least, three independent experiments.
Figure 3.
The expression of the Δlobe A and 3A mutant fails to restore motility and polarity in BW5147α–β– cells.
(A) Migration assays for BW5147α–β– cells stably expressing HA-tagged WT or mutant DOCK2 (V1538A, Δlobe A, or 3A). At least 145 cells were analyzed for each category of cells. Each box plot exhibits the median (central line within each box), the 25th and 75th percentile values (box ends) and the 10th and 90th percentile values (error bars). Statistical analysis was performed by the Kruskal-Wallis H test followed by the Mann-Whitney U test with Bonferroni correction. *P<0.01 for comparison with BW5147α–β– cells. Data are representative of, at least, two independent experiments. (B) The expression levels of HA-tagged WT or mutant DOCK2 (V1538A, Δlobe A, or 3A) in BW5147α–β– cells. Actin expression was included as a loading control. (C) Extracts from BW5147α–β– cells expressing HA-tagged WT or mutant DOCK2 (Δlobe A or 3A) were pulled down with GST-fusion Rac1. Assays were done in Tris-buffered saline-Tween-20 supplemented with 10 mM EDTA (E) or 10 mM MgCl2 plus 30 µM GTPγS (M). Data are representative of two independent experiments. (D, E) BW5147α–β– cells stably expressing WT or mutant DOCK2 (V1538A, Δlobe A, or 3A) were stimulated in suspension with CXCL12, and stained with phalloidin and DAPI. Data indicate the percentage of polarized cells (mean ± SD of at least 160 cells) (D) with representative images (E). *P<0.01 by Student’s t-test for comparison with unstimulated cells. Scale bar, 5 µm.
Figure 4.
Lobe A-mediated DOCK2 dimerization is required to activate Rac effectively during cell migration.
(A, B) FRET analyses for Rac activation in BW5147α–β– cells stably expressing HA-tagged WT or mutant DOCK2 (V1538A or Δlobe A). Cells were retrovirally transduced with Raichu-Rac, and were loaded on stromal cells prepared from the lymph nodes. After 4 hours of incubation, images were taken every 30 seconds, and the emission ratio of 527 nm/475 nm (FRET/CFP ratio) was used to represent the FRET efficiency. The FRET/CFP ratios at the plasma membrane were normalized by dividing by the lowest value in the cells, and cells were judged FRET-positive when the ratio was above 1.6. Data indicate the percentage of FRET-positive cells (B) with representative images (A). For each category of cells, 12–19 cells were analyzed. Scale bar, 5 µm. (C) The association of DOCK2 with Rac in BW5147α–β– cells stably expressing HA-tagged WT or mutant DOCK2 (V1538A or Δlobe A). Cell extracts were incubated with anti-HA affinity matrix in the presence of 10 mM EDTA, and bound Rac was detected with anti-Rac1 antibody.
Figure 5.
DOCK2 dimerization is functionally important for migration of primary T cells.
(A) Flow-cytometric profiles for Dock2−/− T cells infected with recombinant adenovirus encoding GFP-tagged WT or mutant DOCK2 (V1538A, Δlobe A, or 3A). Dotted lines indicate the control profile of uninfected cells. (B) Migration assays for Dock2−/− T cells expressing GFP-tagged WT or mutant DOCK2 (V1538A, Δlobe A, or 3A). At least 140 GFP-positive cells were analyzed for each category of cells. Each box plot exhibits the median (central line within each box), the 25th and 75th percentile values (box ends) and the 10th and 90th percentile values (error bars). Statistical analysis was performed by the Kruskal-Wallis H test followed by the Mann-Whitney U test with Bonferroni correction. *P<0.01 for comparison with the control samples expressing GFP alone.