Morphology of cells expressing membrane-targeted DOCK180, constitutively active Rac1, and constitutively active Cdc42Hs. NIH-3T3 cells were microinjected with pEF–HA–RacV12 (RacV12), pEBG–Cdc42Hs–QL (Cdc42QL), pCA–DOCK-F (DOCK180), or pEBG (GST), incubated with anti-HA antibody for RacV12, anti-GST antibody for Cdc42Hs and GST, or anti-DOCK180 antibody for DOCK180. White bars, 23 μm.

Effect of dominant-negative Rac1 on the DOCK180-induced morphologic change. NIH-3T3 cells were microinjected with expression vectors encoding proteins indicated at left. Cells were fixed and incubated with antibodies, indicated at bottom. White arrowheads (rows 2 and 4) indicate cellular spreading. White bars, 27 μm.

Binding of DOCK180 to Rac1. (A) 293T cells were transfected with pEBG, pEBG–RhoA, pEBG–Rac1, or pEBG–Cdc42Hs, lysed, and GST-tagged proteins were collected by incubation with glutathione–Sepharose, separated by SDS-PAGE, and blotted with anti-DOCK180 antibody. Arrows and numbers at right indicate the position of DOCK180 (1), GST–RhoA (2), GST–Rac1 and Cdc42Hs (3), and GST (4). (B) 293T cells were transfected with pCA–Flag–DOCK180, together with pEB–HA–RacN17 or pEB–HA–RacV12, lysed, and incubated with anti-DOCK180 antibody and protein A–Sepharose for 2 hr. Immune complexes were washed, separated by SDS-PAGE, and blotted with anti-HA-antibody. (IP, NRS) Immunoprecipitation, normal rabbit serum, respectively. (C) DOCK180 purified from 293T cells was incubated with glutathione–Sepharose, which was preloaded with nucleotide-free (−), GDP-bound (GDP), or GTPγS-bound GST–Rac1 (GTP). Proteins bound to beads were analyzed by imunoblotting with anti-His antibody.

Activation of Rac1 by DOCK180. (A) 293T cells were transfected with pEBG–Rac1 (Rac1) or pEBG–RhoA (RhoA) in combination with pCA–Flag–DOCK180 or empty vector (see Materials and Methods). The labeled guanine nucleotides bound to GST-tagged G proteins were separated by TLC and visualized and quantitated with a Molecular Imager. (Ori) Origin of the TLC plate. Data shown were obtained from three independent experiments. Bars, standard deviation. (B) 293T cells were transfected with pEBG–Rac1 in combination with pCA–Flag–DOCK180 or pSR–Vav and processed as in A, except that cells were serum and phosphate starved for 16 hr before ³²P_i labeling. Expression of GST–Rac1 was confirmed by Western blotting (middle). (C) 293T cells were transfected with pEBG–Cdc42Hs with or without pCA–Flag–DOCK180 and processed as in B. (D) 293T cells were transfected with pEBG–Rac1 in combination with pCA–Flag–DOCK180 or pCA–myc–CrkII and pSSRαp130^Cas and processed as in B.

Activation of JNK by DOCK180. (A) 293T cells were transfected with pZipNeo–RasV12, pcDNA3–Cdc42QL, pCA–DOCK180, or empty vector with either pEBG–ERK2 or pEBG–JNK. Thirty-two hours after transfection, medium was changed to DMEM containing 0.5% BSA. After 16 hr starvation, cells were lysed and incubated with glutathione–Sepharose. Beads were washed and incubated in reaction buffer containing [γ-³²P]ATP and either MBP for ERK kinase assay or GST–c-Jun for JNK kinase assay. The reaction mixture was separated by SDS-PAGE. Phosphorylated MBP (phospho-MBP) or GST–c-Jun (phospho-c-jun) was visualized and quantitated with a Molecular Imager (Bio-Rad). Comparable expression of GST–ERK or GST–JNK was examined by Western blotting by use of anti-GST antibody. The graphs show the average and standard deviation of at least three experiments. (B) pCA–DOCK180 and pEBG–JNK were transfected into 293T cells with empty vector, pCMV–RacN17, pCMV–Cdc42HsN17, or pEBG–SEK-DN, and processed as in A. (C) pCA–DOCK180 and pEBG–JNK were transfected into 293T cells with or without pCA–myc–CrkII, and processed as in A.