(A) GULP binds specifically to ACAP1 but not other Arf6-GAPs or Arf6-GEFs. Epitope-tagged versions of Arf6-GEFs or the Arf6-GAPs were expressed in HeLa cells and precipitated with bacterial GST-GULP or GST alone and the binding assessed by immunoblotting. (B) Interaction between endogenous GULP and ACAP1. Lysates from control or GULP-knockdown MEF-1 cells were immunoprecipitated with anti-GULP and immunoblotted for ACAP-1. GULP knockdown and comparable ACAP1 in total cell lysates were confirmed by immunoblotting. (C)GULP directly binds ACAP1 via the PTB domain. Recombinant, bacterially produced His-tagged ACAP1 fragment was incubated with bacterially produced GST-GULP, GST-alone or an unrelated protein GST-RAP. The bound ACAP1 was detected by anti-His immunoblotting. (D) Partial reversal of the ACAP1 mediated inhibition of endogenous Arf6-GTP by GULP and GULP-PTB. Parental LR73 cells or LR73 cells stably expressing GST-tagged full length GULP or GULP-PTB domain were transiently transfected with Flag-ACAP1 or empty vector. The endogenous Arf6-GTP levels were assessed as in 1E.

(A) GULP-PTB is necessary and sufficient for binding to ACAP1. GST-tagged GULP PTB, GULP LZ+C or an unrelated protein RAP was incubated with HeLa cell lysates and the binding of endogenous ACAP1 was assessed by immunoblotting. (B) Schematic diagrams of ACAP1 mutants with an N-terminal Flag tag. (C) GULP binds to the GAP+Ankyrin repeats of ACAP1. Full length ACAP1 or the mutants expressed in HeLa cells were tested for binding to GULP or GULP-PTB. The GST-GULP proteins on the membranes were visualized by Ponceau S staining. (D) GULP-PTB partially rescues ACAP1 mediated inhibition of cell migration. GST alone, GST-tagged GULP or GULP-PTB was co-transfected with ACAP1 into HeLa cells. Cell migration across the Transwell membrane was assessed as in Figure 3A. * indicate statistical significance (unpaired t test, P<0.05, n=4). (E, F, G) Trimeric complex formation among GULP, Arf6 and ACAP1. (E) Arf6 mutants were incubated with GST-GULP bacterially produced bound to glutathione beads. Recombinant His-ACAP1 was then added and the bound ACAP1 assessed. Precipitated ACAP1 levels were compared after setting the ACAP1 signal in the condition of GST-GULP +ACAP1 as 1.0 (lane 1). (F) Indicated amounts of bacterially produced recombinant Arf6^T27N and increasing amount of His-ACAP1 proteins were mixed with bacterially produced GST-GULP bound to glutathione beads. Arf6 levels were compared after setting the Arf6 signal in the GST-GULP +Arf6 T27N only as 1.0 (lane 1). (G) Trimeric complex formation between GULP, ACAP1 and Arf6. Flag-tagged ACAP1 was incubated with GST-GULP and Arf6^T27N. After washing and elution with Flag peptide, a second precipitation using glutathione beads was performed, and the ACAP1 and Arf6 proteins precipitated with GST-GULP were assessed by immunoblotting.

(A) GULP binds GDP-Arf6. GFP-tagged wild-type Arf6 or the mutants expressed in HeLa cells, were precipitated with bacterially produced GST-GULP and assessed for binding. Comparable expression of the Arf6 proteins in total lysates is shown (bottom). (B) GULP binds specifically to Arf6. Q → L or T → N mutants of Arf6-HA, Flag-Rac1, Flag-RhoG or Myc-Cdc42 were expressed in HeLa cells and their precipitation with GST-GULP was assessed by immunoblotting for the individual epitope-tagged GTPases. (C) GULP interacts with endogenous Arf6. GST-tagged full length GULP (GULP-FL), GULP-PTB, GULP-LZ+C, or the Shc-PTB domain were incubated with MEF-2 cell lysates and Arf6 binding was detected by immunoblotting. The GST fusion proteins were visualized by Ponceau S staining. (D) GULP directly binds Arf6 via the PTB domain. Schematic diagram of GULP and the mutants (top). Arf6^T27N and Arf6^Q67L mutants were incubated with GULP (all bacterially produced), and binding assessed by anti-Arf6 immunoblotting. GST-GULP was visualized by Ponceau S staining. ΔN^1–24 mutant lacks the first 24 amino acids GULP-PTB domain. A line indicates the lanes from the same gel that were not run contiguously, but spliced together for presentation. (E) Knockdown of GULP expression decreases endogenous Arf6-GTP in cells. Arf6-GTP levels in two of the GULP knockdown MEF-1 clones and a control clone were assessed by a GST-GGA pull down assay. Comparable Arf6 expression in the cell lines was revealed by immunoblotting. ERK2 immunoblotting revealed equal protein loading. Arf6-GTP levels were compared after setting the ratio of the Arf6-GTP signal to total Arf6 signal in the control clone as 1.0. Relative GULP expression was compared after setting the ratio of GULP signal to ERK2 signal in the control clone as 1.0. (F) Knockdown or overexpression of GULP in LR73 cells affects cellular Arf6-GTP and cellular migration. An arf6-GTP level in cells with GULP overexpression or knockdown was assessed as in E. Cell migration to fibronectin was don in a Transwell assay. * indicates P<0.05 (n=3) compared to the respective control clone.

(A) Reversal of ACAP1 mediated inhibition of cellular migration by GULP is dose dependent. HeLa cells were co-transfected with a GULP plasmid and decreasing amounts of ACAP1 plasmid. A control GFP expressing plasmid was included in all of the transfections. The migration of cells in a Transwell assay toward 20% FBS was assessed. Results were standardized against the condition in which only GFP and a control plasmid were transfected. Expression of transfected ACAP1 and GULP, as well as the GFP and ERK2 levels (to ensure equal loading of cells into the upper chamber of the Transwell) was assessed by immunoblotting. Relative ACAP1 expression levels were compared after setting the ratio of the ACAP1 signal to GFP signal in lane 3 only as 1.0. * P<0.05 (unpaired t test, n=3). (B) GULP specifically regulates ACAP1 inhibition of cellular migration. HeLa cells were co-transfected with the indicated plasmids and cell migration was assessed as in Figure 3A. * P<0.05 (unpaired t test, n=3). (C) GULP regulates ACAP1-mediated inhibition of wound healing. HeLa cells transfected with a GULP plasmid and wild-type ACAP1 or GAP-deficient mutant (ACAP1-GD) were examined in the wound-healing assay. * P<0.05 (unpaired t test, n=3).