RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling endosomes

Abstract

R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.

Keywords: Migration; Ras; Recycling endosome; Small GTPase; Spreading.

Fig. 1. ARNO and activated R-Ras associate with RLIP76 in a complex at recycling endosomes

(A) Co-immunoprecipitations (IP) with GFP antibodies from cells expressing GFP-tagged R-Ras(G38V) and HA-RLIP76 truncated or mutant proteins as indicated. (B) Co-IPs of myc-tagged R-Ras(G38V) with HA-tagged RLIP76 variants with α-R-Ras antibodies or control IgG as indicated. (C) Co-IPs of GFP-R-Ras, HA-RLIP76 variants as indicated, and myc-ARNO, with GFP antibodies. *, Ig heavy chain bands in the IPs. (D) Cells from two independent experiments are shown (I, ii). Secondary antibodies used alone as controls showed no detectable staining, and there were no detectable bleedthrough effects across channels (results not shown). Representative two-color component scatter plots comparing the distribution of correlated pixels for Rab11 and RLIP76 (i) or for R-Ras and RLIP76 (ii) and the calculated Pearson's correlation coefficients are shown on the far right. Bar, 10 μm. n, nucleus.

Fig. 2. ARNO co-localizes with R-Ras to recycling endosomes in a RLIP76-dependent manner

NIH 3T3 cells were transfected with FLAG-ARNO, Rab11-GFP and HA-tagged RLIP76 constructs as indicated. (A) Rab11, green; ARNO, blue. (B) Rab11, green; R-Ras, red. (C) RLIP76-null MEFs transfected with FLAG-ARNO, Rab11-GFP and HA-tagged RLIP76 constructs as indicated. Merged images and lower magnification images showing the selected regions are to the right. Representative two-color component scatter plots comparing the distribution of correlated pixels for ARNO and Rab11 (A), R-Ras and Rab11 (B), or R-Ras and ARNO (C) and the calculated Pearson's correlation coefficients are shown to the far right. Bars, 10 μm. n, nucleus.

Fig. 3. The RLIP76 N-terminus is required for efficient cell spreading upstream of Arf6

(A – D). Cells were transfected as indicated and seeded on fibronectin-coated surfaces for spreading assays. HA-RLIP76 expression levels are shown in the inset panel in (B). (0) = vector control. *, p < 0.02; **, p < 0.004; ***, p < 0.001 (n=3). The average surface areas of GFP-positive cells are shown + SEM (n=3). (C,D) Cells were treated with 1 μM of either QS11 (QS) ArfGAP1 inhibitor or QS11-NC inactive control compound (ctl.), and assayed for spreading. (E) Arf6 activation in cells transfected with the indicated RLIP76 consdtructs, shown as fold increase in the ratio of GTP-Arf6 to total Arf6, normalized to WT (Fold/WT). Representative of four independent experiments.

Fig. 4. The RLIP76 N-terminus and R-Ras interaction are required for fibroblast migration

Primary embryonic fibroblasts derived from RLIP76-null or isogenic wild type mice were transfected as indicated plus GFP. Equal numbers of wild type or RLIP76-null (KO) fibroblasts transfected as shown were seeded onto fibronectin-coated surfaces to form confluent monolayers. A single scratch was made through the monolayer with a pipet tip and GFP-positive cells at the wound margins were imaged over time, from which migration distances were calculated. Average migration distances from the starting point are shown ± SEM. *, p < 0.03; **, p < 0.02 (n=5).