Coordination of Rho and Rac GTPase function via p190B RhoGAP

Abstract

The Rac GTPase regulates Rho signaling in a broad range of physiological settings and in oncogenic transformation [1-3]. Here, we report a novel mechanism by which crosstalk between Rac and Rho GTPases is achieved. Activated Rac1 binds directly to p190B Rho GTPase-activating protein (RhoGAP), a major modulator of Rho signaling. p190B colocalizes with constitutively active Rac1 in membrane ruffles. Moreover, activated Rac1 is sufficient to recruit p190B into a detergent-insoluble membrane fraction, a process that is accompanied by a decrease in GTP-bound RhoA from membranes. p190B is recruited to the plasma membrane in response to integrin engagement [4]. We demonstrate that collagen type I, a potent inducer of Rac1-dependent cell motility in HeLa cells, counteracts cytoskeletal collapse resulting from overexpression of wild-type p190B, but not that resulting from overexpression of a p190B mutant specifically lacking the Rac1-binding sequence. Furthermore, this p190B mutant exhibits dramatically enhanced RhoGAP activity, consistent with a model whereby binding of Rac1 relieves autoinhibition of p190B RhoGAP function. Collectively, these observations establish that activated Rac1, through direct interaction with p190B, modulates subcellular RhoGAP localization and activity, thereby providing a novel mechanism for Rac control of Rho signaling in a broad range of physiological processes.

Figure 1

GTP-bound Rac1 interacts directly with a p190B region that minimally consists of aa382–607. (A) Cartoon of p190B and derivatives used in Figure 1. Numbers refer to amino acids. Known domains in p190B are highlighted in color: N-terminal GTPase domain (grey); C-terminal GAP domain (red); FF domains (blue); RasGAP-binding domain in p190A labeled "Y" (yellow); the function this region in p190B is unknown. (B) Activated Rac1(Q61L) interacts with aa387–1007 of p190B. Aliquots of lysate from Cos7 cells expressing Myc-tagged aa382–1007 of p190B were subjected to GST pulldown assays with activated forms of Ras-like G proteins fused to GST and immobilized on glutathionesepharose beads as previously described [5]. The aa382–1007 fragment is precipitated not only by GST-bound Rnd3 as previously reported [5], but also by GST-bound Rac1(Q61L), in contrast to the other constitutively active Ras-like G proteins tested. (C) The interaction between Rac1 and aa382–1007 of p190B is GTP-dependent and direct. A pulldown assay was conducted with GTPγS- or GDP-loaded wild-type forms of GST-bound RhoA, Rac1, and Cdc42 and recombinant C-terminal FLAG-epitope tagged aa382–1007 of p190B (Figure S2). (D) Comparison by GST-pulldown assay demonstrates that the interaction of activated Rac1 with the aa382–1007 of p190B is as robust as the interaction of activated Rac with wild-type PAK1. (E) GTP-bound Rac1, Rnd3 and RhoA, but not GTP-bound Cdc42, interacts with endogenous p190B from lysates of NIH3T3 cells. Note that, in contrast to GTP-bound Rac1 and Rnd3, the interaction between RhoA-GTP and p190B is mediated solely by the GAP domain [5]. (F) Both activated Rac1 and Rnd3 interact with aa382–607 of p190B in a GST-pulldown assay. All data presented in this work are representative of at least three independent experiments.

Figure 2

Characterization of the interaction between Rac1 and p190B. (A) An intact effector domain of Rac1 is required for the interaction with p190B aa382–1007 in cells. HeLa cells were transfected with mammalian expression constructs encoding GST alone or GST fusion proteins of Rac1(Q61L), Rac1(Q61L, F37A), or Rac1(Q61L, Y40C), as well as with Myc-tagged aa382–1007 fragment of p190B or empty vector control. 24 h after transfection the cells were lysed in Gold lysis buffer. Lysates were incubated with glutathione-sepharose beads for 4 h at 4°C, and bound proteins were processed for SDS-PAGE and western blotting to detect Myc- and GST-tagged proteins. (B) Activated Rac1 interacts with endogenous p190B. HeLa cells expressing GST alone, GST-Rac1(Q61L), or GST-Rac1 effector domain mutants were processed as described in (A) with the exception that western blots were probed with a mAb to p190B. (C) Activated Rac1 recruits aa382–1007 of p190B to membrane ruffles. HeLa cells were transfected as described in (A), and processed for immunofluorescence microscopy to detect GST, GST-tagged Rac1(Q61L), and Myc-tagged 382–1007 of p190B. (D) A Myc-tagged p190B fragment consisting of aa601–1007, which does not bind activated Rac1 (Figure S2A), exhibits reduced localization to membrane ruffles. Asterisks in (C) and (D) indicate untransfected cells that are only detectable upon prolonged exposure due to non-specific staining, and the hatched lines mark their perimeter.

Figure 3

Activated Rac1 recruits p190B into a detergent-insoluble plasma membrane compartment. (A) and (B) Overexpressed p190B colocalizes with Rac1(Q61L) in membrane ruffles of untreated (A), as well as detergent extracted HeLa cells (B). HeLa cells were transfected with Myc-tagged Rac1(Q61L) in combination with full-length p190B, and either fixed in 2% PFA directly (A), or extracted with 0.5% Triton X-100 in MES buffer for 4 min on ice prior to fixation to remove soluble proteins from the cytoplasm (B). The cells were next processed for immunofluorescence microscopy. The p190B mAb used here is not sufficiently sensitive to detect endogenous p190B in HeLa cells by conventional fluorescence microscopy. (C) Endogenous p190B partitions into a detergent-insoluble membrane fraction from cells expressing activated Rac1. The cell fractionation procedure is detailed in Supplementary Experimental Procedures. Note that p190B shifts to the detergent-insoluble fraction in cells expressing activated Rac1(Q61L). (D) Quantification of the p190B in detergent-soluble and Triton-insoluble fractions was performed by densitometry. Data are expressed as mean ± SD from four independent experiments. The shift in p190B distribution between vector control and Rac1(Q61L) transfected cells is statistically significant (*, p<0.01, unpaired two-tailed Student t test), irrespectively of whether the detergent-soluble or detergent-insoluble pools are compared. (E) RBD pull-down assay to determine the levels of membrane-associated GTP-bound RhoA in the triton-soluble, post 100,000× g membrane fraction from cells expressing activated Rac1. For comparison total RhoA in 5% of the cell extract is shown. (F) Quantification of GTP-bound RhoA from four independent RBD pull-down assays. Data are expressed as mean ± SD from four independent experiments. In 4/4 experiments RhoA-GTP levels were reduced in membranes isolated from cells expressing Rac1(Q61L).

Figure 4

The minimal Rac1-binding sequence (RBS) is required for modulation of p190B RhoGAP function. (A) Cartoon of p190B(ΔRBS) and p190B(ΔGAP) mutants. (B) Western blot analysis of wild-type p190B, ΔRBS, or ΔGAP protein levels in HeLa cells transfected with expression constructs. ERK is shown as a loading control. (C) and (D) The ΔRBS mutant is defective in regulating GAP function in response to integrin engagement. HeLa cells were transfected with expression constructs encoding wild-type p190B, ΔRBS, or ΔGAP and plated for 15 h on fibronectin (C), or collagen type-I (D). The cells were then processed for detection of transfected p190B forms (red), polymerized actin (green) and nuclei (blue). Inserts in panels (D) and (D") showing wild-type p190B or ΔGAP transfected cells, respectively, demonstrate the localization of these proteins to peripheral membrane ruffles. Arrows in panel (D') point to ΔRBS expressing cells exhibiting cytoskeletal collapse, which are easily distinguished from rounded mitotic cells (arrowheads) identified by DAPI staining (D', insert). (E) RBD pulldown assay conducted on 293 cells transfected with vector control, p190B, ΔRBS, or ΔGAP expression constructs. Note that the ΔRBS mutant is substantially more potent at hydrolysing RhoA-GTP than wild-type p190B. (F) Model of Rac1-mediated modulation of p190B RhoGAP function. Activated Rac1 interacts directly with the p190B-MD and recruits p190B to peripheral membrane ruffles, where p190B upon undergoing a conformational change exhibits enhanced RhoGAP activity leading to inactivation of RhoA.