ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation.
ABSTRACT: Hepatocyte growth factor (HGF) is a potent signaling factor that acts on epithelial cells, causing them to dissociate and scatter. This migration is coordinated by a number of small GTPases, such as ARF6 and Rac1. Active ARF6 is required for HGF-stimulated migration and intracellular levels of ARF6-GTP and Rac1-GTP increase following HGF treatment. During migration, cross talk between ARF6 and Rac1 occurs through formation of a multi-protein complex containing the ARF-GEF cytohesin-2, the scaffolding protein GRASP/Tamalin, and the Rac1-GEF Dock180. Previously, the role of ARF6 in this process was unclear. We have now found that ARF6 and ARF1 regulate trafficking of GRASP and Dock180 to the plasma membrane following HGF treatment. Trafficking of GRASP and Dock180 is impaired by blocking ARF6-mediated recycling pathways and is required for HGF-stimulated Rac1 activation. Finally, HGF treatment stimulates association of GRASP and Dock180. Inhibition of ARF6 trafficking pathways traps GRASP and Dock180 as a complex in the cell.
Project description:Tubules are the building blocks of epithelial organs and form in response to cues derived from morphogens such as hepatocyte growth factor (HGF). Relatively little is known about signaling pathways that orchestrate the cellular behaviors that constitute tubule development. Here, using three-dimensional cell cultures of Madin-Darby canine kidney cells, we show that the ARF6 GTPase is a critical determinant of tubule initiation in response to HGF. ARF6 is transiently activated during tubulogenesis and perturbing the ARF6 GTP/GDP cycle by inducible expression of ARF6 mutants defective in GTP binding or hydrolysis, inhibits the development of mature tubules. Further, we show that activation of ARF6 is necessary and sufficient to initiate tubule extension. The effect of ARF6 on tubule initiation is two-fold. First, ARF6 regulates the subcellular distribution of the GTPase, Rac1, to tubule extensions. Second, ARF6-induced ERK activation regulates Rac1 activation during tubule initiation through the expression of the receptor for urokinase type plasminogen activator. Thus, we have identified a cellular apparatus downstream of ARF6 activation, which regulates membrane and cytoskeleton remodeling necessary for the early stages of tubule development.
Project description:A common pathobiological feature of malignant gliomas is the insidious infiltration of single tumor cells into the brain parenchyma, rendering these deadly tumors virtually incurable with available therapies. In this study, we report that ADP-ribosylation factor 6 (ARF6), a Ras superfamily small GTPase, is abundantly expressed in invasive human glioma cells. Cellular depletion of ARF6 by small interfering RNA decreased Rac1 activation, impaired HGF-stimulated and serum-stimulated glioma cell migration in vitro, and markedly decreased the invasive capacity of invasive glioma in the brain. Furthermore, ectopic expression of ARF6 in glioma cells promoted cell migration via the activation of Rac1. Upon stimulation of glioma cells with HGF, we show that IQ-domain GTPase-activating protein 1 (IQGAP1) is recruited and overlaps with ARF6 at the leading edge of migrating cells. However, cellular depletion of ARF6 abrogated this recruitment of IQGAP1 and attenuated the formation of surface protrusions. ARF6 forms complexes with Rac1 and IQGAP1 in glioma cells upon HGF stimulation, and knockdown of IQGAP1 significantly inhibits ARF6-induced Rac1 activation and cell migration. Taken together, these data suggest that ARF6-mediated Rac1 activation is essential for glioma cell invasion via a signaling pathway that requires IQGAP1.
Project description:The <i>MET</i> proto-oncogene-encoded receptor tyrosine kinase (MET) and AXL receptor tyrosine kinase (AXL) are independently operating receptor tyrosine kinases (RTKs) that are functionally associated with aggressive and invasive cancer cell growth. However, how MET and AXL regulate the migratory properties of cancer cells remains largely unclear. We report here that the addition of hepatocyte growth factor (HGF), the natural ligand of MET, to serum-starved human glioblastoma cells induces the rapid activation of both MET and AXL and formation of highly polarized MET-AXL clusters on the plasma membrane. HGF also promoted the formation of the MET and AXL protein complexes and phosphorylation of AXL, independent of AXL's ligand, growth arrest-specific 6 (GAS6). The HGF-induced MET-AXL complex stimulated rapid and dynamic cytoskeleton reorganization by activating the small GTPase RAC1, a process requiring both MET and AXL kinase activities. We further found that HGF also promotes the recruitment of ELMO2 and DOCK180, a bipartite guanine nucleotide exchange factor for RAC1, to the MET-AXL complex and thereby stimulates the RAC1-dependent cytoskeleton reorganization. We also demonstrated that the MET-AXL-ELMO2-DOCK180 complex is critical for HGF-induced cell migration and invasion in glioblastoma or other cancer cells. Our findings uncover a critical HGF-dependent signaling pathway that involves the assembly of a large protein complex consisting of MET, AXL, ELMO2, and DOCK180 on the plasma membrane, leading to RAC1-dependent cell migration and invasion in various cancer cells.
Project description:The lack of current treatment and preventable measures for acute kidney injury (AKI) in hospitalized patients results in an increased mortality rate of up to 80% and elevated health costs. Additionally, if not properly repaired, those who survive AKI may develop fibrosis and long-term kidney damage. The molecular aspects of kidney injury and repair are still uncertain. Hepatocyte growth factor (HGF) promotes recovery of the injured kidney by inducing survival and migration of tubular epithelial cells to repopulate bare tubule areas. HGF-stimulated kidney epithelial cell migration requires the activation of ADP-ribosylation factor 6 (Arf6) and Rac1 via the cytohesin family of Arf-guanine-nucleotide exchange factors (GEFs), in vitro. We used an ischemia and reperfusion injury (IRI) mouse model to analyze the effects of modulating this signaling pathway on kidney recovery. We treated IRI mice with either HGF, the cytohesin inhibitor SecinH3, or a combination of both. As previously reported, HGF treatment promoted rapid improvement of kidney function as evidenced by creatinine (Cre) and blood urea nitrogen (BUN) levels. In contrast, simultaneous treatment with SecinH3 and HGF blocks the ability of HGF to promote kidney recovery. Immunohistochemistry showed that HGF treatment promoted recovery of tubule structure, and had enhanced levels of active, GTP-bound Arf6 and GTP-Rac1. SecinH3 treatment, however, caused a dramatic decrease in GTP-Arf6 and GTP-Rac1 levels when compared to kidney sections from HGF-treated IRI mice. Additionally, SecinH3 counteracted the renal reparative effects of HGF. Our results support the conclusion that cytohesin function is required for HGF-stimulated renal IRI repair.
Project description:Focal adhesions (FAs) are dynamic structures that connect the actin cytoskeleton with the extracellular matrix. At least six ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), including ARAP2 (an Arf6 GAP), are implicated in regulation of FAs but the mechanisms for most are not well defined. Although Rac1 has been reported to function downstream of Arf6 to control membrane ruffling and cell migration, this pathway has not been directly examined as a regulator of FAs. Here we test the hypothesis that ARAP2 promotes the growth of FAs by converting Arf6·GTP to Arf6·GDP thereby preventing the activation of the Rho family GTP-binding protein Rac1. Reduced expression of ARAP2 decreased the number and size of FAs in cells and increased cellular Arf6·GTP and Rac1·GTP levels. Overexpression of ARAP2 had the opposite effects. The effects of ARAP2 on FAs and Rac1 were dependent on a functional ArfGAP domain. Constitutively active Arf6 affected FAs in the same way as did reduced ARAP2 expression and dominant negative mutants of Arf6 and Rac1 reversed the effect of reduced ARAP2 expression. However, neither dominant negative Arf6 nor Rac1 had the same effect as ARAP2 overexpression. We conclude that changes in Arf6 and Rac1 activities are necessary but not sufficient for ARAP2 to promote the growth of FAs and we speculate that ARAP2 has additional functions that are effector in nature to promote or stabilize FAs.
Project description:Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.
Project description:A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas.
Project description:Neutrophil chemotaxis is essential in responses to infection and underlies inflammation. In neutrophils, the small GTPase Rac1 has discrete functions at both the leading edge and in the retraction of the trailing structure at the cell's rear (uropod), but how Rac1 is regulated at the uropod is unknown. Here, we identified a mechanism mediated by the trafficking protein synaptotagmin-like 1 (SYTL1 or JFC1) that controls Rac1-GTP recycling from the uropod and promotes directional migration of neutrophils. JFC1-null neutrophils displayed defective polarization and impaired directional migration to N-formyl-methionine-leucyl-phenylalanine in vitro, but chemoattractant-induced actin remodeling, calcium signaling and Erk activation were normal in these cells. Defective chemotaxis was not explained by impaired azurophilic granule exocytosis associated with JFC1 deficiency. Mechanistically, we show that active Rac1 localizes at dynamic vesicles where endogenous JFC1 colocalizes with Rac1-GTP. Super-resolution microscopy (STORM) analysis shows adjacent distribution of JFC1 and Rac1-GTP, which increases upon activation. JFC1 interacts with Rac1-GTP in a Rab27a-independent manner to regulate Rac1-GTP trafficking. JFC1-null cells exhibited Rac1-GTP accumulation at the uropod and increased tail length, and Rac1-GTP uropod accumulation was recapitulated by inhibition of ROCK or by interference with microtubule remodeling. In vivo, neutrophil dynamic studies in mixed bone marrow chimeric mice show that JFC1-/- neutrophils are unable to move directionally toward the source of the chemoattractant, supporting the notion that JFC1 deficiency results in defective neutrophil migration. Our results suggest that defective Rac1-GTP recycling from the uropod affects directionality and highlight JFC1-mediated Rac1 trafficking as a potential target to regulate chemotaxis in inflammation and immunity.
Project description:Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration.
Project description:ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT(1)R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT(1)R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.