Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

BACKGROUND: Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. RESULTS: Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. CONCLUSIONS: Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor

ARF GTPases and their GEFs and GAPs: concepts and challenges

Detailed structural, biochemical, cell biological, and genetic studies of any gene/protein are required to develop models of its actions in cells. Studying a protein family in the aggregate yields additional information, as one can include analyses of their coevolution, acquisition or loss of functionalities, structural pliability, and the emergence of shared or variations in molecular mechanisms. An even richer understanding of cell biology can be achieved through evaluating functionally linked protein families. In this review, we summarize current knowledge of three protein families: the ARF GTPases, the guanine nucleotide exchange factors (ARF GEFs) that activate them, and the GTPase-activating proteins (ARF GAPs) that have the ability to both propagate and terminate signaling. However, despite decades of scrutiny, our understanding of how these essential proteins function in cells remains fragmentary. We believe that the inherent complexity of ARF signaling and its regulation by GEFs and GAPs will require the concerted effort of many laboratories working together, ideally within a consortium to optimally pool information and resources. The collaborative study of these three functionally connected families ( \u3e /=70 mammalian genes) will yield transformative insights into regulation of cell signaling

Actin Up: An Overview of the Rac GEF Dock1/Dock180 and Its Role in Cytoskeleton Rearrangement

Dock1, originally Dock180, was the first identified member of the Dock family of GTPase Exchange Factors. Early biochemical and genetic studies of Dock180 elucidated the functions and regulation of Dock180 and informed our understanding of all Dock family members. Dock180 activates Rac to stimulate actin polymerization in response to signals initiated by a variety of receptors. Dock180 dependent Rac activation is essential for processes such as apoptotic cell engulfment, myoblast fusion, and cell migration during development and homeostasis. Inappropriate Dock180 activity has been implicated in cancer invasion and metastasis and in the uptake of bacterial pathogens. Here, we give an overview of the history and current understanding of the activity, regulation, and impacts of Dock180

The cytohesin coiled-coil domain interacts with threonine 276 to control membrane association.

Cell migration is regulated by a number of small GTPases, including members of the Arf family. Cytohesins, a family of Arf-activating proteins, have been extensively implicated in the regulation of Arfs during migration and cell shape change. Membrane association of both the Arf and its activating protein is a prerequisite for Arf activation. Therefore regulating the extent of cytohesin membrane association is a mechanism for controlling the initiation of cell movement. We have discovered a novel intramolecular interaction that controls the association of cytohesins with membranes. The presence of the coiled-coil domain reduces the association of cytohesin 2 with membranes. We demonstrate that this domain interacts with more C-terminal regions of the protein. This interaction is independent of another previously identified autoinhibitory conformation. A threonine residue (T276) in the cytohesin 2 PH domain is a target for phosphorylation by Akt. Mutation of this threonine to aspartic acid, to mimic phosphorylation, disrupts the binding of the coiled-coil domain to c-terminal regions and promotes membrane association of cytohesin 2. The presence of a second autoinhibitory interaction in the cytohesins suggests that these proteins can act a signal integrators that stimulate migration only after receive multiple pro-migratory signals

The Cytohesin 2 PH domain is phosphorylated at T276 by Akt.

<p>Recombinant GST, GST-cytohesin 2 PH domain, or GST-T276A-cytohesin 2 PH domain were incubated in the presence or absence of recombinant Akt1 as described in Materials and Methods. One tenth of each sample was run out on duplicate gels and either stained with commassie blue or blotted with rabbit anti-Akt substrate antibody.</p

ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

<p>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.</p

Mutation of Threonine 276 to Alanine inhibits cytohesin 2 activation of Arf6.

<p>MDCK cells were transfected with empty vector, cytohesin 2, or cytohesin 2 T276A and active Arf6 was isolated as described in Materials and Methods. A) Representative gel of the pulldown experiments. B) Levels of Arf6 activation in 6 independent pulldown experiments were quantified. Data shown are mean ± standard deviation. The levels of active Arf6 in cytohesin 2 expressing cells were compared to the levels in the vector controls using a paired T-test (N = 6). ** = p<0.01, * = p<0.05, n.s. = no significant difference.</p

Threonine 276 is required for the intramolecular interaction, and for inhibition of membrane binding.

<p>A) Mutation of threonine 276 to aspartic acid promotes the association of cytohesin 2 with membranes. MDCK cells were transfected with constructs encoding mCherry-tagged wild-type or T276D cytohesin 2 and fractionated into cytosol and total membranes. The fractions were Western blotted with mouse anti-mCherry, mouse anti-E-cadherin and mouse anti-actin. B) Mutation of T276 to aspartic acid disrupts the interaction of the coiled-coil domain and the remainder of the protein. 293 cells were transfected with the indicated cytohesin mutants, lysed and half of each lysate incubated with GST or GST-coiled-coil for 2 hours. Expression of cytohesins and bound cytohesins were detected by Western blotting with mouse anti-myc.</p