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

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

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

Scaffolding proteins that bind coiled-coil domain do not disrupt the intramolecular interaction.

<p>MDCK cells were infected with adenoviruses encoding the indicated proteins. The cells were lysed and the lysates incubated with GST-coiled-coil as described above. The lysates and bound proteins were Western blotted with mouse anti-GFP, mouse anti-HA and mouse anti-myc.</p

Model for the relief of the two cytohesin autoinhibitory interactions.

<p>Full activation of cytohesins will require two separate signals to disrupt both interactions.</p