Author: Aroha Patel, Isla Thompson, Liam McKenzie, Noah Rangi
Research Article
Guanine Nucleotide Exchange Factor ARNO Mediates the Activation of ARF and Phospholipase D by Insulin
Isla Thompson¹*, Liam McKenzie², Aroha Patel³ and Noah Rangi⁴
¹Department of Biological Sciences; Victoria University of Wellington; Wellington; New Zealand
²Department of Microbiology; University of Auckland; Auckland; New Zealand
³Department of Virology; University of Otago; Dunedin; New Zealand
4Department of Biotechnology; Massey University; Palmerston North; New Zealand
Published: 18 April 2018
Abstract
Background: Insulin triggers a multitude of cellular responses; including metabolic regulation and membrane trafficking events; mediated by complex signaling cascades. ADP-Ribosylation Factors (ARFs) are small GTPases that regulate vesicular transport and cytoskeletal organization; acting as molecular switches activated by Guanine Nucleotide Exchange Factors (GEFs). Phospholipase D (PLD) generates the lipid second messenger phosphatidic acid and is known to be activated by ARF proteins. ARNO (cytohesin-2) is a GEF for ARF proteins containing a Pleckstrin Homology (PH) domain that binds Phosphatidylinositol 3;4;5-Trisphosphate (PIP3); a product of insulin-stimulated Phosphoinositide 3-Kinase (PI3K).
Objective: This study investigated the hypothesis that ARNO acts as a crucial link between insulin receptor activation and the subsequent activation of ARF and PLD signaling pathways.
Methods: Using cultured insulin-responsive cells (e.g.; 3T3-L1 adipocytes or L6 myotubes); we examined the effects of insulin stimulation on ARF activation (measured by GTP-ARF pull-down assays) and PLD activity (measured by transphosphatidylation assays). The role of PI3K was assessed using specific inhibitors (wortmannin; LY294002). Co-immunoprecipitation and membrane fractionation were used to study ARNO recruitment and interactions. The specific requirement for ARNO was evaluated using siRNA-mediated knockdown. The dependence of PLD activation on ARF was assessed using dominant-negative ARF mutants or pharmacological approaches where applicable.
Results: Insulin stimulation rapidly and transiently increased the levels of GTP-bound ARF (predominantly ARF6) and stimulated PLD activity in a time- and dose-dependent manner. Both ARF activation and PLD stimulation by insulin were completely blocked by PI3K inhibitors; indicating dependence on PIP3 generation. Insulin induced the translocation of endogenous ARNO to the plasma membrane fraction. ARNO knockdown using siRNA significantly attenuated insulin-stimulated ARF activation and subsequent PLD activity. Expression of a dominant-negative ARF mutant also inhibited insulin-stimulated PLD activity.
Conclusion: These findings support a model where insulin; acting via PI3K and PIP3 production; recruits the GEF ARNO to the plasma membrane. Membrane-localized ARNO then activates ARF proteins (particularly ARF6); which subsequently leads to the activation of PLD. This ARNO-ARF-PLD signaling axis represents a key pathway downstream of the insulin receptor; likely contributing to insulin-mediated membrane trafficking events such as GLUT4 transporter translocation and cytoskeletal rearrangements.
Keywords: Insulin signaling; ARNO (Cytohesin-2); ADP-Ribosylation Factor (ARF); ARF6; Phospholipase D (PLD); Guanine Nucleotide Exchange Factor (GEF); Phosphoinositide 3-Kinase (PI3K); Membrane trafficking; Signal transduction.
