Abstract
BackgroundPhospholipase 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.ResultsWild 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.ConclusionsSmall ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2121-4-13) contains supplementary material, which is available to authorized users.
Highlights
Phospholipase D (PLD) is involved in many signaling pathways
Since these proteins were mostly recovered from the supernatant fractions, suggesting that wild type ARNO and the mutants tested are predominantly cytosolic in non-stimulated cells
When digitonin permeablization was performed in the presence of insulin (100 nM), most of wt-ARNO, E156K-ARNO, and ∆CC-ARNO as well as a part of CC-ARNO were recovered from the particulate membrane fraction, suggesting that these ARNO proteins can be recruited to the membrane by insulin to various degrees
Summary
Phospholipase D (PLD) is involved in many signaling pathways. 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. 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. Small GTPases of the ADP-ribosylation factor (ARF) family play a major role in membrane trafficking in eukaryotic cells [1]. ARF activation is facilitated by specific guanine nucleotide exchange factors (ARF-GEFs). The catalytic activity of ARNO for guanine nucleotide exchange is localized in the Sec domain (page number not for citation purposes)
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