Direct Modulation of Phospholipase D Activity by Gβγ

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Abstract
Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gβγ subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Gα and Gβγ subunits, allowing subsequent interaction with distinct effectors. Gβ1γ1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gβ1γ1 and Gβ1γ2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gβ1γ1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gβ1γ1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gβ1γ2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gβγ directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.