Direct Modulation of Phospholipase D Activity by Gβγ

Department of Pharmacology, Vanderbilt University, Нашвилл, Michigan, United States
Molecular Pharmacology (Impact Factor: 4.13). 08/2006; 70(1):311-8. DOI: 10.1124/mol.105.021451
Source: PubMed


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, Gbetagamma subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Galpha and Gbetagamma subunits, allowing subsequent interaction with distinct effectors. Gbeta1gamma1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gbeta1gamma1 and Gbeta1gamma2 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 Gbeta1gamma1 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 Gbeta1gamma1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gbeta1gamma2 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 Gbetagamma directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.

Download full-text


Available from: Anita M Preininger, Nov 07, 2015
  • Source
    • "Although a direct interaction between metazoan PLD and Ga has, to our knowledge, not been described, metazoan PLD activity has been shown to be negatively regulated by Gb 1 c 1 and Gb 1 c 2 in vitro and by Gb 1 c 1 in vivo (Preininger et al., 2006). This suggests that a signaling mechanism common to eukaryotes might exist wherein PLDs are regulated by direct interaction with both Ga and Gbc. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein networks and signaling cascades are key mechanisms for intra- and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The Bimolecular Fluorescence Complementation (BiFC) assay has become a routine tool for in vivo analysis of protein-protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for in planta analysis are still subject to very low signal to noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive in planta analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus (mVenus) split at residue 210, and contains an integrated mTurquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined Double ORF Expression (pDOE) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused mVenus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 "DRY" motif abolishes both of these interactions. This article is protected by copyright. All rights reserved.
    Full-text · Article · Sep 2014 · The Plant Journal
  • Source
    • "PLD2 was described originally as a constitutively active enzyme localized to the plasma membrane (Colley et al., 1997). Subsequent studies have shown that PLD2 can be activated in intact cells by agonists and can be regulated by small GTPbinding proteins (Hiroyama and Exton, 2005), heterotrimeric GTP-binding proteins (Preininger et al., 2006), and PKC isoforms (Chen et al., 2004). Overexpression of PLD2 can result in changes in cytoskeletal organization (Colley et al., 1997). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The phosphatidylcholine-using phospholipase D (PLD) isoform PLD2 is widely expressed in mammalian cells and is activated in response to a variety of promitogenic agonists. In this study, active and inactive hemagglutinin-tagged human PLD2 (HA-PLD2) constructs were stably expressed in an EL4 cell line lacking detectable endogenous PLD1 or PLD2. The overall goal of the study was to examine the roles of PLD2 in cellular signal transduction and cell phenotype. HA-PLD2 confers PLD activity that is activated by phorbol ester, ionomycin, and okadaic acid. Proliferation and Erk activation are unchanged in cells transfected with active PLD2; proliferation rate is decreased in cells expressing inactive PLD2. Basal tyrosine phosphorylation of focal adhesion kinase (FAK) is increased in cells expressing active PLD2, as is phosphorylation of Akt; inactive PLD2 has no effect. Expression of active PLD2 is associated with increased spreading and elongation of cells on tissue culture plastic, whereas inactive PLD2 inhibits cell spreading. Inactive PLD2 also inhibits cell adhesion, migration, and serum-induced invasion. Cells expressing active PLD2 form metastases in syngeneic mice, as do the parental cells; cells expressing inactive PLD2 form fewer metastases than parental cells. In summary, active PLD2 enhances FAK phosphorylation, Akt activation, and cell invasion in EL4 lymphoma cells, whereas inactive PLD2 exerts inhibitory effects on adhesion, migration, invasion, and tumor formation. Overall, expression of active PLD2 enhances processes favorable to lymphoma cell metastasis, whereas expression of inactive PLD2 inhibits metastasis.
    Preview · Article · Sep 2008 · Molecular pharmacology
  • Source
    • "As shown in Fig. 9, platelet aggregation and Rap1 activation by two DAG analogs known to directly activate the PKC pathway (Ha and Exton, 1993b; Cardoso et al., 1997) were inhibited by propranolol, suggesting that PKC may be interposed between PAR1 and PA production. The potent activation of PLD by PKC is well known, in synergy with small GTPases such as Rho and Arf, and Ca 2+ (Henage et al., 2006; Preininger et al., 2006). Ongoing studies focusing on the identification of DAG species produced downstream of PAR1 and PAR4 signaling will enable us to further elucidate the mechanisms by which each receptor triggers platelet activation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Pathological conditions such as coronary artery disease are clinically controlled via therapeutic regulation of platelet activity. Thrombin, through protease-activated receptor (PAR) 1 and PAR4, plays a central role in regulation of human platelet function in that it is known to be the most potent activator of human platelets. Currently, direct thrombin inhibitors used to block platelet activation result in unwanted side effects of excessive bleeding. An alternative therapeutic strategy would be to inhibit PAR-mediated intracellular platelet signaling pathways. To elucidate the best target, we are studying differences between the two platelet thrombin receptors, PAR1 and PAR4, in mediating thrombin's action. In this study, we show that platelet activation by PAR1-activating peptide (PAR1-AP) requires a phospholipase D (PLD)-mediated phosphatidic acid (PA) signaling pathway. We show that this PAR1-specific PA-mediated effect is not regulated through differential granule secretion after PAR-induced platelet activation. Perturbation of this signaling pathway via inhibition of lipid phosphate phosphatase-1 (LPP-1) by propranolol or inhibition of the phosphatidylcholine-derived phosphatidic acid (PA) formation by PLD with a primary alcohol significantly attenuated platelet activation by PAR1-AP. Platelet activation by thrombin or PAR4-AP was insensitive to these inhibitors. Furthermore, these inhibitors significantly attenuated activation of Rap1 after stimulation by PAR1-AP but not thrombin or PAR4-AP. Because PA metabolites such as diacylglycerol play an important role in intracellular signaling, identifying crucial differences in PA regulation of PAR-induced platelet activation may lead to a greater understanding of the role of PAR1 versus PAR4 in progression of thrombosis.
    Full-text · Article · Apr 2007 · Molecular Pharmacology
Show more