Dale J. Powner

Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation.

Phospholipase D (PLD) activity has been implicated in the regulation of membrane trafficking [1,2], superoxide generation and cytoskeletal remodelling [3,4]. Several PLD genes have now been identified and it is probable that different isoforms regulate distinct functions. Defining the subcellular localisation of each isoform would facilitate understanding of their roles. Previous PLD localisation studies have been based largely on enzyme activity measurements, which cannot distinguish between isoforms [2,5]. We have cloned the cDNAs encoding human PLD1a and PLD1b from an HL60 cell cDNA library and expressed them as catalytically active fusion proteins with green fluorescent protein (GFP) in COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates upon cross-linking of the high-affinity immunoglobulin E (IgE) receptor. In unstimulated cells, GFP-PLD1b colocalised with secretory granule and lysosomal markers; it was not found at the plasma membrane or nucleus and did not colocalise with markers for the Golgi. Stimulation or RBL-2H3 cells through IgE receptor cross-linking caused plasma membrane recruitment of GFP-PLD1b. Inhibition of IgE-receptor-stimulated, PLD-catalysed phosphatidate formation suppressed secretion of granule and lysosomal contents, but did not affect translocation of GFP-PLD1b. These experiments suggest that PLD1 plays a role in regulated exocytosis rather than endoplasmic reticulum (ER) to Golgi membrane transport.

Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain.

The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.

The regulation of phospholipase D by inositol phospholipids and small GTPases

Phospholipase D1 and D2 (PLD1, PLD2) both have PX and PH domains in their N‐terminal regions with these inositol lipid binding domains playing key roles in regulating PLD activity and localisation. The activity of PLD1 is also regulated by protein kinase C and members of the Rho and Arf families of GTPases. Each of these proteins binds to unique sites; however, there appears to be little in vitro discrimination between individual family members. In agonist‐stimulated cells, however, there is specificity, with, for example in RBL‐2H3 cells, antigen stimulating the activation of PLD1 by association with Arf6, Rac1 and protein kinase Cα. PLD2 appears to be less directly regulated by GTPases and rather is primarily controlled through interaction with phosphatidylinositol 4‐phosphate 5‐kinase that generates the activating phosphatidylinositol 4,5‐bisphosphate.

Exocytosis of CTLA-4 Is Dependent on Phospholipase D and ADP Ribosylation Factor-1 and Stimulated during Activation of Regulatory T Cells

CTLA-4 is an essential protein in the regulation of T cell responses that interacts with two ligands found on the surface of APCs (CD80 and CD86). CTLA-4 is itself poorly expressed on the T cell surface and is predominantly localized to intracellular compartments. We have studied the mechanisms involved in the delivery of CTLA-4 to the cell surface using a model Chinese hamster ovary cell system and compared this with activated and regulatory human T cells. We have shown that expression of CTLA-4 at the plasma membrane (PM) is controlled by exocytosis of CTLA-4-containing vesicles and followed by rapid endocytosis. Using selective inhibitors and dominant negative mutants, we have shown that exocytosis of CTLA-4 is dependent on the activity of the GTPase ADP ribosylation factor-1 and on phospholipase D activity. CTLA-4 was identified in a perinuclear compartment overlapping with the cis-Golgi marker GM-130 but did not colocalize strongly with lysosomal markers such as CD63 and lysosome-associated membrane protein. In regulatory T cells, activation of phospholipase D was sufficient to trigger release of CTLA-4 to the PM but did not inhibit endocytosis. Taken together, these data suggest that CTLA-4 may be stored in a specialized compartment in regulatory T cells that can be triggered rapidly for deployment to the PM in a phospholipase D- and ADP ribosylation factor-1-dependent manner.

Phospholipase D2 stimulates integrin-mediated adhesion via phosphatidylinositol 4-phosphate 5-kinase Iγb

Cellular adhesion can be regulated by, as yet, poorly defined intracellular signalling events. Phospholipase D enzymes generate the messenger lipid phosphatidate and here we demonstrate that suppression of this reaction inhibits cellular adhesion. This effect was reversed by the addition of cell-permeable analogues of either phosphatidate or phosphatidylinositol 4,5-bisphosphate. By contrast, neither diacylglycerol nor lysophosphatidic acid were able to reverse this effect suggesting that phosphatidate itself acts directly on a target protein(s) to regulate adhesion rather than as the result of its conversion to either of these metabolite lipids. Antibodies that block β1 and β2 integrin-substrate interactions inhibited adhesion stimulated by both phosphatidate and phosphatidylinositol 4,5-bisphosphate indicating that these lipids regulate β1 and β2 integrin-mediated adhesion. In vivo, these lipids can be generated by phospholipase D2 and phosphatidylinositol 4-phosphate 5-kinase Iγb, respectively, and over-expression of catalytically-functional forms of these enzymes dose-dependently stimulated adhesion while siRNA depletion of PLD2 levels inhibited adhesion. Furthermore the ability of over-expressed phospholipase D2 to stimulate adhesion was inhibited by a dominant-negative version of phosphatidylinositol 4-phosphate 5-kinase Iγb. Consistent with this, phosphatidylinositol 4-phosphate 5-kinase Iγb-mediated adhesion was dependent upon phospholipase D2s product, phosphatidate indicating that phosphatidylinositol 4-phosphate 5-kinase Iγb is downstream of, and necessary for, phospholipase D2s regulation of adhesion. It is likely that this phospholipase D2-generated phosphatidate directly stimulates phosphatidylinositol 4-phosphate 5-kinase Iγb to generate phosphatidylinositol 4,5-bisphosphate as this mechanism has previously been demonstrated in vitro. Thus, our data indicates that during the initial stages of adhesion, phospholipase D2-derived phosphatidate stimulates phosphatidylinositol 4-phosphate 5-kinase Iγb to generate phosphatidylinositol 4,5-bisphosphate and that consequently this inositol phospholipid promotes adhesion through its regulation of cell-surface integrins.

Phospholipase D activity is essential for actin localization and actin-based motility in Dictyostelium

PLD (phospholipase D) activity catalyses the generation of the lipid messenger phosphatidic acid, which has been implicated in a number of cellular processes, particularly the regulation of membrane traffic. In the present study, we report that disruption of PLD signalling causes unexpectedly profound effects on the actin-based motility of Dictyostelium. Cells in which PLD activity is inhibited by butan-1-ol show a complete loss of actin-based structures, accompanied by relocalization of F-actin into small clusters, and eventually the nucleus, without a visible fall in levels of F-actin. Addition of exogenous phosphatidic acid reverses the effects of butan-1-ol, confirming that these effects are caused by inhibition of PLD. Loss of motility correlates with complete inhibition of endocytosis and a reduction in phagocytosis. Inhibition of PLD caused a major decrease in the synthesis of PtdIns(4,5)P2, which could again be reversed by exogenously applied phosphatidic acid. Thus the essential role of PLD signalling in both motility and endocytosis appears to be mediated directly via regulation of PtdIns(4)P kinase activity. This implies that localized PLD-regulated synthesis of PtdIns(4,5)P2 is essential for Dictyostelium actin function.

Tetraspanin CD9 in cell migration

Tetraspanin CD9 is associated with integrin adhesion receptors and it was reported that CD9 regulates integrin-dependent cell migration and invasion. Pro- and anti-migratory effects of CD9 have been linked to adhesion-dependent signalling pathways, including phosphorylation of FAK (focal adhesion kinase) and activation of phosphoinositide 3-kinase, p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). In the present paper, we describe a novel mechanism whereby CD9 specifically controls localization of talin1, one of the critical regulators of integrin activation, to focal adhesions: CD9-deficiency leads to impaired localization of talin1 to focal adhesions and correlates with increased motility of breast cancer cells.

Assays to Study Phospholipase D Regulation by Inositol Phospholipids and ADP‐Ribosylation Factor 6

Phospholipase D (PLD) is an enzyme implicated in the regulation of both exocytic and endocytic vesicle trafficking as well as many other processes. Consistent with this, the small GTPase Arf6 and regulated changes in inositol phospholipids levels are two factors that regulate both PLD and vesicle trafficking. Here we describe three methodologies through which the activation of PLD by Arf6 and inositol phospholipids may be investigated. The first method described is an in vitro protocol that allows the analysis of purified proteins or cell lysates. Furthermore, this protocol can be used to analyze the effects of different inositol phospholipids by changing the composition of the substrate vesicle. The major advantage of this protocol lies in the ability to analyze the effects of direct interactions on PLD activation by using pure proteins and lipids. The other two methods are in vivo protocols for the analysis of PLD activation in response to extracellular stimuli. Modification of cellular composition using overexpression/deletion or knockout of specific genes can be utilized with these protocols to characterize PLD activation pathways. The first of these methods uses the detection of radiolabeled PLD products and can be used for most cell types whereas the second of these two protocols is used to measure PLD products when radiolabeling of cells is not possible, such as freshly isolated cells that will not survive long enough to attain radiochemical equilibrium.

Determination of phospholipase D, lysophospholipase D and DG kinase signaling pathways in disease states by mass spectrometry.

Phospholipase D (PLD) catalyzes the hydrolysis of the ester bond between the phosphate at the sn-3 position on the glycerol backbone of phospholipids and the headgroup base, generally choline. In mammalian cells PLD1 and 2 (E.C. 3.1.4.4) are the best characterized, hydrolyzing phosphatidylcholine (PtdCho) to generate free choline and phosphatidic acid (PtdOH) (Exton, 2002; McDermott et al., 2004). Of the products, PtdOH is now recognized to have messenger properties. This enzymatic reaction is stimulated by the occupation of receptors that also activate other signaling pathways in cells, notably the phospholipase C and PtdIns-3-kinases; this points to the importance of considering the activation of parallel, potentially interacting signaling processes rather than considering activation of single pathways in isolation in an agoniststimulated cell. Analysis of the physiological regulation of PLD1 and 2, by a number of groups, has pointed to the importance of phosphoinositides, small GTPases and protein kinase C (E.C. 2.7.11.13) (Exton, 2002; McDermott et al., 2004). PLD1 is most clearly regulated by the ARF family of GTPases (Brown et al., 1993; Cockcroft et al., 1994), with ARF6 being the most regularly reported, presumably because of its role in cytoskeletal change (Powner et al., 2002). Each member of the Rho family of GTPases (Rho, Rac, Cdc42) is able to activate PLD1 (Cai and Exton, 2001; Malcolm et al., 1994), though mutagenesis and surface plasmon resonance binding studies demonstrate that they bind to the same C-terminal region of the enzyme (Powner