The primary known function of phospholipase D (PLD) is to generate phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine. However, the functional role of PA is not well understood. We report here evidence that links the activation of PLD by insulin and the subsequent generation of PA to the activation of the Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A (BFA), an inhibitor of the activation of ADP-ribosylation factor proteins, inhibited insulin-dependent production of PA and MAPK phosphorylation. The addition of PA reversed the inhibition of MAPK activation by BFA. Overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocked insulindependent activation of PLD and phosphorylation of MAPK. Real time imaging analysis showed that insulin induced Raf-1 translocation to cell membranes by a process that was inhibited by BFA. PA addition reversed the effects of BFA on Raf-1 translocation. However, PA did not activate Raf-1 in vitro or in vivo, suggesting that the primary function of PA is to enhance the recruitment of Raf-1 to the plasma membrane where other factors may activate it. Finally, we found that the recruitment of Raf-1 to the plasma membrane was transient, but Raf-1 remained bound to endocytic vesicles.Growth factor-mediated activation of PLD 1 has been well documented and occurs in response to a broad class of mitogens, including insulin, platelet-derived growth factor, epidermal growth factor, vasopressin, and phorbol esters (1-4). Activation of PLD occurs through interaction with the small G-proteins of the ADP-ribosylation factor (ARF) (5, 6) and Rac/Rho families (7) as well as with protein kinase C (PKC) (8, 9). The relative contribution of these factors to the activation of PLD is highly dependent on the cell type and signaling model examined. For example, stimulation of Rat-1 fibroblasts overexpressing the human insulin receptor (HIRcB cells) with insulin activates PLD exclusively through the ARF pathway (10), whereas the activation of PLD by insulin in adipocytes appears to be primarily Rho-mediated (11). Activation of PLD has been implicated in a wide variety of intracellular and extracellular processes, including actin polymerization, coatomer assembly, vesicle transport, neutrophil activation, and platelet aggregation (12-16).Activated PLD catalyzes the hydrolysis of phosphatidylcholine to generate PA. However, the downstream consequences of PA generation are not well understood. Although it is clear that the principal effects of PA in some systems may be mediated by its conversion to diacylglycerol (DAG) or lysophosphatidic acid (LPA), PA may also be a potent second messenger. Several laboratories have identified putative targets for PA in growth factor signal transduction, including a protein tyrosine phosphatase (17), phospholipase C-␥ (18), and Ras-GAP (19). However, the physiological relevance of these interactions has not been established.Recently, Ghosh et al. (20) reported that PA interacts directly with the serine-threonine kinase Raf-1...

The activation of ARF proteins is stimulated by insulin and this process plays an important role in insulin-mediated regulation of PLD.

ADP-ribosylation factor 1 (ARF1) is a key regulator of transport in the secretory system. Like all small GTPases, deactivation of ARF1 requires a GTPaseactivating protein (GAP) that promotes hydrolysis of GTP to GDP on ARF1. Structure-function analysis of a GAP for ARF1 revealed that its activity in vivo requires not only a domain that catalyzes hydrolysis of GTP on ARF1 but also a non-catalytic domain. In this study, we show that the non-catalytic domain of GAP is required for its recruitment from cytosol to membranes and that this domain mediates the interaction of GAP with the transmembrane KDEL receptor. Blocking its interaction with the KDEL receptor leaves the GAP cytosolic and prevents the deactivation in vivo of Golgi-localized ARF1. Thus, these findings suggest that the KDEL receptor plays a critical role in the function of GAP by regulating its recruitment from cytosol to membranes, where it can then act on its membrane-restricted target, the GTP-bound form of ARF1.

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Background: Phospholipase 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.

We show here that A10 cells express the phospholipase D (PLD) isoforms PLD1b and PLD2. The activation of PLD in these cells by angiotensin II (AngII), endothelin-1 (ET-1), and platelet-derived growth factor (PDGF) was found to be sensitive to inhibitors of the activation of ADP-ribosylation factor (ARF) but not to blockers of Rho protein function. PDGF, AngII, and ET-1 induced the binding of ARF proteins to cell membranes in a permeabilized cell assay. Cells permeabilized and depleted of ARF were no longer sensitive to stimulation with AngII, ET-1, or PDGF, but the addition of recombinant myristoylated human ARF1 restored agonist-dependent PLD activity. Expression of dominant negative ARF mutants blocked receptor-dependent activation of PLD. PLD activity was also potently stimulated by treatment with phorbol esters, but this activity was only partially inhibited by brefeldin A or by the overexpression of ARF dominant negative mutants. Transient expression of catalytically inactive mutants of PLD2, but not PLD1, inhibited significantly PDGF- and AngII-dependent PLD activity. We conclude: 1) the activation of PLD by cell surface receptors occurs primarily by an ARF-dependent mechanism in A10 cells, whereas the activation of PLD by protein kinase C-dependent pathways is only partially dependent on the regulation of ARF proteins; and 2) cell surface receptors, such as AngII and PDGF, signal primarily via PLD2 in A10 cells.

ith significant resources and money being invested in the identification of lead compounds in the drug screening process, it is critical that researchers have a highly efficient, reliable, and often automated method of culturing cells for various cellular assays. As such, the culture plate and its ability to integrate with automated systems is key to streamlining an effective high-throughput process. W However, the commonly experienced "edge effect" can often impact on data consistency. The evaporation of medium from wells during incubation is especially prominent in the wells closest to the perimeter of the plate (the edge wells) and results in well-to-well variations. As medium evaporates, concentrations are consequently altered, and cell growth is adversely affected.Differential evaporation across the plate results in variability; a volume loss as small as 10% concentrates media components and metabolites enough to alter cell physiology and, in some cases, this can be quite severe. Furthermore, plates are often not optimized for imaging purposes, making it difficult to obtain clearly focused fields. As a result, the plate cannot be used efficiently.In an attempt to alleviate the edge effect, researchers often decide not to culture cells in the outermost wells but to fill these with sterile water, and use only the inner wells of each plate for cell cultures. By rendering these wells unusable, throughput and, therefore, efficiency, is substantially reduced. The additional difficulties of obtaining clearly focused field images also hinders throughput since multiple screenings may need to be performed in order to obtain usable results. Tina Marwood (tina.marwood@ thermofisher.com) is research manager, Chandrasekaran Vasudevan is platform manager, detection and analysis, and Thomas Brevig is director, global research, labware and specialtyplastics. Web: www.thermoscientific.com.

ADP-ribosylation factors (ARF) are small G proteins that play key roles in vesicular transport processes. We have studied the distribution of ARF1 in live cells using chimeras of ARF1 mutants (wild type (wt) ARF1; Q71L-ARF1 (reduced GTPase); T31N (low affinity for GTP); and (Delta)Nwt (deletion of amino acids 2–18)) with green fluorescent protein (GFP). Confocal microscopy studies showed that the wt and Q71L proteins were localized in the Golgi and cytoplasm. The (Delta)Nwt and the T31N mutants were exclusively cytoplasmic. The behavior of the wt and Q71L proteins was studied in detail. About 15% of wt-ARF1-GFP was bound to the Golgi. Bound wt-ARF1-GFP dissociated rapidly after addition of Brefeldin A (BFA). This process did not appear to be a consequence of BFA-induced disappearance of the Golgi. Photobleaching recovery showed that essentially all the ARF-GFP was mobile, although it diffused very slowly. In contrast, about 40–50% of the Q71L mutant was found in the Golgi, and its rate of dissociation in the presence of BFA was slow and biphasic. Q71L-ARF1-GFP diffused more slowly than the wt. We conclude that ARF1 proteins exist in a dynamic equilibrium between Golgi-bound and cytosolic pools, and that the translocation of ARF in live cells requires the hydrolysis of GTP by the Golgi-bound protein.