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.