No abstract

Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive.Stimulation of cells by particular agonists which occupy either heterotrimeric G-protein-coupled receptors or those with an intrinsic tyrosine kinase activity induce an increase in the mass of diradylglycerols (collectively diacylglycerol, alkyl, acylglycerol and alkenyl, acylglycerol; DRG), 1 in particular sn-1,2-diacylglycerol (DAG), the physiological activator of protein kinase C (PKC) (1). DAG is produced, together with inositol 1,4,5-trisphosphate which stimulates the elevation of intracellular free calcium concentration, by phospholipase C (PLC)-catalyzed phosphatidylinositol 4,5-bisphosphate hydrolysis. Agonist stimulation of this pathway is rapidly desensitized, DAG generation has been demonstrated to be rapid, but transient, declining toward basal levels within 1-2 min (2, 3). However, there is frequently a second sustained phase of DAG generation. This phase has been associated with an increase in the activation of phospholipase D (PLD)-catalyzed phosphatidylcholine (PC) hydrolysis, producing phosphatidate (PA) which can be converted to DAG by the action of phosphatidate phosphohydrolase. It has also been proposed that DAG can be derived from other pathways, e.g. through a PC-PLC pathway, although the evidence for stimulation of this pathway in mammalian cells remains mostly circumstantial (4, 5).Cells contain multiple species of DAG, however, a limited subset of these change following stimulation. Comparison of the acyl chain DAG structures with those of the cellular phospholipids indicated that the initial phase of DAG increase was predominantly from inositol phospholipids, while the sustained phase, which was accompanied by an increase in choline release, was probably produced from PC (6 -9). The initial phase of DAG generation was ma...

Metalloproteinases (MMP) produced by both cancer and normal stromal fibroblast cells play a critical role in the metastatic spread of tumours, however little is known of the regulation of their release. In this report we demonstrate that breast cancer cells in culture release apparently full length soluble EMMPRIN that promotes the release of pro-MMP2 from fibroblasts. The generation of MMP2 is mediated by activation of phospholipase A 2 and 5-lipoxygenase. These results suggest that the production of soluble EMMPRIN, phospholipase A 2 and 5-lipoxygenase activities are sites for potential therapeutic intervention.

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA It is becoming increasingly recognized that the compartmentalization of proteins within specific regions of the cell is pivotal to the appropriate functioning of many, if not all, signaling pathways. For example, the localization of the RII subunits of PKA 1 to distinct intracellular sites, achieved by binding to A-Kinase-Anchor Proteins (AKAPs), allows for the localized activation of this protein kinase and functionally compartmentalized signaling (1). Activation of signaling processes themselves can lead to changes in the organization of intracellular proteins. This can take the form of protein-protein interactions, of which the SH2 and SH3 modules provide mechanistic and structural examples (2, 3). However, more recently, lipid-directed re-organization of protein components within cells has been recognized, such as that seen in the binding of phosphoinositides to pleckstrin homology (PH) and FYVE domain modules, as well as the interaction of C2 domains with phospholipids (4, 5). To appreciate the ways in which various signaling processes are connected in the cell, it is important to appreciate the range of protein modules that allow for interaction with intracellular anchors.Stimulation of many cells with a plethora of growth factors, cytokines, and hormones activates phospholipase D (PLD). This enzyme catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) which is presumed to mediate downstream signaling effects such as secretion, vesicle trafficking, cytoskeletal reorganization, apoptosis, and mitogenesis (6 -8). PA has been reported to bind to and activate Raf-1, and a neutrophil PA-regulated protein kinase has been described (9, 10). In addition, PA has been shown to activate "long form" cAMP phosphodiesterases (11)(12)(13)(14). However, how PA specifically interacts with signaling proteins is not yet clear. In particular, no module akin to either the pleckstrin homology or the FYVE domains involved in selectively binding 3-phosphorylated phosphoinositides or the C2 domains that are able to bind