Phospholipase D (PLD) hydrolyzes the phosphodiester bond of the glycerolipid phosphatidylcholine, resulting in the production of phosphatidic acid and free choline. Phosphatidic acid is widely considered to be the intracellular lipid mediator of many of the biological functions attributed to PLD. However, phosphatidic acid is a tightly regulated lipid in cells and can be converted to other potentially bioactive lipids, including diacylglycerol and lysophosphatidic acid. PLD activities have been described in multiple organisms, including plants, mammals, bacteria and yeast. In mammalian systems, PLD activity regulates the actin cytoskeleton, vesicle trafficking for secretion and endocytosis, and receptor signaling. PLD is in turn regulated by phosphatidylinositol-4,5-bisphosphate, protein kinase C and ADP Ribosylation Factor and Rho family GTPases. This review focuses on the lipid precursors and products of mammalian PLD metabolism, especially phosphatidic acid and the roles this lipid performs in the mediation of the functions of PLD.

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A role for sphingolipids in the yeast heat stress response has been suggested by the isolation of suppressors of mutants lacking these lipids, which are unable to grow at elevated temperatures. The current study examines the possible role of sphingolipids in the heat adaptation of yeast cells as monitored by growth and viability studies. The suppressor of long chain base auxotrophy (SLC, strain 7R4) showed a heat-sensitive phenotype that was corrected by transformation with serine palmitoyltransferase. Thus, the deficiency in sphingolipids and not the suppressor mutation was the cause of the heat-sensitive phenotype of the SLC strain 7R4. The ability of sphingolipids to rescue the heatsensitive phenotype was examined, and two endogenous yeast sphingoid backbones, phytosphingosine and dihydrosphingosine, were found to be most potent in this effect. Next, the effect of heat stress on the levels of the three major classes of sphingolipids was determined. The inositol phosphoceramides showed no change over a 1.5-h time course. However, the four detected species of sphingoid bases increased after 15 min of heat stress from 1.4-to 10.8-fold. The largest increases were seen in two sphingoid bases, C 20 phytosphingosine and C 20 dihydrosphingosine, which increased 6.4-and 10.8-fold over baseline, respectively. At 60 min of heat stress two species of yeast ceramide increased by 9.2-and 10.6-fold over baseline. The increase seen in the ceramides was partially decreased by Fumonisin B1, a ceramide synthase inhibitor. Therefore, heat stress induces accumulation of sphingoid bases and of ceramides, probably through de novo synthesis. Taken together, these results demonstrate that sphingolipids are involved in the yeast heat stress adaptation.Saccharomyces cerevisiae has been shown to respond to a transfer of 25-37 or 39°C with the physiology defined as a heat stress response (1, 2), which appears to involve two phases. The initial phase of the response is the gaining of thermotolerance, and an increase in trehalose accumulation is proposed as a marker for this event (3). This is accompanied by the induction of heat shock proteins (4) and a G 1 arrest in cell cycle that lasts for a period of approximately 1 h (5). Once thermotolerance is gained, the second phase of the response occurs when the yeast begin to grow at the increased temperature. At this point, trehalose is degraded in an HSP70-dependent process (6), and the cells begin to cycle and resume growth. Therefore, the ability of yeast to grow under increased temperature provides for an overall assessment of the heat stress response. However, the mechanisms that mediate adaptation and growth under the heat-stressed state are not fully defined.The isolation of suppressors of mutants lacking sphingolipids in yeast (Table I) has suggested a possible role for sphingolipids in the heat stress response. The initial mutation is a Ura disruption knockout of the serine palmitoyltransferase (SPT) gene (LCB1) (7), which catalyzes the first step of sphingolipid biosynthesis (Fig. 1...

storage before transplantation correlates clinically with inThe injury resulting from cold ischemia and warm recreased primary graft nonfunction, graft rejection, and rate perfusion during liver transplantation is a major clinical of re-transplantation. 4,5 problem that limits graft success. Kupffer cell activation Reperfusion injury is difficult to study clinically, but has plays a pivotal role in reperfusion injury, and Kupffer been investigated in animal models, including orthotopic cell products, including free radicals and tumor necrosis liver transplantation in rats. These studies have shown that factor a (TNF-a), are implicated as damaging agents.hepatic reperfusion following ischemia induces Kupffer cell However, the second messengers and signaling pathactivation, superoxide formation, and elevated plasma levels ways that are activated by the stress of hepatic ischemia/ of tumor necrosis factor a (TNF-a). 6,7 Pretreatment with reperfusion remain unknown. The purpose of this study methyl palmitate inhibits Kupffer cell activation and imis to assess the activation of the three known vertebrate mitogen activated protein kinase (MAPKs) and the acti-proves transplant survival threefold, thereby supporting a vating protein 1 (AP-1) transcription factor in response role for Kupffer cells in reperfusion injury. 8 In addition, treatto ischemia and reperfusion in the transplanted rat ment with agents that suppress TNF-a release from activated liver. There was a potent, sustained induction of c-jun Kupffer cells decrease transplant failure.9 Nisoldipine, a Ca 2/ N-terminal kinase (JNK), but not of the related MAPKs channel blocker, reduces plasma levels of TNF-a and inextracellular signal-regulated kinases (ERK) or p38, creases transplant survival time. 10,11 Similarly, pentoxifylupon reperfusion after transplantation. TNF-a messen-line, a methylxanthine which suppresses TNF-a messenger ger RNA (mRNA) levels and transcription factors AP-1 RNA (mRNA) accumulation in response to lipopolysacchaand nuclear factor-kB (NF-kB) were induced in the liver ride, has a protective effect on liver grafts.12 Taken together, after 60 minutes of reperfusion. Finally, there was an these data suggest an important role for TNF-a in mediating elevation of ceramide, but not diacylglycerol or sphingo-reperfusion injury. sine, in the transplanted liver. Ceramide is a second mes-TNF-a is a pleiotropic cytokine that induces cellular effects senger generated by TNF-a treatment and is an activator ranging from proliferation to apoptosis. TNF-a is a potent of JNK. Because JNK activation preceded the elevations activator of activating protein 1 (AP-1) and NF-kB transcripin ceramide and TNF-a mRNA, these results suggest that tion factors and of the c-jun N-terminal kinase (JNK, also increased hepatic TNF-a and ceramide may perpetuate known as stress-activated protein kinase, SAPK).13-15 JNK is JNK induction, but that they are not the initiating sig-a member of the vertebrate mitogen activated protein kinase nals of JNK activation during reperfus...

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We have identified the yeast sphingosine resistance gene (YSR2) of Saccharomyces cerevisiae as encoding a protein that specifically dephosphorylates dihydrosphingosine 1-phosphate (DHS-1-P), and we refer to this protein as dihydrosphingosine-1-phosphate phosphatase. Overexpression of YSR2 conferred sphingosine resistance to the dihydrosphingosine-1-P lyase-defective mutant (JS16) of S. cerevisiae, which is hypersensitive to sphingosine. The ysr2⌬ deletion mutant of S. cerevisiae accumulated DHS-1-P compared with its wild type strain upon labeling with D-erythro-[4,5-3 H]dihydrosphingosine, whereas overexpression of YSR2 increased dephosphorylation of DHS-1-P. An epitopetagged fusion protein (YSR2-Flag) was partially purified and found to specifically dephosphorylate DHS-1-P to yield dihydrosphingosine. YSR2 failed to dephosphorylate ceramide 1-phosphate or phosphatidic acid. Functionally, the mutant bearing the ysr2⌬ deletion decreased labeling of sphingolipids and increased labeling of glycerolipids dramatically following in vivo labeling with D-erythro-[ 3 H]dihydrosphingosine, but it slightly affected labeling of sphingolipids with inositol. Taken together, these results identify YSR2 as dihydrosphingosine-1-phosphate phosphatase. They also raise the intriguing possibility that phosphorylation followed by dephosphorylation is required for incorporation of exogenous long chain sphingoid bases into sphingolipids.Sphingolipids are important components of eukaryotic cell membranes. Animals develop different diseases due to genetically or environmentally altered sphingolipid metabolism (1, 2). Sphingolipids have structural functions in maintaining cell membrane integrity, and they act as anchors to proteins (3). In addition, metabolites of sphingolipids such as ceramide, sphingosine, and sphingosine 1-phosphate (S-1-P) 1 have been demonstrated to be involved as bioeffector molecules and second messengers in key events including cell growth, differentiation, cell senescence, apoptosis, and stress responses (1-5). S-1-P has a proliferative effect on certain quiescent cells and has been shown to trigger intracellular calcium mobilization (5). Platelet-derived growth factor induces an elevation in cellular levels of sphingosine and S-1-P and activates sphingosine kinase in quiescent fibroblasts. These studies imply that S-1-P participates in the mitogenic action of this and other growth factors (6).In the yeast Saccharomyces cerevisiae, sphingolipids have been demonstrated to be essential for cell viability, based on studies of long chain sphingoid base auxotrophs that are defective in serine palmitoyltransferase, the first enzyme in the de novo pathway of sphingolipid synthesis (7). Importantly, at least some sphingolipid-mediated cell events are conserved between mammalian cells and S. cerevisiae (8). For example, ceramide induces S. cerevisiae growth suppression and cell cycle arrest, and the mammalian counterpart of ceramide-activated protein phosphatase has been identified in S. cerevisiae (9).In this report, we ...

The activation of sphingomyelinase and generation of ceramide have been implicated as important regulatory pathways in cell growth and apoptosis. Bacterial sphingomyelinase has been used in many cell systems to mimic the activation of endogenous sphingomyelinase. These studies, however, have been complicated by the inability of exogenously applied bacterial sphingomyelinase to perform many of the effects of short chain cell permeable ceramides, indicating that there may be a distinct signal transducing pool of sphingomyelin not accessible to exogenous sphingomyelinase or that endogenous ceramide is not sufficient to induce these changes. We cloned the Bacillus cereus sphingomyelinase gene by polymerase chain reaction and subcloned it into a mammalian expression vector under the control of an inducible promoter. Upon stable transfection and induction of B. cereus sphingomyelinase, there were increases in neutral sphingomyelinase activity, cellular ceramide levels, cleavage of the death substrate poly(ADP-ribosyl)polymerase, and cell death. In contrast, exogenously applied B. cereus sphingomyelinase, despite causing higher elevations in ceramide levels, was unable to induce poly(ADP-ribosyl)polymerase cleavage or cell death. These results support the existence of a signal transducing pool of sphingomyelin that is distinct from the pool accessible to exogenous sphingomyelinase.

Sphingolipids are essential eukaryotic membrane lipids that are structurally and metabolically conserved through evolution. Sphingolipids have also been proposed to regulate eukaryotic stress responses as novel second messengers. Here we show that, in Saccharomyces cerevisiae, phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-dependent proteolysis via the endocytosis vacuolar degradation and 26 S proteasome pathways. Inactivation of serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress. Addition of phytosphingosine bypasses the requirement for serine palmitoyltransferase and restores proteolysis. Phytosphingosine-induced proteolysis requires multiubiquitin chain formation through the stress-responsive lysine 63 residue of ubiquitin. We propose that heat stress increases phytosphingosine and activates ubiquitin-dependent proteolysis.Sphingolipids are complex lipids containing a sphingoid base, which is a long chain amino base. Sphingolipids comprise indispensable structural components of all known eukaryotic plasma membranes and also regulate signal transduction elements including protein kinase C (1). Sphingosine, ceramide, sphingosine 1-phosphate, and other sphingolipid derivatives are also known to play central roles in apoptosis, cellular senescence, cell cycle regulation, inflammation, tumor development, and intracellular calcium mobilization (2)(3)(4).Despite the growing number of cellular functions regulated by sphingolipids and their derivatives, the molecular mechanisms by which these regulatory functions are executed in mammalian cells are poorly understood (5-7). Being ubiquitous and essential components of eukaryotic plasma membranes, sphingolipids are evolutionarily conserved from yeast to humans (8, 9). The yeast Saccharomyces cerevisiae has several advantages as a model system to study sphingolipid-mediated cellular regulation. First, the basic structure and metabolism of sphingolipids are conserved between yeast and mammals, and yet yeast has only three major species of sphingolipids, whereas mammalian cells may contain more than 300 distinct molecular species (10). Second, yeast is a genetically tractable organism whose genome has been completely sequenced (11). Lastly, many yeast genes encoding sphingolipid biosynthetic and metabolic enzymes have been recently identified (12). The controlled expression of these genes can be exploited to modulate intracellular levels of certain sphingolipids and their derivatives.The LCB1 and LCB2 genes encode serine palmitoyltransferase, which catalyzes the first committed step in yeast sphingolipid biosynthesis, the condensation of L-serine and palmitoyl-CoA to produce 3-ketodihydrosphingosine (KDS) 1 (13,14). Recently, heat stress was shown to increase cellular levels of sphingoid bases (dihydrosphingosine (DHS) and phytosphingosine (PHS)) and ceramides with little effect on the levels of complex sphingolipids (15,16). This in...