The amyloid precursor protein interacts with Go heterotrimeric protein within a cell compartment specialized in signal transduction. (1/1232)

The function of the beta-amyloid protein precursor (betaAPP), a transmembrane molecule involved in Alzheimer pathologies, is poorly understood. We recently reported the presence of a fraction of betaAPP in cholesterol and sphingoglycolipid-enriched microdomains (CSEM), a caveolae-like compartment specialized in signal transduction. To investigate whether betaAPP actually interferes with cell signaling, we reexamined the interaction between betaAPP and Go GTPase. In strong contrast with results obtained with reconstituted phospholipid vesicles (Okamoto et al., 1995), we find that incubating total neuronal membranes with 22C11, an antibody that recognizes an N-terminal betaAPP epitope, reduces high-affinity Go GTPase activity. This inhibition is specific of Galphao and is reproduced, in the absence of 22C11, by the addition of the betaAPP C-terminal domain but not by two distinct mutated betaAPP C-terminal domains that do not bind Galphao. This inhibition of Galphao GTPase activity by either 22C11 or wild-type betaAPP cytoplasmic domain suggests that intracellular interactions between betaAPP and Galphao could be regulated by extracellular signals. To verify whether this interaction is preserved in CSEM, we first used biochemical, immunocytochemical, and ultrastructural techniques to unambiguously confirm the colocalization of Galphao and betaAPP in CSEM. We show that inhibition of basal Galphao GTPase activity also occurs within CSEM and correlates with the coimmunoprecipitation of Galphao and betaAPP. The regulation of Galphao GTPase activity by betaAPP in a compartment specialized in signaling may have important consequences for our understanding of the physiopathological functions of betaAPP.  (+info)

Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes. (2/1232)

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.  (+info)

SEC14-dependent secretion in Saccharomyces cerevisiae. Nondependence on sphingolipid synthesis-coupled diacylglycerol production. (3/1232)

The SEC14 gene in Saccharomyces cerevisiae encodes a phosphatidylinositol transfer protein required for secretory protein movement from the Golgi. Mutation of SAC1, a gene of unknown function, restores secretory flow in sec14-1(ts) strains. The existing model for the bypass of the sec14-1(ts) defect by sac1-22 involves stimulation of sphingolipid biosynthesis and, in particular, the synthesis of mannosyl-diinositolphosphoryl-ceramide with concomitant increases in Golgi diacylglycerol levels. To test this model, we disrupted IPT1, the mannosyl-diinositolphosphoryl-ceramide synthase of S. cerevisiae. Disruption of the IPT1 gene had no effect on the ability of sac1-22 to bypass sec14-1(ts). Furthermore, sphingolipid analysis of sec14-1(ts) and sec14-1(ts) sac1-22 strains showed that mannosyl-diinositolphosphoryl-ceramide synthesis was not stimulated in the bypass mutant. However, the sec14-1(ts) strain had elevated mannosyl-monoinositolphosphoryl-ceramide levels, and the sec14-1(ts) sac1-22 strain showed an 8-fold increase in phosphatidylinositol 4-phosphate along with a decrease in phosphatidylinositol 4,5-bisphosphate. Cellular diacylglycerol levels, measured by [14C]acetate incorporation, did not differ between the sec14-1(ts) and the sec14-1 sac1-22 bypass strains, although disruption of IPT1 in the bypass strain resulted in reduced levels. These data indicate that phosphatidylinositol 4-phosphate, rather than mannosyl-diinositolphosphoryl-ceramide, accumulates in the sec14-1(ts) sac1-22 bypass strain, and that Golgi diacylglycerol accumulation is not required for bypass of the sec14-1(ts) growth and secretory phenotypes.  (+info)

Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis. (4/1232)

This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type 2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2 receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, beta-chloro-L-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and beta-chloro-L-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis.  (+info)

Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters. (5/1232)

Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.  (+info)

Induction and suppression of endothelial cell apoptosis by sphingolipids: a possible in vitro model for cell-cell interactions between platelets and endothelial cells. (6/1232)

Because sphingosine (Sph) is actively incorporated into platelets and rapidly converted to sphingosine 1-phosphate (Sph-1-P), which is then released extracellularly, it is important to study the effects of Sph and Sph-1-P on endothelial cells from the viewpoint of platelet-endothelial cell interaction. In this study, we found that Sph, as well as ceramide, induces apoptosis in human umbilical vein endothelial cells (HUVECs). In contrast, Sph-1-P acts as a HUVEC survival factor; this bioactive lipid was shown to protect HUVECs from apoptosis induced by the withdrawal of growth factors and to stimulate HUVEC DNA synthesis. In metabolic studies, [3H]Sph, incorporated into HUVECs, was converted to [3H]Cer and further to [3H]sphingomyelin in a time-dependent manner, whereas [3H]Sph-1-P formation from [3H]Sph was weak and transient. These findings in HUVECs are very different from those of platelets, which possess a highly active Sph kinase but lack Sph-1-P lyase. As a result, platelets abundantly store Sph-1-P, whereas HUVECs contain much less Sph-1-P. Finally, HUVECs, in contrast to platelets, failed to release Sph-1-P extracellularly, indicating that HUVECs themselves are not able to supply the survival factor Sph-1-P, but receive it from activated platelets. Our results suggest that platelets may maintain the integrity of endothelial cells by incorporating Sph and releasing Sph-1-P.  (+info)

Metabolism and selected functions of sphingolipids in the yeast Saccharomyces cerevisiae. (7/1232)

Our knowledge of sphingolipid metabolism and function in Saccharomyces cerevisiae is growing rapidly. Here we discuss the current status of sphingolipid metabolism including recent evidence suggesting that exogenous sphingoid long-chain bases must first be phosphorylated and then dephosphorylated before incorporation into ceramide. Phenotypes of strains defective in sphingolipid metabolism are discussed because they provide hints about the undiscovered functions of sphingolipids and are one of the major reasons for studying this model eukaryote. The long-chain base phosphates, dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate, have been hypothesized to play roles in heat stress resistance, perhaps acting as signaling molecules. We evaluate the data supporting this hypothesis and suggest future experiments needed to verify it. Finally, we discuss recent clues that may help to reveal how sphingolipid synthesis and total cellular sphingolipid content are regulated.  (+info)

Modulation of nanotube formation by structural modifications of sphingolipids. (8/1232)

Galactosylceramides (GalCers) containing nervonoyl (24:1(Delta15(cis))) acyl chains have the capacity to assemble into nanotubular microstructures in excess water (. Biophys. J. 69:1976-1986). To define the structural parameters that modulate nanotube formation, GalCer derivatives were synthesized that contained cis monounsaturated acyl chains with the formula X:1((X-9)). X indicates the total acyl carbon number (24, 22, 20, or 18), and 1 indicates a single cis double bond, the location of which is designated by the superscript (X-9). Deep etching of freeze-fractured 24:1(Delta15(cis)) GalCer dispersions followed by replica production and transmission electron microscopic analysis confirmed nanotube morphology (25-30-nm diameter). Control experiments revealed that tubule formation was promoted by cooling through the main enthalpic phase transition coupled with repetitive freeze-thaw cycling. Imparting a negative charge to the sugar headgroup of 24:1(Delta15)GalCer via sulfate dramatically altered mesomorpholgy and resulted in myelinic-like, multilamellar structures. Removal of the sugar headgroup (24:1(Delta15)Cer) resulted in flattened cylindrical structures with a cochleate appearance. Compared to these large-scale changes in morphology, more subtle changes were induced by structural changes in the acyl chain of 24:1(Delta15)GalCer. 22:1(Delta13)GalCer dispersions consisted of long, smooth tubules (35-40-nm diameters) with a strong tendency to self-align into bundle-like aggregates. In contrast, the microstructures formed by 20:1(Delta11)GalCer resembled helical ribbons with a right-handed twist. Ribbon widths averaged 30-35 nm, with helical pitches of 80-90 nm. 18:1(Delta9)GalCer displayed a variety of morphologies, including large-diameter multilamellar cylinders and liposome-like structures, as well as stacked, plate-like arrays. The results are discussed within the context of current theories of lipid tubule formation.  (+info)