Autotaxin (ATX), or nucleotide pyrophosphatase-phosphodiesterase 2, is a secreted lysophospholipase D that promotes cell migration, metastasis, and angiogenesis. ATX generates lysophosphatidic acid (LPA), a lipid mitogen and motility factor that acts on several G protein-coupled receptors. Here we report that ATX-deficient mice die at embryonic day 9.5 (E9.5) with profound vascular defects in yolk sac and embryo resembling the G␣ 13 knockout phenotype. Furthermore, at E8.5, ATX-deficient embryos showed allantois malformation, neural tube defects, and asymmetric headfolds. The onset of these abnormalities coincided with increased expression of ATX and LPA receptors in normal embryos. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development, indicate that ATX is the major LPA-producing enzyme in vivo, and suggest that the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through G␣ 13 .Autotaxin (ATX), also known as ectonucleotide pyrophosphatase-phosphodiesterase 2, belongs to the nucleotide pyrophosphatase (NPP) family of ectoenzymes and exoenzymes, originally defined by their ability to hydrolyze nucleotides in vitro (8,15,44). Full-length ATX is cleaved along the classical export pathway and secreted as a catalytically active glycoprotein (21, 52). ATX was initially isolated as an autocrine motility factor for melanoma cells (45) and later found to promote metastasis and tumor vascularization in nude mice as well as eliciting an angiogenic response in Matrigel assays (31, 32). Hence, ATX may contribute to tumor progression by providing an invasive and/or angiogenic microenvironment for both malignant and stromal cells, a notion supported by growing evidence that ATX expression is upregulated in various invasive and metastatic cancers (4,18,22,28,43,55).The physiological substrate of ATX had remained elusive until it was discovered that ATX is identical to lysophospholipase D (lysoPLD), a secreted enzyme present in plasma and conditioned media that converts lysophosphatidylcholine (LPC) into bioactive lysophosphatidic acid (LPA) (11,47,48). LPA stimulates cell proliferation, migration, and survival by acting on specific G protein-coupled receptors (GPCRs) that are linked to multiple G proteins, including G q/11 , G i/o , and G 12/13 (20,30). LPA promotes wound healing in vivo and has been implicated in tumor progression, inflammation, vascular disease, and neural development (5,23,28,42,51). It has now become clear that LPA production, rather than nucleotide metabolism, accounts for the growth factor-like effects of ATX observed in cell culture. Strikingly, the other NPP family members lack intrinsic lysoPLD activity despite the similarity between their catalytic domain and that of ATX (14), implying that ATX/NPP2 is a unique lysoPLD with no functional redundancy within the NPP family.In addition to converting LPC into LPA, ATX can also hydrolyze sphingosyl-phosphorycholine (...

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (K i ϳ 10 ؊7 M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand. Autotaxin (ATX)1 is a member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of ecto-enzymes that hydrolyze phosphodiester bonds in various nucleotides and nucleotide derivatives (1-3). ATX, also termed NPP2, was originally isolated as an autocrine motility factor for melanoma cells (4, 5) and later found to enhance the invasive and metastatic potential of Ras-transformed NIH3T3 cells in nude mice and to induce an angiogenic response in Matrigel plug assays (6, 7). ATX mRNA is overexpressed in various human cancers, adding support to a link between ATX and tumor progression (8). Expression analysis has further suggested a normal physiological role for ATX in neurogenesis, oligodendrocyte differentiation, and myelination (9, 10).The mode of action of ATX/NPP2 has long been elusive because the biological effects of ATX could not be explained by nucleotide hydrolysis. The surprise came when it was discovered that ATX is identical to plasma lysophospholipase D (lyso-PLD) and acts by hydrolyzing lysophospatidylcholine (LPC) into lysophosphatidic acid (LPA) (11, 12), a lipid mediator that signals cell proliferation, migration, and survival via specific G protein-coupled receptors (13). It has now become clear that de novo production of LPA can fully account for the biological effects of ATX observed in cell culture. The lysophospholipid substrate range of ATX has recently been broadened by showing that the enzyme can also hydrolyze sphingosylphosphorylcholine (SPC) to yield sphingosine 1-phosphate (S1P) (14), a bioactive lipid with signaling properties very similar to those of LPA while acting on distinct receptors (15-17). The physiological significance of the SPC-to-S1P conversion is debatable, however, because the reported K m of ATX for SPC (14) is 3 orders of magnitude higher than the normal SPC levels in plasma and serum (18). Rather than through SPC hydrolysis, S1P is thought to originate largely from the phosphorylation o...

The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma Furthermore, ALP was found accumulated in isolated rafts and disruption of rafts also prevented the inhibition of PC synthesis and apoptosis induction in S49 cells. In summary, ALP is internalized by raft-dependent endocytosis to inhibit PC synthesis, which triggers apoptosis.

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]–sphingosylphosphorylcholine (C6-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca2+ ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-β-cyclodextrin, which is indicative of a loss of tight SM–cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C6- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-zeta (PKC-zeta) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3beta and -theta; isotypes, but not by 14-3-3zeta. Far-Western blotting revealed that 14-3-3 binds PKC-zeta directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3beta facilitated PKC-zeta binding. Together, the results suggest that 14-3-3 binds both PKC-zeta (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-zeta mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to complex dissociation. 14-3-3beta and -θ were substrates for PKC-zeta, whereas 14-3-3zeta was not. Phosphorylation of 14-3-3beta by PKC-zeta negatively regulated their physical association. 14-3-3beta with its putative PKC-zeta phosphorylation sites mutated enhanced co-precipitation between PKC-zeta and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-zeta weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-zeta to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-zeta.

It is mostly accepted that P-glycoprotein (Pgp) , known as drug transporter, is contributed to multidrug resistance. In view of the multitude of factors involved in drug resisrance of tumor cells, the molecular mechanism on MDR still remains poorly understood. From point of membrane lipid-protein interaction, here, physical state change of membrane lipids in sensitive A549549 and resistant A5495qg/DDP cells to cisplatin were studied and compared. The properties of membrane lipids in two cell lines were examined by various approaches of fluorescence assay, flow cytometry, NMR and gas chromatography. The results obtained as follows: 1. Result of anisotropy measurements of the fluorescence dye DPH indicated that fluorescence polarization(P) of the sensitive A549549 cells and the resistant A54954q/DDP cells were 0.162 and 0.194 respectively which means lipid fluidity of the cell membrane of the former increased compared with the later. 2.Fluorescence intensity of MC540 (Merocyanine 540). monitored by flow cytometry, were 93.5 and 49.5 for sensitive A549549 cells and resistant A54954g/DDP cells respectively, which would mean that membrane lipid packing of A549549 cells are looser than the resistant A54954g/DDP. 3. Measurement of proton NMR spectra showed that a significant decrease in the methylene/methyl and glutamine-glutamate/creatine-creatinine ratios, a significant increase in the choline/methyl ratio. 4. Result of fatty acid analysis, monitored by gas chromatography, of the membrane lipids showed a 21.6%, 27%, 31.8% increase in the ratio of C18:118,1, C18:Z18,2, c18:318:3 fatty acid respectively in A549549 cells compared with A54954g/DDP cells, and unsaturated degree of plasma membrane fatty acid is 69.13 for A549549 cell, and 55.08 for A54954q/DDP. This would provide the evidence for physical state difference of the membrane lipids between the two cell lines with respect to the difference of fatty acid composition. Rhodamine 123 is often used to detect the mukidrug resistance,the efflux rate of Rhodamine 123 of A54954g/DDP cells was much faster than that of A549549 cells showing that A54954g/DDP cells was obviously more resistant to Rhodamine 123. Taken together, We suppose that physical state changes of the membrane lipids in the two cell lines might be related to the resistance of A54954g/DDP cells to cisplatin.1430 Effect of Chlorpromazine on hypotonic and isotonic hemolysis -, M. Bennouna Nouveau quartier N47 Martil, MarocWe have studied the interaction of the amphipath chlorpromazine (CPZ) with the rat erythrocytes membrane: Effect of concentration, pH and time on hypotonic and isotonic hemolysis. The protective effect of CPZ against hypotonic hemolysis was examined at various pHs. Fresh blood was employed in the investigation, which used a colorimetric method for the determination of hemolysis. CPZ reduced hypotonic hemolysis at low concentrations but had an incrementally opposite effect at high concentrations, this drug at concentration of 2.10-4 M-10-4 M-8. 10-6 M-10-8 M at p H 5,7,8, 10 respectively ca...

14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-zeta and facilitate coupling of PKC-zeta to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-zeta activity. Using recombinant 14-3-3 proteins and purified PKC-zeta in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-zeta activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-zeta by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-zeta with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-zeta. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-zeta activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-zeta, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.