A search for novel genes that are up-regulated during development and differentiation of the epithelial cells of the intestinal mucosa led to the isolation of the Dri 42 cDNA clone (Dri, differentially expressed in rat intestine). The nucleotide sequence of the full-length cDNA has shown that it encodes a 35.5-kDa protein with one consensus sequence for N-linked glycosylation and alternating hydrophilic and hydrophobic domains. To determine the intracellular localization of Dri 42, polyclonal antibodies were raised in hens against a bacterially produced Dri 42-glutathione S-transferase fusion protein. Immunofluorescence detection with these antibodies has shown specific staining of the endoplasmic reticulum (ER) in the relatively undifferentiated fetal rat intestinal cell line FRIC B and in sections of rat small intestine. ER membrane localization of Dri 42 was confirmed by laser confocal microscopy of polarized Madin-Darby canine kidney cells overexpressing a Dri 42-chloramphenicol acetyltransferase (CAT) fusion protein by transfection. Pulse labeling experiments on transiently transfected cells demonstrated that the protein does not acquire Golgi modifications up to 4 h after synthesis, thus indicating that Dri 42 is an ER resident protein. The transmembrane disposition of Dri 42 was studied using in vitro insertion of Dri 42-CAT fusion proteins into microsomal membranes. The fusion proteins consisted of several different lengths of truncated Dri 42 and a reporter protein, CAT, that was linked in-frame after each hydrophobic segment. Hydrophobic segments H1, H3, and H5 had a signal/anchor function, and membrane insertion of Dri 42 is achieved co-translationally by the action of a series of alternating insertion signals and halt transfer signals, resulting in the exposure of both termini of the protein to the cytosolic side. The functional implications of the structure and localization of Dri 42, whose primary sequence does not share significant homology to any previously described protein, are discussed (Barila, 1996).
A Mg2+-independent phosphatidate phosphohydrolase was purified from rat liver plasma membranes in two distinct forms, an anionic protein and a cationic protein. Both forms of the enzyme are able to dephosphorylate phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. When assayed at a constant molar ratio of lipid to Triton X-100 of 1:500, the apparent Km values of the anionic phosphohydrolase for the lipid substrates were 3.5, 1.9, 0.4, and 4.0 microM, respectively. The relative catalytic efficiency of the enzyme for phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate was 0.16, 0.14, 0.48, and 0.04 liter (min x mg)-1, respectively. The hydrolysis of phosphatidate is inhibited competitively by ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. The Ki(app) values were 5.5, 5.9, and 4.0 microM, respectively. The hydrolysis of phosphatidate by the phosphohydrolase conforms to a surface dilution kinetic model. It is concluded that the enzyme is a lipid phosphomonoesterase that can modify the balance of phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate relative to diacylglycerol, ceramide, monoacylglycerol, and sphingosine, respectively. The enzyme could thus play an important role in regulating cell activation and signal transduction (Waggoner, 1996).
Two human cDNA clones were obtained encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (Mr = 32,158) and PAP-2b (Mr = 35,119), both of which contain six putative transmembrane domains. Both enzymes are glycosylated and cleaved by N-glycanase and endo-beta-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b share 47% identical sequence and are the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively. Furthermore, the sequences of both PAPs were 34% to 39% identical to that of Drosophila Wunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings suggest critical roles for PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyze lysophosphatidate and ceramide-1-phosphate in addition to phosphatidate, the hydrolysis of sphingosine-1-phosphate was detected only for PAP-2b. PAP-2b is expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a is relatively specific, being extremely low in the placenta and thymus. In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation (Kai, 1997).
Phosphatidic acid (PA) and diacylglycerol (DG) are lipids involved in signal transduction and in structural membrane-lipid biosynthesis in cells. Phosphatidic acid phosphatase (PAP) catalyzes the conversion of PA to DG. This enzyme exists in at least two isoforms, one of which (PAP1) is presumed to be cytosolic and membrane associated and the other (PAP2) to be an integral membrane protein. Homology search of the GenBank database using a murine sequence probe enabled the cloning of several putative human isoenzymes. Two isoforms, presumed to be alternative splice variants from a single gene, designated as PAP2-alpha1 and PAP2-alpha2, have been cloned and expressed. The PAP2-alpha1 and PAP2-alpha2 have a 84% and a 72% overall match, respectively, with the published mouse PAP amino acid sequence. The area of alternative exon usage was confined to the coding region at amino acids 20 to 70. Ectopic expression of PAP2-alpha1 and PAP2-alpha2 cDNAs in ECV304 endothelial cells led to a 6- to 8-fold and a 2-fold increase in PAP activity, respectively, in cell-free extracts using an in vitro assay that measured the conversion of [14C]PA to [14C]DG. The increase in PAP activity in PAP2-alpha-transfected cells correlated with a >50% decrease in the steady-state PA level. Northern analysis showed that PAP2-alpha mRNA expression was suppressed in several tumor tissues, notably those derived from the lower alimentary tract. Subsequent analysis of colon tumor tissue derived from four donors confirmed lower expression of PAP2-alpha than in matching normal colon tissue. Considering these data and previous demonstrations that certain transformed cell lines have lower PAP activity, it is suggested that human PAP cDNAs may be candidates for gene therapy for certain tumors (Leung, 1998).
Phosphatidic acid (PA), lysophosphatidic acid, ceramide 1-phosphate (C1P), and sphingosine 1-phosphate (S1P) are lipid mediators generated by phospholipases, sphingomyelinases, and lipid kinases. The major pathway for degradation of these lipids is dephosphorylation catalyzed by members of two classes (types 1 and 2) of phosphohydrolase activities (PAPs). cDNAs encoding two type 2 PAPs, PAP-2a and PAP-2b, have been expressed by transient transfection and shown to catalyze hydrolysis of PA, C1P, and S1P. A third type 2 PAP enzyme, PAP-2c, has been identified that exhibits 54% and 43% sequence homology to PAPs 2a and 2b. Expression of HA epitope-tagged PAP-2a, -2b, and 2c in HEK293 cells produced immunoreactive proteins and increased membrane-associated PAP activity. Sf9 insect cells contain very low endogenous PAP activity. Recombinant expression of the three PAP enzymes produces dramatic increases in membrane-associated PAP activity. Expression of PAP-2a but not PAP-2b or -2c resulted in high levels of cell surface PAP activity in intact insect cells. Kinetic analysis of PAP-2a, -2b, and -2c activity against PA, lysophosphatidic acid, C1P, and S1P revealed differences in substrate specificity and susceptibility to inhibition by sphingosine, Zn2+, and propranol (Roberts, 1998).
Sphingosine and sphingosine-1-phosphate (SPP) are interconvertible sphingolipid metabolites with opposing effects on cell growth and apoptosis. Based on sequence homology with LBP1, a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast, this study reports the cloning, identification, and characterization of a mammalian SPP phosphatase (mSPP1). This hydrophobic enzyme, which contains the type 2 lipid phosphohydrolase conserved sequence motif, shows substrate specificity for SPP. Partially purified Myc-tagged mSPP1 was also highly active at dephosphorylating SPP. When expressed in yeast, mSPP1 can partially substitute for the function of LBP1. Membrane fractions from human embryonic kidney HEK293 cells transfected with mSPP1 markedly degraded SPP but not lysophosphatidic acid, phosphatidic acid, or ceramide-1-phosphate. Enforced expression of mSPP1 in NIH 3T3 fibroblasts not only decreased SPP and enhanced ceramide levels, it also markedly diminished survival and induced the characteristic traits of apoptosis. Collectively, these results suggest that SPP phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate (Mandala, 2000).
The wunen gene of Drosophila encodes a multipass membrane-spanning protein that negatively regulates primordial germ cell migration. A mouse gene has been cloned that encodes a protein homologous to wunen and to the Type 2 phosphatidic acid phosphatases. This gene encodes a 251-amino-acid protein that most closely resembles the human Type 2 phosphatidic acid phosphatase PAP-2c. Northern blot analysis reveals the presence of a single 1.9-kb Ppap2c transcript. The Ppap2c gene was localized to the central portion of mouse Chromosome 10 by interspecific backcross analysis (Zhang, 2000a).
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 when compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation is inhibited by extracellular Ca2+ and this inhibition is diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations does not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts show relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreases 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 (Jasinska, 1999).
Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. The Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzes the dephosphorylation of DGPP. This reaction is Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and is inhibited by Mn2+ ions. PAP2 exhibits a high affinity for DGPP. DGPP inhibits the ability of PAP2 to dephosphorylate PA, and PA inhibits the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae is inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 shows homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, this work indicates that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. A model is proposed in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol (Dillon, 2003).
Phosphatidic acid phosphatases (PAPs) catalyze the conversion of phosphatidic acid to diacylglycerol and inorganic phosphate and have been postulated to function both in lipid biosynthesis and in cellular signal transduction. In Drosophila melanogaster, the Type 2 phosphatidic acid phosphatase protein encoded by the wunen gene, negatively regulates primordial germ cell migration. A mouse Ppap2c gene encodes the Type 2 phosphatidic acid phosphatase Pap2c. To analyze the in vivo role of the Ppap2c gene, a null mutation was constructed by gene targeting. Ppap2c(-/-) homozygous mutant mice are viable, fertile, and exhibit no obvious phenotypic defects. These data demonstrate that the Ppap2c gene is not essential for embryonic development or fertility in mice (Zhang, 2000b).
Bioactive phospholipids, which include sphingosine-1-phosphate, lysophosphatidic acid, ceramide and their derivatives regulate a wide variety of cellular functions in culture such as proliferation, apoptosis and differentiation. The availability of these lipids and their products is regulated by the lipid phosphate phosphatases (LPPs). Mouse embryos deficient for LPP3 fail to form a chorio-allantoic placenta and yolk sac vasculature. A subset of embryos also show a shortening of the anterior-posterior axis and frequent duplication of axial structures that are strikingly similar to the phenotypes associated with axin deficiency, a critical regulator of Wnt signaling. Loss of LPP3 results in a marked increase in beta-catenin-mediated TCF transcription, whereas elevated levels of LPP3 inhibit beta-catenin-mediated TCF transcription. LPP3 also inhibits axis duplication and leads to mild ventralization in Xenopus embryo development. Although LPP3 null fibroblasts show altered levels of bioactive phospholipids, consistent with loss of LPP3 phosphatase activity, mutant forms of LPP3, specifically lacking phosphatase activity, are able to inhibit beta-catenin-mediated TCF transcription and also suppress axis duplication, although not as effectively as intact LPP3. These results reveal that LPP3 is essential to formation of the chorio-allantoic placenta and extra-embryonic vasculature. LPP3 also mediates gastrulation and axis formation, probably by influencing the canonical Wnt signaling pathway. The exact biochemical roles of LPP3 phosphatase activity and its undefined effect on beta-catenin-mediated TCF transcription remain to be determined (Escalante-Alcalde, 2003).
Outgrowth of axons in the central nervous system is governed by specific molecular cues. Molecules detected so far act as ligands that bind to specific receptors. A membrane-associated lipid phosphate phosphatase is described that was named plasticity-related gene 1 (PRG-1). PRG-1 facilitates axonal outgrowth during development and regenerative sprouting. PRG-1 is specifically expressed in neurons and is located in the membranes of outgrowing axons. There, it acts as an ecto-enzyme and attenuates phospholipid-induced axon collapse in neurons and facilitates outgrowth in the hippocampus. Thus, a novel mechanism is proposed by which axons are able to control phospholipid-mediated signaling and overcome the growth-inhibiting, phospholipid-rich environment of the extracellular space (Bräuer, 2003).
Lysophosphatidic acid (LPA) is an extracellular signaling mediator with a broad range of cellular responses. Three G-protein-coupled receptors (Edg-2, -4, and -7) have been identified as receptors for LPA. In this study, the ectophosphatase lipid phosphate phosphatase 1 (LPP1) has been shown to down-regulate LPA-mediated mitogenesis. Furthermore, using degradation-resistant phosphonate analogs of LPA and stereoselective agonists of the Edg receptors it has been demonstrated that the mitogenic and platelet aggregation responses to LPA are independent of Edg-2, -4, and -7. Specifically, LPA degradation is insufficient to account for the decrease in LPA potency in mitogenic assays, and the stereoselectivity observed at the Edg receptors is not reflected in mitogenesis. Additionally, RH7777 cells, which are devoid of Edg-2, -4, and -7 receptor mRNA, have a mitogenic response to LPA and LPA analogs. Finally, the ligand selectivity of the platelet aggregation response is consistent with that of mitogenesis, but not with Edg-2, -4, and -7 (Hooks, 2001).
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