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Phosphatidic acid phosphatase from mammalian tissues: discovery of channel-like proteins with unexpected functions1
Biochimica et Biophysica Acta 1348 Ž1997. 56–62
Chapter IVB
Phosphatidic acid phosphatase from mammalian tissues: discovery of channel-like proteins with unexpected functions 1 Hideo Kanoh ) , Masahiro Kai, Ikuo Wada Department of Biochemistry, Sapporo Medical UniÕersity, School of Medicine, West-17, South-1, Chuo-Ku Sapporo 060, Japan Received 20 February 1997; revised 24 April 1997; accepted 29 April 1997
Abstract Phosphatidic acid phosphatase ŽPAP. has long been known as a key enzyme involved in both glycerolipid biosynthesis and cellular signal transduction. The cDNA cloning of a plasma membrane-bound type 2 PAP has revealed the existence of a novel glycoprotein with six transmembrane domains. The type 2 PAP now represents an enzyme family consisting of Drosophila Wunen and rat Dri 42, which participate in germ cell migration and epithelial differentiation, respectively. Such novel functions of the type 2 PAP suggest the unexpected importance of lipids andror their metabolic enzymes. q 1997 Elsevier Science B.V. Keywords: Phosphatidic acid phosphatase; cDNA cloning; Signal transduction; Cell differentiation; Cell migration; Plasma membrane; Wunen Ž Drosophila.; Dri 42 Žrat.
1. Introduction Phosphatidic acid phosphatase ŽPAP. was first described by Kennedy’s group in chicken and rat livers w1x. At that time diacylglycerol had already been established by the same group as an immediate biosynthetic precursor of triacylglycerol and membrane phospholipids. The discovery of PAP thus completed the well-known scheme of the main biosynthetic
Abbreviations: PAP, phosphatidic acid phosphatase Corresponding author. Fax: q81 11 6125861; E-mail: [email protected] 1 Note added in proof: the catalytic mechanism of PAP-2 has been proposed to be common to those of a variety of enzymes including haloperoxidases, bacterial acid phosphatases and mammalian glucose-6-phosphatases ŽNeuwald, A.F., Protein Sci., in press, and Stuckey, J. and Carman, G.M., Protein Sci. 6 Ž1997. 469–472.. )
pathway of mammalian glycerolipids. Since then a number of experiments have described the properties of PAP present in a wide range of subcellular fractions w2–4x, including cytosol, microsomes, mitochondria, nuclei, and plasma membranes. However, molecular mechanisms of the PAP action and its physiological significance have remained largely unknown until recently due in part to the lack of knowledge on the molecular properties of the enzyme. We have recently experienced the rapid expansion of the studies of cellular signal transduction. Diacylglycerol, phosphatidic acid and related lipids, long known to serve as classical metabolic intermediates, are now generally recognized as signal mediators liberated by different phospholipases. The novel function of such lipids accelerates the progress of studies on PAP. Indeed, most of the recent work on PAP is concerned with signal transduction rather than with
0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 0 9 4 - 5
H. Kanoh et al.r Biochimica et Biophysica Acta 1348 (1997) 56–62
phospholipid biosynthesis. The role of PAP in signal transduction is widely considered to contribute to the control of the balance between the two lipid second messengers, diacylglycerol and phosphatidic acid. Brindley’s group was the first to describe the existence of two types of PAP, which are different from each other with respect to subcellular localization and enzymological properties w2x. After attempts of enzyme purification by several groups, we have finally succeeded in the cDNA cloning of the plasma membrane-bound type 2 PAP ŽPAP-2. w5x. The sequence comparison with genes independently cloned in different research areas demonstrated that PAP-2 is directly involved in such unexpected cellular functions as germ cell migration w6x and differentiation of intestinal mucosa w7x. We can expect further understanding of novel functions of PAP previously unimaginable to lipid enzymologists. The functions of lipids andror their metabolic enzymes appear to be much more important than previously anticipated.
2. Regulation and functions Jamal et al. w2x found that there exist two forms of PAP in rat liver homogenates. The type 1 enzyme present in the cytosol and endoplasmic reticulum is dependent of Mg 2q and inhibited by SH-reactive reagents like N-ethylmaleimide Ž NEM. . The type 2 enzyme, on the other hand, is tightly bound to plasma membranes, independent on Mg 2q, and insensitive to NEM. This work represents significant progress of PAP research, since the two types of PAP can be readily distinguished simply by pretreating crude enzymes by NEM. The type 1 enzyme was considered to be involved in glycerolipid synthesis, since the translocation of soluble PAP to microsomes had been repeatedly observed when cellular triacylglycerol synthesis was stimulated w3,8,9x. The translocation of the type 1 enzyme appeared to be regulated by protein kinaseŽs. in view of the effects of okadaic acid on rat hepatocytes w10x. The type 2 enzyme, on the other hand, was generally not affected by cellular metabolic changes, and was postulated to participate in cellular signal transduction mediated by phospholipase D w11x. Other workers w12,13x reported the presence at the cell surface of an ecto-PAP, which hydrolyzes exogenous short-chain phosphatidic acid w12x or
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lysophosphatidic acid w13x. Some properties of the ecto-PAP, such as NEM-insensitivity and hydrolysis of lysophosphatidic acid, are similar to those of the type 2 enzyme. It therefore remains possible that the ecto-PAP and the type 2 enzyme may be the same protein. The role of PAP in signal transduction has attracted attention, since phosphatidic acid liberated by the action of phospholipase D is always metabolized albeit variably to diacylglycerol. The experiments using PAP inhibitors like propranolol w14x and sphingosine w15,16x confirmed that cellular PAP acts in signal-stimulated cells, but the exact regulatory mechanisms operating for the two types of PAP remain to be investigated. The activities of the type 2 enzyme and of phospholipase D were decreased in a coordinate manner in oncogenically transformed fibroblasts w17x. The effects of a novel inhibitor of the type 1 PAP, bromoenol lactone w18x, showed that this PAP isoform participates in triacylglycerol synthesis in mouse macrophages. These observations appear to support the different functions ascribed to the two PAP isoforms discussed above. However, there are several important findings which show the participation of the type 1 PAP in signal transduction. For example, the cytosolic type 1 PAP was translocated to membranes when the polymorphonuclear leukocytes were stimulated with inflammatory substances w19x. In A431 cells, in particular, it was demonstrated that the activity of the type 1 PAP associated with the epidermal growth factor receptor was reduced upon cell activation resulting in its reassociation with protein kinase C e w20x. It is therefore reasonable to assume at the present stage of investigation that both type 1 and type 2 PAP can participate in cellular signal transduction depending on the different cells and stimuli.
3. Substrate specificity Interpretation of the regulatory function of PAP in signal transduction is complicated by the broad substrate specificity of the enzyme. Waggoner et al. w21x found that the type 2 PAP purified from rat liver plasma membranes can hydrolyze lysophosphatidic acid, ceramide-1-phosphate and sphingosine-1-phosphate in addition to phosphatidic acid. We have also
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confirmed that two human type 2 PAP isozymes cloned possess similarly a broad specificity, although the two isozymes show different preferences toward substrates ŽKai et al., J. Biol. Chem., in press.. The pleiotropic effects of lysophosphatidic acid are well documented w22x. The products of agonist-stimulated sphingomyelinase w23,24x and related lipids, such as ceramide, sphingosine and sphingosine-1-phosphate, are also known to act as second messengers in a variety of signal transductions. PAP can thus participate in the metabolism of signal molecules derived from both glycerolipids and sphingolipids. Rat liver plasma membranes are described to be enriched with ceramide-1-phosphate phosphatase w25x, which may also act as a type 2 PAP.
4. Enzyme purification Recent attempts to purify PAP from mammalian tissues have been confined to the type 2 enzyme because this isoform is unexpectedly stable when detergent-solubilized from membranes w3,26x. We highly purified PAP from porcine thymus membranes and interpreted, wrongly as recognized by ourselves later w5x, the results as the purification of an 83-kDa enzyme w26x. The type 2 PAP of a similar molecular mass Ž83 kDa. was subsequently obtained from rat liver membranes w27x. However, the PAP purified from rat liver plasma membranes by Brindley’s group w28x is a 51–53 kDa glycosylated protein. Furthermore, the PAP from the same materials was recently identified as a 31 kDa protein w29x. These variable results reflect in part the difficulty of purification of the enzymes involved in phospholipid biosynthesis. The results may also suggest the presence of multiple forms of the type 2 PAP. In our attempts to further characterize the porcine enzyme, we had to reevaluate the previous enzyme purification. By analyzing partial amino acid sequences and using antipeptide antibody raised against the determined sequence, we found that the PAP activity in the final enzyme preparation from the porcine thymus membranes could be accounted for by a minor 35 kDa glycosylated protein and not by the predominant 83 kDa band w5x. This reevaluation led us to the successful cDNA cloning of the enzyme as described below. It has not been finally settled
whether all of the different forms of PAP-2 purified by several groups are indeed the enzyme proteins.
5. cDNA cloning-discovery of a novel channel-like glycoprotein with six transmembrane domains After partially sequencing the 35-kDa enzyme identified as described, it was easy to clone the cDNA, since the amino acid sequences of the porcine protein were highly conserved in the mouse hic53 clone previously reported as a H 2 O 2-inducible gene w30x. Polymerase chain reaction amplification of the hic53 clone, however, yielded a novel cDNA encoding a 31 894 Da polypeptide ŽFig. 1.. We confirmed by cDNA expression experiments that the clone indeed encodes the type 2 PAP and that the encoded protein becomes 35 kDa due to N-glycosylation w5x. We also predicted that the enzyme consisted of six putative transmembrane regions.
Fig. 1. Sequence comparison of type 2 phosphatidic acid phosphatase gene family members. The amino acid sequences of mouse PAP-2ŽmPAP. w5x, rat Dri 42 w7x and Drosophila Wunen w6x are aligned. Dashes indicate gaps inserted to maximize alignment and identical amino acids are shaded. ) N-Glycosylation site conserved in mouse PAP and rat Dri 42.
H. Kanoh et al.r Biochimica et Biophysica Acta 1348 (1997) 56–62
Once the cDNA encoding a novel protein is cloned, modern biochemistry allows us to deduce abundant information without doing any benchwork, i.e. through data base searches. It was soon noticed that the mouse 35-kDa PAP shares 34% identical sequence with Wunen protein, which was discovered as a repulsive gene responsible for guiding the germ cell migration in Drosophila embryo w6x. We have confirmed that the Wunen cDNA indeed encodes PAP activity when transfected into mammalian cells Ž Wada et al., unpublished.. Another unexpected discovery obtained from the database search is that both mouse 35-kDa PAP and Drosophila Wunen are highly similar to the product of Dri 42 gene w7x, sharing 48.1 and 34.4% identical sequences, respectively Ž Fig. 1.. Dri42 was described to be up-regulated during the epithelial differentiation in rat intestinal mucosa w7x. We have independently cloned two human PAP-2
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isozymes designated 2a and 2b Ž Kai et al., J. Biol. Chem., in press.. PAP-2a is the human homolog of the mouse 35-kDa enzyme, but the 2b isozyme was unexpectedly found to be the human homolog of the rat Dri gene product. We have evidence that the human PAP-2b is located, like the 2a enzyme, in the plasma membranes, whereas the Dri 42 was described to reside in the endoplasmic reticulum w7x. The reason for this important discrepancy is presently unknown. The sequences of these homologs, now representing type 2 PAP gene family members, are compared in Fig. 1. Since the mode of the transmembrane disposition of Dri 42 was characterized in detail w7x, the membrane topology of the mouse 35kDa PAP and Wunen can also be predicted Ž Fig. 2. . We also obtained some evidence that both of the Nand C-termini of mouse PAP are cytoplasmically oriented as depicted in Fig. 2 ŽWada et al., unpub-
Fig. 2. A model for the arrangement of mouse PAP-2 across the cell-surface membrane. A membrane topology of the mouse PAP-2 is deduced from a previously reported model for the Dri 42 gene product w7x. The transmembrane segments are predicted by PHDhtm program of The PredictProtein server at The European Molecular Biology Laboratory. Amino acids conserved in common in mouse PAP-2 w5x, Drosophila Wunen w6x and rat Dri 42 w7x are dotted and bold-faced. I, inner segment; TM, transmembrane segment; O, outer segment; and CHO, oligosaccharide.
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lished.. As shown in Fig. 2, the major portion of PAP-2 is occupied by the six transmembrane domains as has already been demonstrated for rat Dri 42 w7x. The enzyme has three extracellular regions and its cytoplasmic portion consists of the N- and C-termini and two short juxtamembrane segments. From the model of PAP-2 at the cell-surface membranes depicted in Fig. 2, we should certainly imagine a kind of channel protein rather than lipid phosphatases. Indeed, Dri 42 was postulated to serve as a subunit of a channel protein w7x. The possibility thus exists that PAP-2 can serve as a channel of unknown substances in addition to metabolizing phosphatidic acid. The sequences of the PAP-2 gene family proteins did not show significant similarities to those of a number of phosphatases deposited in the data bases. However, we detected as shown in Fig. 3 that the amino acid clusters most highly conserved among bacterial nonspecific acid phosphatases are also conserved significantly in all of the PAP-2 proteins. Such a highly conserved sequence may be involved in the catalytic action of the non-specific phosphatases. In
the case of PAP this conserved sequence is located in the second and third extracellular segments. This might indicate that the type-2 PAP is an ecto-PAP previously described. Furthermore, a homology exists between the sequences of the mouse PAP and yeast pyrophosphatidic acid Ž DGPP. phosphatase w31x ŽCarman, G.M., this issue.. Identification of the catalytic site of PAP-2 is required in order to understand the mechanisms of actions of these PAP family members. In this context we observed that the two human PAP isozymes, 2a and 2b, were not inactivated by diisopropylfluorophosphate and p-brom ophena cylbromide, thus suggesting that both enzymes do not contain reactive serine and histidine, respectively, at their active sites Žunpublished. . We already demonstrated that the PAP activity can be accounted for by a single 35-kDa protein w5x. However, this finding does not preclude the possibility that PAP-2 is operating at the cell-surface membranes as homooligomers. During the earlier enzyme purification experiments w3,26,27x, the PAP-2 activity behaved as a polypeptide much larger than 35 kDa in
Fig. 3. Short PAP-2 ecto-regions exposed on the membranes show a significant similarity to the conserved sequence of class A bacterial acid phosphatases. CRSeq database was scanned for the presence of the conserved PAP-2 sequence motif by the BEAUTYŽBLAST Enhanced Alignment Utility. program w32x. Alignment to cluster 3292.5 which is the most conserved region of class A bacterial acid phosphatases and apyrase is presented. Conserved amino acids between the two gene families are boxed. Accession number for PHOC_MORMO Ž Proteus morganii ., PHON_PROST Ž ProÕidencia stuartii ., PHON_SALTY Ž Salmonella typhimurium. and PPA_ZYMMO Ž Zymomonas mobilis . are P28581, P26975, P26976 and P14924, respectively.
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gel filtration or in density gradient centrifugation. This may indicate the multimeric structure of the native PAP-2.
lar structure in the near future following, for example, the strategy of low stringency screening of cDNA library using the PAP-2 cDNA as a probe.
6. Future developments
Acknowledgements
PAP has long been known to be a key enzyme in glycerolipid biosynthesis, and recently also has attracted attention as the enzyme metabolizing lipid second messengers liberated in cellular signal transduction. However, our cDNA cloning work demonstrated that PAP-2 may be directly involved in cell migration and differentiation phenomena as exemplified by Drosophila Wunen and rat Dri genes. In the case of Wunen, in particular, PAP appears to regulate the modes of cell–cell contacts during the cell migration process. In such a case, the role of PAP should become easier to understand if we can assume that this type of PAP is an ecto-enzyme metabolizing exogenous lipid substrates. This is an interesting subject of further studies. We have to also consider the broad substrate specificity of the type 2 enzymes. All of the substrates and the reaction products of the PAP-2 can potentially serve as potent lipid mediators. It is therefore necessary to define which type of lipids is involved in the regulatory functions exerted by PAP-2. A rather vague but intriguing question is: can the lipids be so important as to control specific and fundamental cellular functions? In view of the channel-like appearance of the PAP-2 molecules Ž Fig. 2. , the possibility remains that these enzymes might have additional non-catalytic functions like interacting with specific ligands provided in the extracellular space. These intriguing possibilities are to be explored further. It is recognized that we know little of the molecular structure of the soluble type 1 PAP. As found by Brindley’s group, this PAP isoform appears to act under catalytic mechanisms quite distinct from those of the type 2 enzymes. After realizing the broad and unexpected functions of PAP-2 as discussed in this paper, the type 1 PAP becomes even more attractive, since PAP-1 may be the enzyme principally involved in glycerolipid metabolism and in signal transduction mediated by phospholipase D. Although no successful enzyme purification has been described for the type 1 enzyme, we can probably elucidate its molecu-
This work has been supported in part by Grants-inAid for Scientific Research from The Ministry of Education, Science, Sports and Culture of Japan, and by Yamanouchi Foundation for Metabolic Disorders. We thank Drs. Ken Howard and G.M. Carman for helpful discussion. Dedicated to Professor E.P. Kennedy in commemoration of his pioneering work on this enzyme. References w1x S.W. Smith, S.B. Weiss, E.P. Kennedy, The enzymatic dephosphorylation of phosphatidic acid, J. Biol. Chem. 228 Ž1957. 915–922. w2x Z. Jamal, A. Martin, A. Gomez-Munoz, D.N. Brindley, Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol, J. Biol. Chem. 266 Ž1991. 2988–2996. w3x C.P. Day, S.J. Yeaman, Physical evidence for the presence of two forms of phosphatidate phosphohydrolase in rat liver, Biochim. Biophys. Acta 1127 Ž1992. 87–94. w4x M. Balboa, J. Balsinde, E.A. Dennis, P.A. Insel, A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells, J. Biol. Chem. 270 Ž1995. 11738–11740. w5x M. Kai, I. Wada, S. Imai, F. Sakane, H. Kanoh, Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase Žtype 2. bound to plasma membranes, J. Biol. Chem. 271 Ž1996. 18931–18938. w6x N. Zhang, J. Zhang, K.J. Purcell, Y. Cheng, K. Howard, The Drosophila protein Wunen repels migrating germ cells, Nature ŽLondon. 385 Ž1997. 64–67. w7x D. Barila, M. Plateroti, F. Nobili, A.O. Muda, Y. Xie, T. Morimoto, G. Perozzi, The Dri 42 gene, whose expression is up- regulated during epithelial differentiation, encodes a novel endoplasmic reticulum resident transmembrane protein, J. Biol. Chem. 271 Ž1996. 29928–29936. w8x A. Gomez-Munoz, E.H. Hamza, D.N. Brindley, Effects of sphingosine, albumin and unsaturated fatty acids on the activation and translocation of phosphatidate phosphohydrolases in rat liver hepatocytes, Biochim. Biophys. Acta 1127 Ž1992. 49–56. w9x O. Aridor-Piterman, Y. Lavie, M. Liscovitch, Bimodal distribution of phosphatidic acid phosphohydrolase in NG10815 cells, Eur. J. Biochem. 204 Ž1992. 561–568.
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w10x A. Gomez-Munoz, G.M. Hatch, A. Martin, Z. Jamal, D.E. Vance, D.N. Brindley, Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes, FEBS Lett. 301 Ž1992. 103–106. w11x J.H. Exton, Signaling through phosphatidylcholine breakdown, J. Biol. Chem. 265 Ž1990. 1–4. w12x D.K. Perry, V.L. Stevens, T.S. Widlanski, J.D. Lambeth, A novel ecto-phosphatidic acid phosphohydrolase activity mediates activation of neutrophil superoxide generation by exogenous phosphatidic acid, J. Biol. Chem. 268 Ž1993. 25302–25310. w13x M. Xie, M.G. Low, Identification and characterization of an ecto-Žlyso.phosphatidic acid phosphatase in PAM212 keratinocytes, Arch. Biochem. Biophys. 312 Ž1994. 254–259. w14x C.S.T. Hii, Y.S. Edwards, A.W. Murray, Phorbol esterstimulated hydrolysis of phosphatidylcholine and phosphatidyl-ethanolamine by phospholipase D in HeLa cells, J. Biol. Chem. 266 Ž1991. 20238–20243. w15x Y. Lavie, O. Piterman, M. Liscovitch, Inhibition of phosphatidic acid phosphohydrolase activity by sphingosine, FEBS Lett. 277 Ž1990. 7–10. w16x T.J. Mullmann, M.I. Siegel, R.W. Egan, M.M. Billah, Sphingosine inhibits phosphatidate phosphohydrolase in human neutrophils by a protein kinase C-independent mechanism, J. Biol. Chem. 266 Ž1991. 2013–2016. w17x A. Martin, A. Gomez-Munoz, D.W. Waggoner, J.C. Stone, D.N. Brindley, Decreased activities of phosphatidate phosphohydrolase and phospholipase D in ras and tyrosine kinase Ž fps . transformed fibroblasts, J. Biol. Chem. 268 Ž1993. 23924–23932. w18x J. Balsinde, E.A. Dennis, Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages, J. Biol. Chem. 271 Ž1996. 31937–31941. w19x A.P. Truett, S.B. Bocckino, J.J. Murray, Regulation of phosphatidic acid phosphohydrolase activity during stimulation of human polymorphonuclear leukocytes, FASEB J. 6 Ž1992. 2720–2725. w20x Y. Jiang, Zh. Lu, Q. Zang, D.A. Foster, Regulation of phosphatidic acid phosphohydrolase by epidermal growth factor, J. Biol. Chem. 271 Ž1996. 29529–29532. w21x D.W. Waggoner, A. Gomez-Munoz, J. Dewald, D.N. Brindley, Phosphatidate phosphohydrolase catalyzes the hydroly-
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sis of ceramide-1-phosphate, lysophosphatidate, and sphingosine-1- phosphate, J. Biol. Chem. 271 Ž1996. 16506– 16509. K. Jalink, P.L. Hordijk, W.H. Moolenaar, Growth factor-like effects of lysophosphatidic acid, a novel lipid mediator, Biochim. Biophys. Acta 1198 Ž1994. 185–196. Y.A. Hannun, The sphingomyelin cycle and the second messenger function of ceramide, J. Biol. Chem. 269 Ž1994. 3125–3128. R. Kolesnick, D.W. Golde, The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling, Cell 77 Ž1994. 325–328. O. Boudker, A.H. Futerman, Detection and characterization of ceramide-1-phosphate phosphatase activity in rat liver plasma membrane, J. Biol. Chem. 268 Ž1993. 22150–22155. H. Kanoh, S. Imai, K. Yamada, F. Sakane, Purification and properties of phosphatidic acid phosphatase from porcine thymus membranes, J. Biol. Chem. 267 Ž1992. 25309– 25314. I.N. Fleming, S.J. Yeaman, Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolaseŽPAP2. from rat liver, Biochem. J. 308 Ž1995. 983–989. D.W. Waggoner, A. Martin, J. Dewald, A. Gomez-Munoz, D.N. Brindley, Purification and characterization of a novel plasma membrane phosphatidate phosphohydrolase from rat liver, J. Biol. Chem. 270 Ž1995. 19422–19429. E.A. Siess, M.M. Hofstetter, Identification of phosphatidate phosphohydrolase purified from rat liver membranes on SDS-polyacrylamide gel electrophoresis, FEBS Lett. 381 Ž1996. 169–173. K. Egawa, M. Yoshiwara, M. Shibanuma, K. Nose, Isolation of a novel ras-recision gene that is induced by hydrogen peroxide from a mouse osteoblastic cell line MC3T3-E1, FEBS Lett. 372 Ž1995. 74–77. W.-I. Wu, Y. Liu, B. Riedel, J.B. Wissing, A.S. Fischl, G.M. Carman, Purification and characterization of diacylglycerol pyrophosphate phosphatase from Saccharomyces cereÕisiae, J. Biol. Chem. 271 Ž1996. 1868–1876. K.C. Worley, B.A. Wiese, R.F. Smith, BEAUTY: An enhanced BLAST-based search tool that integrates mutiple biological information resources into sequence similarity search results, Genome Res. 5 Ž1995. 173–184.
Chapter IVB
Phosphatidic acid phosphatase from mammalian tissues: discovery of channel-like proteins with unexpected functions 1 Hideo Kanoh ) , Masahiro Kai, Ikuo Wada Department of Biochemistry, Sapporo Medical UniÕersity, School of Medicine, West-17, South-1, Chuo-Ku Sapporo 060, Japan Received 20 February 1997; revised 24 April 1997; accepted 29 April 1997
Abstract Phosphatidic acid phosphatase ŽPAP. has long been known as a key enzyme involved in both glycerolipid biosynthesis and cellular signal transduction. The cDNA cloning of a plasma membrane-bound type 2 PAP has revealed the existence of a novel glycoprotein with six transmembrane domains. The type 2 PAP now represents an enzyme family consisting of Drosophila Wunen and rat Dri 42, which participate in germ cell migration and epithelial differentiation, respectively. Such novel functions of the type 2 PAP suggest the unexpected importance of lipids andror their metabolic enzymes. q 1997 Elsevier Science B.V. Keywords: Phosphatidic acid phosphatase; cDNA cloning; Signal transduction; Cell differentiation; Cell migration; Plasma membrane; Wunen Ž Drosophila.; Dri 42 Žrat.
1. Introduction Phosphatidic acid phosphatase ŽPAP. was first described by Kennedy’s group in chicken and rat livers w1x. At that time diacylglycerol had already been established by the same group as an immediate biosynthetic precursor of triacylglycerol and membrane phospholipids. The discovery of PAP thus completed the well-known scheme of the main biosynthetic
Abbreviations: PAP, phosphatidic acid phosphatase Corresponding author. Fax: q81 11 6125861; E-mail: [email protected] 1 Note added in proof: the catalytic mechanism of PAP-2 has been proposed to be common to those of a variety of enzymes including haloperoxidases, bacterial acid phosphatases and mammalian glucose-6-phosphatases ŽNeuwald, A.F., Protein Sci., in press, and Stuckey, J. and Carman, G.M., Protein Sci. 6 Ž1997. 469–472.. )
pathway of mammalian glycerolipids. Since then a number of experiments have described the properties of PAP present in a wide range of subcellular fractions w2–4x, including cytosol, microsomes, mitochondria, nuclei, and plasma membranes. However, molecular mechanisms of the PAP action and its physiological significance have remained largely unknown until recently due in part to the lack of knowledge on the molecular properties of the enzyme. We have recently experienced the rapid expansion of the studies of cellular signal transduction. Diacylglycerol, phosphatidic acid and related lipids, long known to serve as classical metabolic intermediates, are now generally recognized as signal mediators liberated by different phospholipases. The novel function of such lipids accelerates the progress of studies on PAP. Indeed, most of the recent work on PAP is concerned with signal transduction rather than with
0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 0 9 4 - 5
H. Kanoh et al.r Biochimica et Biophysica Acta 1348 (1997) 56–62
phospholipid biosynthesis. The role of PAP in signal transduction is widely considered to contribute to the control of the balance between the two lipid second messengers, diacylglycerol and phosphatidic acid. Brindley’s group was the first to describe the existence of two types of PAP, which are different from each other with respect to subcellular localization and enzymological properties w2x. After attempts of enzyme purification by several groups, we have finally succeeded in the cDNA cloning of the plasma membrane-bound type 2 PAP ŽPAP-2. w5x. The sequence comparison with genes independently cloned in different research areas demonstrated that PAP-2 is directly involved in such unexpected cellular functions as germ cell migration w6x and differentiation of intestinal mucosa w7x. We can expect further understanding of novel functions of PAP previously unimaginable to lipid enzymologists. The functions of lipids andror their metabolic enzymes appear to be much more important than previously anticipated.
2. Regulation and functions Jamal et al. w2x found that there exist two forms of PAP in rat liver homogenates. The type 1 enzyme present in the cytosol and endoplasmic reticulum is dependent of Mg 2q and inhibited by SH-reactive reagents like N-ethylmaleimide Ž NEM. . The type 2 enzyme, on the other hand, is tightly bound to plasma membranes, independent on Mg 2q, and insensitive to NEM. This work represents significant progress of PAP research, since the two types of PAP can be readily distinguished simply by pretreating crude enzymes by NEM. The type 1 enzyme was considered to be involved in glycerolipid synthesis, since the translocation of soluble PAP to microsomes had been repeatedly observed when cellular triacylglycerol synthesis was stimulated w3,8,9x. The translocation of the type 1 enzyme appeared to be regulated by protein kinaseŽs. in view of the effects of okadaic acid on rat hepatocytes w10x. The type 2 enzyme, on the other hand, was generally not affected by cellular metabolic changes, and was postulated to participate in cellular signal transduction mediated by phospholipase D w11x. Other workers w12,13x reported the presence at the cell surface of an ecto-PAP, which hydrolyzes exogenous short-chain phosphatidic acid w12x or
57
lysophosphatidic acid w13x. Some properties of the ecto-PAP, such as NEM-insensitivity and hydrolysis of lysophosphatidic acid, are similar to those of the type 2 enzyme. It therefore remains possible that the ecto-PAP and the type 2 enzyme may be the same protein. The role of PAP in signal transduction has attracted attention, since phosphatidic acid liberated by the action of phospholipase D is always metabolized albeit variably to diacylglycerol. The experiments using PAP inhibitors like propranolol w14x and sphingosine w15,16x confirmed that cellular PAP acts in signal-stimulated cells, but the exact regulatory mechanisms operating for the two types of PAP remain to be investigated. The activities of the type 2 enzyme and of phospholipase D were decreased in a coordinate manner in oncogenically transformed fibroblasts w17x. The effects of a novel inhibitor of the type 1 PAP, bromoenol lactone w18x, showed that this PAP isoform participates in triacylglycerol synthesis in mouse macrophages. These observations appear to support the different functions ascribed to the two PAP isoforms discussed above. However, there are several important findings which show the participation of the type 1 PAP in signal transduction. For example, the cytosolic type 1 PAP was translocated to membranes when the polymorphonuclear leukocytes were stimulated with inflammatory substances w19x. In A431 cells, in particular, it was demonstrated that the activity of the type 1 PAP associated with the epidermal growth factor receptor was reduced upon cell activation resulting in its reassociation with protein kinase C e w20x. It is therefore reasonable to assume at the present stage of investigation that both type 1 and type 2 PAP can participate in cellular signal transduction depending on the different cells and stimuli.
3. Substrate specificity Interpretation of the regulatory function of PAP in signal transduction is complicated by the broad substrate specificity of the enzyme. Waggoner et al. w21x found that the type 2 PAP purified from rat liver plasma membranes can hydrolyze lysophosphatidic acid, ceramide-1-phosphate and sphingosine-1-phosphate in addition to phosphatidic acid. We have also
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confirmed that two human type 2 PAP isozymes cloned possess similarly a broad specificity, although the two isozymes show different preferences toward substrates ŽKai et al., J. Biol. Chem., in press.. The pleiotropic effects of lysophosphatidic acid are well documented w22x. The products of agonist-stimulated sphingomyelinase w23,24x and related lipids, such as ceramide, sphingosine and sphingosine-1-phosphate, are also known to act as second messengers in a variety of signal transductions. PAP can thus participate in the metabolism of signal molecules derived from both glycerolipids and sphingolipids. Rat liver plasma membranes are described to be enriched with ceramide-1-phosphate phosphatase w25x, which may also act as a type 2 PAP.
4. Enzyme purification Recent attempts to purify PAP from mammalian tissues have been confined to the type 2 enzyme because this isoform is unexpectedly stable when detergent-solubilized from membranes w3,26x. We highly purified PAP from porcine thymus membranes and interpreted, wrongly as recognized by ourselves later w5x, the results as the purification of an 83-kDa enzyme w26x. The type 2 PAP of a similar molecular mass Ž83 kDa. was subsequently obtained from rat liver membranes w27x. However, the PAP purified from rat liver plasma membranes by Brindley’s group w28x is a 51–53 kDa glycosylated protein. Furthermore, the PAP from the same materials was recently identified as a 31 kDa protein w29x. These variable results reflect in part the difficulty of purification of the enzymes involved in phospholipid biosynthesis. The results may also suggest the presence of multiple forms of the type 2 PAP. In our attempts to further characterize the porcine enzyme, we had to reevaluate the previous enzyme purification. By analyzing partial amino acid sequences and using antipeptide antibody raised against the determined sequence, we found that the PAP activity in the final enzyme preparation from the porcine thymus membranes could be accounted for by a minor 35 kDa glycosylated protein and not by the predominant 83 kDa band w5x. This reevaluation led us to the successful cDNA cloning of the enzyme as described below. It has not been finally settled
whether all of the different forms of PAP-2 purified by several groups are indeed the enzyme proteins.
5. cDNA cloning-discovery of a novel channel-like glycoprotein with six transmembrane domains After partially sequencing the 35-kDa enzyme identified as described, it was easy to clone the cDNA, since the amino acid sequences of the porcine protein were highly conserved in the mouse hic53 clone previously reported as a H 2 O 2-inducible gene w30x. Polymerase chain reaction amplification of the hic53 clone, however, yielded a novel cDNA encoding a 31 894 Da polypeptide ŽFig. 1.. We confirmed by cDNA expression experiments that the clone indeed encodes the type 2 PAP and that the encoded protein becomes 35 kDa due to N-glycosylation w5x. We also predicted that the enzyme consisted of six putative transmembrane regions.
Fig. 1. Sequence comparison of type 2 phosphatidic acid phosphatase gene family members. The amino acid sequences of mouse PAP-2ŽmPAP. w5x, rat Dri 42 w7x and Drosophila Wunen w6x are aligned. Dashes indicate gaps inserted to maximize alignment and identical amino acids are shaded. ) N-Glycosylation site conserved in mouse PAP and rat Dri 42.
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Once the cDNA encoding a novel protein is cloned, modern biochemistry allows us to deduce abundant information without doing any benchwork, i.e. through data base searches. It was soon noticed that the mouse 35-kDa PAP shares 34% identical sequence with Wunen protein, which was discovered as a repulsive gene responsible for guiding the germ cell migration in Drosophila embryo w6x. We have confirmed that the Wunen cDNA indeed encodes PAP activity when transfected into mammalian cells Ž Wada et al., unpublished.. Another unexpected discovery obtained from the database search is that both mouse 35-kDa PAP and Drosophila Wunen are highly similar to the product of Dri 42 gene w7x, sharing 48.1 and 34.4% identical sequences, respectively Ž Fig. 1.. Dri42 was described to be up-regulated during the epithelial differentiation in rat intestinal mucosa w7x. We have independently cloned two human PAP-2
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isozymes designated 2a and 2b Ž Kai et al., J. Biol. Chem., in press.. PAP-2a is the human homolog of the mouse 35-kDa enzyme, but the 2b isozyme was unexpectedly found to be the human homolog of the rat Dri gene product. We have evidence that the human PAP-2b is located, like the 2a enzyme, in the plasma membranes, whereas the Dri 42 was described to reside in the endoplasmic reticulum w7x. The reason for this important discrepancy is presently unknown. The sequences of these homologs, now representing type 2 PAP gene family members, are compared in Fig. 1. Since the mode of the transmembrane disposition of Dri 42 was characterized in detail w7x, the membrane topology of the mouse 35kDa PAP and Wunen can also be predicted Ž Fig. 2. . We also obtained some evidence that both of the Nand C-termini of mouse PAP are cytoplasmically oriented as depicted in Fig. 2 ŽWada et al., unpub-
Fig. 2. A model for the arrangement of mouse PAP-2 across the cell-surface membrane. A membrane topology of the mouse PAP-2 is deduced from a previously reported model for the Dri 42 gene product w7x. The transmembrane segments are predicted by PHDhtm program of The PredictProtein server at The European Molecular Biology Laboratory. Amino acids conserved in common in mouse PAP-2 w5x, Drosophila Wunen w6x and rat Dri 42 w7x are dotted and bold-faced. I, inner segment; TM, transmembrane segment; O, outer segment; and CHO, oligosaccharide.
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lished.. As shown in Fig. 2, the major portion of PAP-2 is occupied by the six transmembrane domains as has already been demonstrated for rat Dri 42 w7x. The enzyme has three extracellular regions and its cytoplasmic portion consists of the N- and C-termini and two short juxtamembrane segments. From the model of PAP-2 at the cell-surface membranes depicted in Fig. 2, we should certainly imagine a kind of channel protein rather than lipid phosphatases. Indeed, Dri 42 was postulated to serve as a subunit of a channel protein w7x. The possibility thus exists that PAP-2 can serve as a channel of unknown substances in addition to metabolizing phosphatidic acid. The sequences of the PAP-2 gene family proteins did not show significant similarities to those of a number of phosphatases deposited in the data bases. However, we detected as shown in Fig. 3 that the amino acid clusters most highly conserved among bacterial nonspecific acid phosphatases are also conserved significantly in all of the PAP-2 proteins. Such a highly conserved sequence may be involved in the catalytic action of the non-specific phosphatases. In
the case of PAP this conserved sequence is located in the second and third extracellular segments. This might indicate that the type-2 PAP is an ecto-PAP previously described. Furthermore, a homology exists between the sequences of the mouse PAP and yeast pyrophosphatidic acid Ž DGPP. phosphatase w31x ŽCarman, G.M., this issue.. Identification of the catalytic site of PAP-2 is required in order to understand the mechanisms of actions of these PAP family members. In this context we observed that the two human PAP isozymes, 2a and 2b, were not inactivated by diisopropylfluorophosphate and p-brom ophena cylbromide, thus suggesting that both enzymes do not contain reactive serine and histidine, respectively, at their active sites Žunpublished. . We already demonstrated that the PAP activity can be accounted for by a single 35-kDa protein w5x. However, this finding does not preclude the possibility that PAP-2 is operating at the cell-surface membranes as homooligomers. During the earlier enzyme purification experiments w3,26,27x, the PAP-2 activity behaved as a polypeptide much larger than 35 kDa in
Fig. 3. Short PAP-2 ecto-regions exposed on the membranes show a significant similarity to the conserved sequence of class A bacterial acid phosphatases. CRSeq database was scanned for the presence of the conserved PAP-2 sequence motif by the BEAUTYŽBLAST Enhanced Alignment Utility. program w32x. Alignment to cluster 3292.5 which is the most conserved region of class A bacterial acid phosphatases and apyrase is presented. Conserved amino acids between the two gene families are boxed. Accession number for PHOC_MORMO Ž Proteus morganii ., PHON_PROST Ž ProÕidencia stuartii ., PHON_SALTY Ž Salmonella typhimurium. and PPA_ZYMMO Ž Zymomonas mobilis . are P28581, P26975, P26976 and P14924, respectively.
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gel filtration or in density gradient centrifugation. This may indicate the multimeric structure of the native PAP-2.
lar structure in the near future following, for example, the strategy of low stringency screening of cDNA library using the PAP-2 cDNA as a probe.
6. Future developments
Acknowledgements
PAP has long been known to be a key enzyme in glycerolipid biosynthesis, and recently also has attracted attention as the enzyme metabolizing lipid second messengers liberated in cellular signal transduction. However, our cDNA cloning work demonstrated that PAP-2 may be directly involved in cell migration and differentiation phenomena as exemplified by Drosophila Wunen and rat Dri genes. In the case of Wunen, in particular, PAP appears to regulate the modes of cell–cell contacts during the cell migration process. In such a case, the role of PAP should become easier to understand if we can assume that this type of PAP is an ecto-enzyme metabolizing exogenous lipid substrates. This is an interesting subject of further studies. We have to also consider the broad substrate specificity of the type 2 enzymes. All of the substrates and the reaction products of the PAP-2 can potentially serve as potent lipid mediators. It is therefore necessary to define which type of lipids is involved in the regulatory functions exerted by PAP-2. A rather vague but intriguing question is: can the lipids be so important as to control specific and fundamental cellular functions? In view of the channel-like appearance of the PAP-2 molecules Ž Fig. 2. , the possibility remains that these enzymes might have additional non-catalytic functions like interacting with specific ligands provided in the extracellular space. These intriguing possibilities are to be explored further. It is recognized that we know little of the molecular structure of the soluble type 1 PAP. As found by Brindley’s group, this PAP isoform appears to act under catalytic mechanisms quite distinct from those of the type 2 enzymes. After realizing the broad and unexpected functions of PAP-2 as discussed in this paper, the type 1 PAP becomes even more attractive, since PAP-1 may be the enzyme principally involved in glycerolipid metabolism and in signal transduction mediated by phospholipase D. Although no successful enzyme purification has been described for the type 1 enzyme, we can probably elucidate its molecu-
This work has been supported in part by Grants-inAid for Scientific Research from The Ministry of Education, Science, Sports and Culture of Japan, and by Yamanouchi Foundation for Metabolic Disorders. We thank Drs. Ken Howard and G.M. Carman for helpful discussion. Dedicated to Professor E.P. Kennedy in commemoration of his pioneering work on this enzyme. References w1x S.W. Smith, S.B. Weiss, E.P. Kennedy, The enzymatic dephosphorylation of phosphatidic acid, J. Biol. Chem. 228 Ž1957. 915–922. w2x Z. Jamal, A. Martin, A. Gomez-Munoz, D.N. Brindley, Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol, J. Biol. Chem. 266 Ž1991. 2988–2996. w3x C.P. Day, S.J. Yeaman, Physical evidence for the presence of two forms of phosphatidate phosphohydrolase in rat liver, Biochim. Biophys. Acta 1127 Ž1992. 87–94. w4x M. Balboa, J. Balsinde, E.A. Dennis, P.A. Insel, A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells, J. Biol. Chem. 270 Ž1995. 11738–11740. w5x M. Kai, I. Wada, S. Imai, F. Sakane, H. Kanoh, Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase Žtype 2. bound to plasma membranes, J. Biol. Chem. 271 Ž1996. 18931–18938. w6x N. Zhang, J. Zhang, K.J. Purcell, Y. Cheng, K. Howard, The Drosophila protein Wunen repels migrating germ cells, Nature ŽLondon. 385 Ž1997. 64–67. w7x D. Barila, M. Plateroti, F. Nobili, A.O. Muda, Y. Xie, T. Morimoto, G. Perozzi, The Dri 42 gene, whose expression is up- regulated during epithelial differentiation, encodes a novel endoplasmic reticulum resident transmembrane protein, J. Biol. Chem. 271 Ž1996. 29928–29936. w8x A. Gomez-Munoz, E.H. Hamza, D.N. Brindley, Effects of sphingosine, albumin and unsaturated fatty acids on the activation and translocation of phosphatidate phosphohydrolases in rat liver hepatocytes, Biochim. Biophys. Acta 1127 Ž1992. 49–56. w9x O. Aridor-Piterman, Y. Lavie, M. Liscovitch, Bimodal distribution of phosphatidic acid phosphohydrolase in NG10815 cells, Eur. J. Biochem. 204 Ž1992. 561–568.
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