Cloning and Characterization of Two Human Isozymes of Mg2+-independent Phosphatidic Acid Phosphatase

Abstract

We obtained two human cDNA clones encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (M r ‫؍‬ 32,158) and -2b (M r ‫؍‬ 35, 119), both of which contained six putative transmembrane domains. Both enzymes were glycosylated and cleaved by N-glycanase and endo-␤-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b shared 47% identical sequence and were judged to be 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 -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 unexpectedly suggest critical roles of PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyzed lysophosphatidate and ceramide-1-phosphate in addition to phosphatidate, the hydrolysis of sphingosine-1-phosphate was detected only for PAP-2b. PAP-2b was expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a was relatively variable, 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.Phosphatidic acid phosphatase (PAP) 1 (EC 3.1.3.4) supplies diacylglycerol (DG) in glycerolipid biosynthesis by dephosphorylating phosphatidic acid (PA) (1). Since both DG (2) and PA (3) are potent signaling molecules, PAP plays an important role in cellular signal transduction in addition to lipid biosynthesis (4). In signaling systems mediated by phospholipase D (5), liberated PA is generally converted, albeit to different extents, to DG by the action of PAP (6 -8), thus suggesting that PAP contributes to the control of the relative balance of the two lipid second messengers.There exist two forms of mammalian PAP that can be distinguished with respect to subcellular localization and enzymologic properties (9). So far, distinct functions have been ascribed to the two forms of PAP. The type 1 PAP, a Mg 2ϩ -dependent and N-ethylmaleimide-sensitive enzyme, is considered to be primarily involved in lipid synthesis based on its translocation from the cytosol to microsomes upon stimulation of cellular triacylglycerol synthesis (10 -12). In this context, a recent work showed using a novel enzyme inhibitor that the type 1 PAP indeed participated in triacylglycerol synthesis in mouse macrophages (13). On the other hand, the type 2 PAP, a Mg 2ϩ -independent and N-ethylmaleimide-insensitive enzyme, has been postulated to participate in cellular signal transduction mediated by phospholipase D. The finding that the activities of the type 2 PAP and phospholipase D were decreased in a c...

Translocation of diacylglycerol kinase α to the nuclear matrix of rat thymocytes and peripheral T‐lymphocytes

“…This antibody was previously shown to be specific to rat [5] and human [7] DGKc~. Anti-rat protein kinase Ca and anti-rat TcR antibodies were obtained from Gibco BRL and Serotech, Oxford, UK, respectively.…”

“…The aliquots of the supernatant and pellet were treated with SDS sample buffer [22] and were subjected to SDS-polyacrylamide gel electrophoresis (9% acrylamide). The resolved proteins were transferred onto nitrocellulose membranes (Scleicher and Schuell) as previously described [7,9]. The membrane was probed with the anti-DGKet antibody, and the immunoreactive bands were visualized using an ECL system (Amersham Corp.) as described previously [7,9].…”

“…The resolved proteins were transferred onto nitrocellulose membranes (Scleicher and Schuell) as previously described [7,9]. The membrane was probed with the anti-DGKet antibody, and the immunoreactive bands were visualized using an ECL system (Amersham Corp.) as described previously [7,9]. In some experiments thymocytes were homogenized by passing through a 30 gauge needle 10 times in homogenization buffer (5 mM HEPES, 5 mM MgCI2, 1 mM phenylmethylsulfonyl fluoride, and 10 gg/ml each of leupeptin and pepstatin, pH 7.5).…”

Lipid phosphate phosphatase inhibitors locally amplify lysophosphatidic acid LPA1 receptor signalling in rat brain cryosections without affecting global LPA degradation

“…LPA is rapidly dephosphorylated by a family of integral membrane proteins known as lipid phosphate phosphatases (LPPs) [16-18]. The LPP family (also known as PAP2) comprises four members (LPP 1 , LPP 2 , LPP 3 and a splice variant LPP 1a ) which dephosphorylate their lipid substrates, namely LPA, PA, sphingosine 1-phosphate (S1P), and ceramide 1-phosphate [19-21]. All the LPP subtypes are expressed in the brain [18,19,22] but very little is known about their functional roles.…”

“…The LPP family (also known as PAP2) comprises four members (LPP 1 , LPP 2 , LPP 3 and a splice variant LPP 1a ) which dephosphorylate their lipid substrates, namely LPA, PA, sphingosine 1-phosphate (S1P), and ceramide 1-phosphate [19-21]. All the LPP subtypes are expressed in the brain [18,19,22] but very little is known about their functional roles. A hallmark of LPP activity is that it does not require Mg 2+ and is resistant to the alkylating agent, N-ethylmaleimide (NEM) [23].…”