Cloning, Expression, and Characterization of a Novel Phospholipase D Complementary DNA from Rat Brain

Abstract

Here we report the cloning and characterization of cDNA for a different type of PLD (rat PLD2 (rPLD2)) from rat brain. We synthesized highly degenerate amplimers corresponding to the conserved regions of eukaryote PLDs and performed polymerase chain reaction on a rat brain cDNA library. Using the amplified sequence as the probe, we cloned a rat brain cDNA clone that contained an open reading frame of 933 amino acids with an M r of 105,992. The deduced amino acid sequence showed significant similarity to hPLD1 with a large deletion in the middle of the sequence. When the sequence was expressed in the fission yeast Schizosaccharomyces pombe, PLD activity was greatly increased. The activity was markedly stimulated by phosphatidylinositol 4,5-bisphosphate, but not by ADPribosylation factor 1 and RhoA. Rat brain cytosol known to stimulate small GTP-binding protein-dependent PLD did not stimulate rPLD2 expressed in S. pombe. The transcript was detected at significant levels in brain, lung, heart, kidney, stomach, small intestine, colon, and testis, but at low levels in thymus, liver, and muscle. Only a negligible level was found in spleen and pancreas. Thus rPLD2 is a novel type of PLD dependent on phosphatidylinositol 4,5-bisphosphate, but not on the small GTP-binding proteins ADP-ribosylation factor 1 and RhoA. Phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid and choline (1). A variety of signal molecules such as hormones, neurotransmitters, and growth factors are known to induce the activation of PLD in a wide range of cell types. Hence PLD is implicated in a broad spectrum of physiological processes and diseases, including metabolic regulation, inflammation, secretion, mitogenesis, oncogenesis, neural and cardiac stimulation, diabetes, and senescence (for reviews, see Ref.2). Despite its crucial importance in signal transduction, the molecular structure and characteristics of PLD enzyme are only poorly understood.Multiple PLD isoforms exist in mammalian tissues. Several factors were reported to stimulate PLD activity in vitro, including unsaturated fatty acid (3), phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (4), monomeric GTP-binding proteins (G proteins) such as ADP-ribosylation factor 1 (ARF1) (5, 6) and RhoA (7, 8), protein kinase C (9), and calmodulin (10). Massenburg et al. (11) showed that two major forms of PLD activity in rat brain membranes can be separated into ARF-dependent and oleate-dependent enzymes, clearly indicating that these are distinct isoforms. Both oleate-dependent and ARF-dependent types of PLD were recently highly purified from pig lung and brain, respectively (12, 13). In addition, there may be multiple forms of small G protein-dependent PLD including ARF-sensitive, RhoA-sensitive, and ARF-, RhoA-sensitive PLDs. Siddiqi et al. (14) reported that the cytosolic fraction of HL-60 cells contained a soluble PLD activated by ARF, but not RhoA. Malcolm et al. (8) showed rat liver plasma membrane PLD to be sensitive to RhoA, but not to ARF. PL...

Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase

“…In the absence of further evidence, some workers have suggested that the purified activity may have been related to PLD2 (40). Two related polyphosphoinositolactivated PLD isoforms have been cloned on the basis of yeast PLD sequences (262,266) and are known as PLD1 (120 kDa) (81, 82, 122, 187) and PLD2 (106 kDa) (43,130). The catalytic site contains two H(x) K(x) 4 D (HKD) domains separated by variable nonactive site sequences.…”

Regulation of Phospholipid Synthesis in the Yeast cki1Δ eki1Δ Mutant Defective in the Kennedy Pathway

“…1) (1,4,(13)(14)(15)(16)(17). In the CDP-DAG pathway, PC is synthesized from CDP-DAG via the reactions catalyzed by PS synthase (18 -20), PS decarboxylase (21-23), PE methyltransferase (24, 25), and phospholipid methyltransferase (24,26). In the CDP-choline branch of the Kennedy pathway, PC is synthesized from choline via the reactions catalyzed by choline kinase (27), phosphocholine cytidylyltransferase (28), and choline phosphotransferase (29,30).…”

“…However, unlike mutants defective in the CDP-DAG pathway (23)(24)(25)(26)(43)(44)(45)(46), these Kennedy pathway mutants do not exhibit any auxotrophic requirements (36,47). Moreover, even in the absence of the Kennedy pathway, the cki1⌬ eki1⌬ (47) and cpt1 ept1 (36) mutants have an essentially normal complement of phospholipids including PC.…”

Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes

“…In reconstitution systems, the catalytic activity of recombinant hPLD1, purified PLD, or enriched PLD fractions was synergistically increased by the presence of ARF, rho small GTPases, and protein kinase C␣ (PKC␣). Another protein with PLD activity and 60% homology to PLD1 was recently cloned in mouse, rat, and human tissues and named PLD2 [7][8][9][10]. Mouse PLD2 was found to be insensitive to small GTPases but required PIP2 for maximal activation [8].…”

“…Another protein with PLD activity and 60% homology to PLD1 was recently cloned in mouse, rat, and human tissues and named PLD2 [7][8][9][10]. Mouse PLD2 was found to be insensitive to small GTPases but required PIP2 for maximal activation [8]. In contrast, human PLD2 was found to be sensitive to ARF, although to a lesser extent than hPLD1 [10].…”