Characterization of a Rat Brain Phospholipase D Isozyme

Abstract

We have recently cloned a cDNA encoding a phospholipase D (PLD) from rat brain and named it rPLD1. It shows 90% amino acid identity with the human PLD isoform hPLD1b. We have expressed rPLD1 as a histidine-tagged fusion protein in insect (Sf9) cells using the expression vector pBlueBacHis and purified the recombinant protein to homogeneity by Ni 2؉ -agarose affinity chromatography. Phosphatidylinositol 4,5-P 2 and phosphatidylinositol 3,4,5-P 3 activated the PLD equipotently, but other acidic phospholipids were ineffective. The activity of rPLD1 was dependent on both Mg 2؉ and Ca 2؉ . It was specific for phosphatidylcholine and showed a broad dependence on pH with optimum activity at pH 6.5-7.5. The enzyme was inhibited by oleate and activated by the small G proteins ARF3 and RhoA in the presence of guanosine 5-3-O-(thio)triphosphate. Protein kinase C (PKC)-␣ and -␤II, but not PKC-␥, -␦, -⑀, or -, activated rPLD1 in a manner that was stimulated by phorbol ester but did not require ATP. Neither synergistic interactions between ARF3 and RhoA nor between these G proteins and PKC-␣ or -␤II were observed. Recombinant PKC-␣ and -␤II phosphorylated purified rPLD1 to high stoichiometry in vitro, and the phosphorylated PLD exhibited a mobility shift upon electrophoresis. Phosphorylation of the PLD by PKC was correlated with inhibition of its catalytic activity. rPLD1 bound to concanavalin A-Sepharose beads, and its electrophoretic mobility was altered by treatment with endoglycosidase F. The amount of PLD bound to the beads was decreased in a concentration-dependent manner when tunicamycin was added to the Sf9 expression system. Tunicamycin also decreased membrane localization of rPLD1. These results suggest that rPLD1 is a glycosylated protein and that it is negatively regulated by phosphorylation by PKC in vitro.Phospholipase D type 1 (PLD1) 1 plays an important role in signal transduction in a variety of cells. PLD catalyzes the hydrolysis of phosphatidylcholine (PC), the major phospholipid of membranes, to phosphatidic acid and choline in response to a variety of hormones, neurotransmitters, growth factors, and cytokines (1). Phosphatidic acid (PA), the direct product of PLD action, has been implicated in increased DNA synthesis, activation of protein kinases and a protein-tyrosine phosphatase, stimulation of the respiratory burst in neutrophils, stimulation of c-fos and c-myc transcription, activation of certain enzymes of inositol phospholipid metabolism, and effects on actin polymerization and the GTPase-activating proteins of small G proteins (1-4). PA can be further metabolized by PA phosphohydrolase to yield diacylglycerol and by phospholipase A 2 to form the intercellular messenger lysophosphatidic acid. Diacylglycerol derived from PC via PA can result in prolonged activation of certain PKC isozymes (1, 5). Thus, agonist-induced stimulation of PLD through its activation of PKC could play a role in long term cellular responses such as proliferation and differentiation.Regulation of PLD activity is not fully und...

Involvement of Tyrosine Phosphorylation and Protein Kinase C in the Activation of Phospholipase D by H2O2 in Swiss 3T3 Fibroblasts

“…We have also observed that rPLD1 and PKC␣ are associated in Sf9 cells and that the interaction is enhanced by treatment of the cells with phorbol ester. 2 Furthermore, direct interaction between these proteins is indicated by in vitro findings (25,36). The association of rPLD1 with the PDGF receptor is somewhat surprising, although the fact that it is not enhanced by PDGF is to be expected since this PLD isoform has no recognizable SH2 or PTB domains (24,36).…”

“…We have cloned and characterized a rat brain phospholipase D isozyme (rPLD1) (24,25). In the present study, we have investigated the effect of hydrogen peroxide as a pharmacologic agent on tyrosine phosphorylation and activation of rPLD1 in Swiss 3T3 fibroblasts.…”

A Novel Phosphatidic Acid-selective Phospholipase A1That Produces Lysophosphatidic Acid

“…In mammalian cells, the molecular identities of the two isozymes of PLD, PLD 1 and PLD 2 , have been elucidated. Among these two isozymes, PLD 1 is activated by PMA both in vivo and in vitro through an activation of protein kinase C␣ (39). Although information about PLD isozyme(s) in Sf9 insect cells is limited (40), the observation that PMA stimulated LPA formation in Sf9-mPA-PLA 1 cells (Fig.…”

Association of the N- and C-terminal Domains of Phospholipase D

“…We have cloned a PLD1 isoform from rat brain (rPLD1) and have studied its properties in vivo and in vitro (7,9,26,27). Our previous study of this isoform showed that N-terminal or C-terminal fragments containing only one of the HKD domains did not have PLD activity when expressed in COS7 cells (10).…”