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...

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O2 consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease.

Phospholipase D (PLD) has been suggested to play an important role in a variety of cellular functions. PLD activity has been shown to be significantly elevated in many tumours and transformed cells, suggesting the possibility that PLD might be involved in tumorigenesis. In this study, we have established stable cell lines overexpressing PLD1 and PLD2 from fibroblast cells. These cells, but not control cells, showed altered growth properties and anchorage-independent growth in soft agar. Both PLD1 and PLD2 also induced an up-regulation of the activity of matrix metalloprotease-9 as detected by zymograms. Furthermore, both PLD1 and PLD2 transformants, but not vector-transfectants, induced undifferentiated sarcoma when transplanted into nude mice. Both PLD1- and PLD2-mediated cell cycle distributions in stable cell lines revealed an increased fraction of cells in the S phase compared with control cells. Interestingly, the level of cyclin D3 protein, known as an activator of G(1) to S phase transition in the cell cycle, was aberrantly high in cells overexpressing PLD1 and PLD2 compared with control cells. These results suggest that overexpression of PLD isozymes may play an important role in neoplastic transformation.

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