Presence of a phospholipase D (PLD) distinct from PLD1 or PLD2 in human neutrophils: immunobiochemical characterization and initial purification

Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8-8.0 was determined. PIP(2), EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-beta-glucopyranoside, divalent cations, GTPgammaS and H(2)O(2). The apparent K(m) for the butanol substrate was 0.1 mM and the V(max) was 6.0 nmol mg(-1) h(-1). Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of approximately 90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme's regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granulocyte PLD isoform.     

Genome-wide analysis of the phospholipase D family in Oryza sativa and functional characterization of PLDβ1 in seed germination

Phospholipase D （PLD） plays a critical role in plant growth and development, as well as in hormone and stress responses. PLD encoding genes constitute a large gene family that are present in higher plants. There are 12 members of the PLD family in Arabidopsis thaliana and several of them have been functionally characterized; however, the members of the PLD family in Oryza sativa remain to be fully described. Through genome-wide analysis, 17 PLD members found in different chromosomes have been identified in rice. Protein domain structural analysis reveals a novel subfamily, besides the C2-PLDs and PXPH-PLDs, that is present in rice - the SP-PLD. SP-PLD harbors a signal peptide instead of the C2 or PXPH domains at the N-terminus. Expression pattern analysis indicates that most PLD-encoding genes are differentially expressed in various tissues, or are induced by hormones or stress conditions, suggesting the involvement of PLD in multiple developmental processes. Transgenic studies have shown that the suppressed expression office PLDβ1 results in reduced sensitivity to exogenous ABA during seed germination. Further analysis of the expression of ABA signaling-related genes has revealed that PLDβ1 stimulates ABA signaling by activating SAPK, thus repressing GAmyb exoression and inhibiting seed germination.     

Design and synthesis of isoform-selective phospholipase D (PLD) inhibitors. Part I: Impact of alternative halogenated privileged structures for PLD1 specificity

This Letter describes the synthesis and structure–activity-relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of alternative halogenated piperidinyl benzimidazolone privileged structures, in combination with a key (S)-methyl group, novel PLD inhibitors with low nM potency and unprecedented levels of PLD1 isoform selectivity (～1700-fold) over PLD2 were developed.     

Crystal structure of a phospholipase D family member

The first crystal structure of a phospholipase D (PLD) family member has been determined at 2.0 A resolution. The PLD superfamily is defined by a common sequence motif, HxK(x)4D(x)6GSxN, and includes enzymes involved in signal transduction, lipid biosynthesis, endonucleases and open reading frames in pathogenic viruses and bacteria. The crystal structure suggests that residues from two sequence motifs form a single active site. A histidine residue from one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoenzyme intermediate, whereas a histidine residue from the other motif appears to function as a general acid in the cleavage of the phosphodiester bond. The structure suggests that the conserved lysine residues are involved in phosphate binding. Large-scale genomic sequencing revealed that there are many PLD family members. Our results suggest that all of these proteins may possess a common structure and catalytic mechanism.     

Characterization of microsomal and mitochondrial phospholipase D activities and cloning of a phospholipase D alpha cDNA from strawberry fruits.

Phospholipase D alpha (PLD, EC 3.1.4.4)) is a key enzyme involved in membrane deterioration that occurs during fruit ripening and senescence. The biochemical and molecular characteristics of PLD was studied in strawberry (Fragaria ananassa Duch) fruits, which are non-climacteric fruits. PLD activity was primarily associated with the mitochondrial and microsomal fractions and showed increased activity during development. Optimal pH levels of activity were observed at 5.5 and 6.5 for mitochondrial PLD and at 5 and 7 for microsomal PLD. Calcium enhanced microsomal PLD activity at 1-40 microM levels. PLD activity followed Michaelis-Menten kinetics. Lineweaver-Burk analysis gave Km values in the range of 114 and 277 microM using dipalmitoylphosphatidylcholine (DPPC) as substrate for mitochondrial and microsomal PLD, respectively. The Vmax value for the microsomal PLD was nearly 12-fold higher than that of mitochondrial PLD. A 2874 bp full-length cDNA for PLD alpha was amplified from strawberry fruit mRNA using RT-PCR and 5'- and 3'-RACE encoding an 810 amino acid-polypeptide. The predicted strawberry PLD sequence showed the characteristic C2 domain and the phospholipase domains conferring calcium sensitivity and the enzyme activity, respectively. The strawberry PLD alpha showed a high degree of similarity to other PLD alphas from plants. The implications of PLD regulation during ripening of fruits are discussed.     

Characterization of functional domains of human EB1 family proteins

EB1 family proteins are evolutionarily conserved proteins that bind microtubule plus-ends and centrosomes and regulate the dynamics and organization of microtubules. Human EB1 family proteins, which include EB1, EBF3, and RP1, also associate with the tumor suppressor protein adenomatous polyposis coli (APC) and p150glued, a component of the dynactin complex. The structural basis for interaction between human EB1 family proteins and their associated proteins has not been defined in detail. EB1 family proteins have a calponin homology (CH) domain at their N terminus and an EB1-like C-terminal motif at their C terminus; the functional importance of these domains has not been determined. To better understand functions of human EB1 family proteins and to reveal functional similarities and differences among these proteins, we performed detailed characterizations of interactions between human EB1 family proteins and their associated proteins. We show that amino acids 1-133 of EB1 and EBF3 and the corresponding region of RP1, which contain a CH domain, are necessary and sufficient for binding microtubules, thus demonstrating for the first time that a CH domain contributes to binding microtubules. EB1 family proteins use overlapping but different regions that contain the EB1-like C-terminal motif to associate with APC and p150glued. Neither APC nor p150glued binding domain is necessary for EB1 or EBF3 to induce microtubule bundling, which requires amino acids 1-181 and 1-185 of EB1 and EBF3, respectively. We also determined that the EB1 family protein-binding regions are amino acids 2781-2820 and 18-111 of APC and p150glued, respectively.     

Overexpression of protein kinase C-epsilon and its regulatory domains in fibroblasts inhibits phorbol ester-induced phospholipase D activity

In fibroblasts, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) stimulates phospholipase D (PLD)-mediated hydrolysis of both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) by PKC-alpha-mediated nonphosphorylating and phosphorylating mechanisms. Here we have used NIH 3T3 fibroblasts overexpressing holo PKC-epsilon and its regulatory, catalytic, and zinc finger domain fragments to determine if this isozyme also regulates PLD activity. Overexpression of holo PKC-epsilon inhibited the stimulatory effects of PMA (5-100 nM) on both PtdCho and PtdEtn hydrolysis. Overexpression of PKC-epsilon also was found to inhibit platelet-derived growth factor-induced PLD activity. Expression of the catalytic unit of PKC-epsilon had no effect on PMA-induced PLD activity. In contrast, expression of both the regulatory domain fragment and the zinc finger domain of PKC-epsilon resulted in significant inhibition of PMA-stimulated PtdCho and PtdEtn hydrolysis. Interestingly, although PKC-alpha also mediates the stimulatory effect of PMA on the synthesis of PtdCho by a phosphorylation mechanism, overexpression of holo PKC-epsilon or its regulatory domain fragments did not affect PMA-induced PtdCho synthesis. These results indicate that the PKC-epsilon system can act as a negative regulator of PLD activity and that this inhibition is mediated by its regulatory domain.     

The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs

A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia. WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation. However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP, we still lack the exact nature of its binding site. We have now identified and characterized the N-WASP WH1 binding motif in WIP in vitro and in vivo using Shigella and vaccinia systems. The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain, binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand. Interaction of the WH1 domain of N-WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif. The WH1 binding motif we have identified is conserved in WIP, CR16, WICH, and yeast verprolin.     

Purification and characterization of canine myocardial cytosolic phospholipase A2. A calcium-independent phospholipase with absolute f1-2 regiospecificity for diradyl glycerophospholipids

Recently, we identified a novel calcium-independent, plasmalogen-selective phospholipase A2 activity in canine myocardial cytosol which represents the major measurable phospholipase A2 activity in myocardial homogenates (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303). We now report the 154,000-fold purification of this phospholipase A2 to homogeneity through utilization of sequential anion exchange, chromatofocusing, affinity, Mono Q, and hydroxylapatite chromatographies. The purified enzyme had a molecular mass of 40 kDa, possessed a specific activity of 227 mumol/mg min, had a pH optimum of 6.4, and catalyzed the regiospecific cleavage of the sn-2 fatty acid from diradyl glycerophospholipids. The purified polypeptide was remarkable for its ability to selectively hydrolyze plasmenylcholine in homogeneous vesicles (subclass rank order: plasmenylcholine greater than alkyl-ether choline glycerophospholipid greater than phosphatidylcholine) as well as in mixed bilayers comprised of equimolar plasmenylcholine/phosphatidylcholine. Purified myocardial phospholipase A2 also possessed selectivity for hydrolysis of phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic or palmitic acid. Taken together, these results constitute the first purification of a calcium-independent phospholipase with absolute regiospecificity for cleavage of the sn-2 acyl linkage in diradyl glycerophospholipids and demonstrate that myocardial phospholipase A2 has kinetic characteristics which are anticipated to result in the selective hydrolysis of sarcolemmal phospholipids during myocardial ischemia.     

Quantitative profiling of Arabidopsis polar glycerolipids in response to phosphorus starvation. Roles of phospholipases D zeta1 and D zeta2 in phosphatidylcholine hydrolysis and digalactosyldiacylglycerol accumulation in phosphorus-starved plants

Phosphorus is an essential macronutrient that often limits plant growth and development. Under phosphorus-limited conditions, plants undergo substantial alterations in membrane lipid composition to cope with phosphorus deficiency. To characterize the changes in lipid species and to identify enzymes involved in plant response to phosphorus starvation, 140 molecular species of polar glycerolipids were quantitatively profiled in rosettes and roots of wild-type Arabidopsis (Arabidopsis thaliana) and phospholipase D knockout mutants pld zeta1, pld zeta2, and pld zeta1 pld zeta2. In response to phosphorus starvation, the concentration of phospholipids was decreased and that of galactolipids was increased. Phospholipid lost in phosphorus-starved Arabidopsis rosettes was replaced by an equal amount of galactolipid. The concentration of phospholipid lost in roots was much greater than in rosettes. Disruption of both PLD zeta1 and PLD zeta2 function resulted in a smaller decrease in phosphatidylcholine and a smaller increase in digalactosyldiacylglycerol in phosphorus-starved roots. The results suggest that hydrolysis of phosphatidylcholine by PLD zetas during phosphorus starvation contributes to the supply of inorganic phosphorus for cell metabolism and diacylglycerol moieties for galactolipid synthesis.     

1-Butanol interferes with phospholipase D1 and protein kinase Calpha association and inhibits phospholipase D1 basal activity

1-Butanol is commonly used as a substrate for phospholipase D (PLD) activity measurement. Surprisingly we found that, in the presence of 30 mM 1-butanol (standard PLD assay conditions), PLD1 activity in COS-7 cells was lost after incubation for 2 min. In contrast, in the presence of the protein kinase C (PKC) inhibitor staurosporine or dominant negative PKCalpha D481E, the activity was sustained for at least 30min. The binding between PLD1 and PKCalpha was also lost after 2 min incubation with 30 mM 1-butanol while staurosporine and D481E maintained the binding. 1-Butanol at 2 mM did not inhibit PLD1 basal activity or PLD1 binding to PKCalpha, and staurosporine and PKCalpha D481E produced a constant increase in PLD1 basal activity of 2-fold. These results indicate that 1-butanol is inhibitory to PLD1 activity by reducing its association with PKCalpha, and that the concentration of 1-butanol is an important consideration in assaying basal PLD1 activity.     

Characterization of interactions of Na+/H+ exchanger regulatory factor-1 with the parathyroid hormone receptor and phospholipase C.

The Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) is a molecular scaffold important for the signaling of the G-protein coupled receptor for the parathyroid hormone (PTH1R). The two PDZ (PSD-95, Discs-large, ZO1) domains of NHERF1 through association with the C-termini of PTH1R and phospholipase C enhance the signaling pathway associated with PTH. To examine these interactions, we have produced the individual PDZ1 and PDZ2 domains as well as the tandem PDZ1-PDZ2 domains (PDZ12) of NHERF1 and have characterized the binding affinities of the C-terminal motifs of PTH1R and PLCbeta using fluorescence anisotropy. Circular dichroism indicates that the PDZ1 and PDZ2 are properly folded. Based on fluorescence anisotropy we find that the C-terminus of PTH1R, containing ETVM, has similar affinities (approximately 10 microm) for both PDZ1 and PDZ2. The PTH1R displayed reduced binding affinity for the tandem PDZ12 (16 microm) compared with the individual domains or a solution of equal molar concentrations of PDZ1 and PDZ2 (5.8 microm), suggesting negative cooperativity between the PDZ domains or intervening region. The C-termini of PLCbeta (both beta1 and beta2 isozymes were examined, containing DTPL and ESRL, respectively) displayed a diminished affinity for PDZ2 (approximately 30 microm) over that of PDZ1 (approximately 8 microm). Finally, we demonstrate trans PDZ1-PDZ2 association that is enhanced in the presence of the C-terminus of PTH1R or PLCbeta, suggesting oligomerization of NHERF as a mode for enhancing the signaling associated with PTH.