Regulation of phospholipase D

Structural studies of plant and bacterial members of the phospholipase D (PLD) superfamily are providing information about the role of the conserved HKD domains in the structure of the catalytic center and the catalytic mechanism of mammalian PLD isozymes (PLD1 and PLD2). Mutagenesis and sequence comparison studies have also defined the presence of pleckstrin homology and phox homology domains in the N-terminus and have demonstrated that a conserved sequence at the C-terminus is required for catalysis. The N- and C-terminal regions of PLD1 also contain interaction sites for protein kinase C, which can directly activate the enzyme through a non-phosphorylating mechanism. Small G proteins of the Rho and ADP-ribosylation factor families also directly regulate the enzyme, with RhoA binding to a sequence in the C-terminus. Certain tyrosine kinases and members of the Ras subfamily of small G proteins can activate the enzyme, but the mechanisms appear to be indirect. The mechanisms by which agonists activate PLD in vivo probably involve multiple pathways.     

Regulation of phospholipase D by tyrosine kinases

Activation of phospholipase D (PLD) represents part of an important signalling pathway in mammalian cells, Phospholipase D catalyzed hydrolysis of phospholipids generates phosphatidic acid (PA) which is subsequently metabolized to lyso-PA (LPA) or diacylglycerol (DAG). While DAG is an endogenous activator of protein kinase C (PKC), PA and LPA have been recognized as second messengers as well, Activation of PLD in response to an external stimulus may involve PKC, Ca²⁺, G-proteins and/or tyrosine kinases. In this review, we will address the role of protein tyrosine phosphorylation in growth factor-, agonist- and oxidant-mediated activation of PLD. Furthermore, a possible link between PKC, Ca²⁺, G-proteins and tyrosine kinases is discussed to indicate the complexity involved in the regulation of PLD in mammalian cells.     

Regulation of phospholipase D by protein kinase C

In nearly all mammalian cells and tissues examined, protein kinase C (PKC) has been shown to serve as a major regulator of a phosphatidylcholine-specific phospholipase D (PLD) activity, At least 12 distinct isoforms of PKC have been described so far; of these enzymes only the α- and β-isoform were found to regulate PLD activity, While the mechanism of this regulation has remained unknown, available evidence suggests that both phosphorylating and non-phosphorylating mechanisms may be involved. A phosphatidylcholine-specific PLD activity was recently purified from pig lung, but its possible regulation by PKC has not been reported yet. Several cell types and tissues appear to express additional forms of PLD which can hydrolyze either phosphatidylethanolamine or phosphatidylinositol. It has also been reported that at least one form of PLD can be activated by oncogenes, but not by PKC activators, Similar to activated PKC, some of the primary and secondary products of PLD-mediated phospholipid hydrolysis, including phosphatidic acid, 1,2-diacylglycerol, choline phosphate and ethanolamine, also exhibit mitogenic/co-mitogenic effects in cultured cells. Furthermore, both the PLD and PKC systems have been implicated in the regulation of vesicle transport and exocytosis. Recently the PLD enzyme has been cloned and the tools of molecular biology to study its biological roles will soon be available. Using specific inhibitors of growth regulating signals and vesicle transport, so far no convincing evidence has been reported to support the role of PLD in the mediation of any of the above cellular effects of activated PKC.     

ARF-regulated phospholipase D: a potential role in membrane traffic

Phospholipase D activity is stimulated rapidly upon occupation of cell-surface receptors. One of the intracellular regulators of phospholipase D activity has been identified as ADP ribosylation factor (ARF). ARF is a small GTP binding protein whose function has been elucidated in vesicular traffic. This review puts into context the connection between the two fields of signal transduction and vesicular transport.     

Phospholipase D in cellular senescence

Cellular senescence appears to be an important part of organismal aging. Cellular senescence is characterized by flattened enlarged morphology, inhibition of DNA replication in response to growth factors, inability to phosphorylate the pRb tumor suppressor protein, inability to produce c-fos or AP-1 and overexpression of a variety of genes, notably p21 (CIP-1/WAF-1) and p16^INK. It is now clear that certain early mitotic signals become defective with the onset of senescence. Among these is the PLD/PKC pathway. Evidence suggests that activation of PLD and PKC is critical for mitogenesis. Recent data suggest that the defect in PLD/PKC in cellular senescence is a result of elevated cellular ceramide levels which inhibit PLD activation. It appears that the elevated ceramide is a result of neutral sphingomyelinase activation. Ceramide acts to inhibit the activation of PLD by possibly three mechanisms, inhibiting activation by Rho, translocation to the membrane and gene expression. Addition of ceramide to young cells not only inhibits PLD but also recapitulates all the standard measures of cellular senescence as described above.     

Sites on phospholipase D2 phosphorylated by PKCalpha

The phosphorylation sites in phospholipase D2 (PLD2) induced by activation of protein kinase Calpha (PKCalpha) in COS 7 cells were analyzed by mass spectrometry. Ser134, 146, and 243, and Thr72, 99/100, and 252 were identified. These sites were mutated to Ala and the double mutation of Ser243 and Thr252 eliminated the phosphorylation. However, the PLD2 activity, and the binding between PKCalpha and PLD2 were unaffected by the mutations. We conclude that phosphorylation of these residues is not required for PLD2 activation by PKCalpha, and that protein-protein interaction between PLD2 and PKCalpha is sufficient to activate PLD2.     

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.     

Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity

Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [³H]phosphatidic acid ([³H]PA) and [³H]phosphatidylethanol ([³H]PEth) after labeling the muscle strips with [³H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [³H]PA and [³H]PEth in a concentration-dependent manner (>105–400% vs. controls in the presence of 10^–6 to 10^–4 M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10^–6 M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10^–8 to 10^–6 M) and GFX (10^–8 to 10^–6 M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.     

Role of phospholipase D in the activation of protein kinase D by lysophosphatidic acid

Protein kinase D was auto-phosphorylated at Ser916 and trans-phosphorylated at Ser744/Ser748 in Rat-2 fibroblasts treated with lysophosphatidic acid. Both phosphorylations were inhibited by 1-butanol, which blocks phosphatidic acid formation by phospholipase D. The phosphorylations were also reduced in Rat-2 clones with decreased phospholipase D activity. Platelet-derived growth factor-induced protein kinase D phosphorylation showed a similar requirement for phospholipase D, but that induced by 4beta-phorbol 12 myristate 13-acetate did not. Propranolol an inhibitor of diacylglycerol formation from phosphatidic acid blocked the phosphorylation of protein kinase D, whereas dioctanoylglycerol induced it. The temporal pattern of auto-phosphorylation of protein kinase D closely resembled that of phospholipase D activation and preceded the trans-phosphorylation by protein kinase C. These results suggest that protein kinase D is activated by lysophosphatidic acid through sequential phosphorylation and that diacylglycerol produced by PLD via phosphatidic acid is required for the autophosphorylation that occurs prior to protein kinase C-mediated phosphorylation.     

A point mutation at phenylalanine 663 abolishes protein kinase C alpha's ability to translocate to the perinuclear region and activate phospholipase D1

Previous research showed that protein kinase C alpha (PKC alpha) translocated to the perinuclear region and activated phospholipase D1, but the mechanism involved was not clear. Here, we provide evidence that Phe 663 (the 10th amino acid from C-terminus) of PKC alpha is essential for its translocation. A point mutation (F663D) completely blocked PKC alpha's binding to and activation of phospholipase D1. Further studies showed that deletion of the C-terminal nine amino acids of PKC alpha did not alter its translocation to the perinuclear region but deletion of the C-terminal 10 amino acids and the F663D mutation abolished this translocation. The F663D mutant was found to be resistant to dephosphorylation, which might account for its inability to translocate to the perinuclear region and activate PLD1, since dephosphorylation of PKC alpha is required for its relocation from plasma membrane to the perinuclear region.     

Tyrosine kinase regulates phospholipase D activation at a point downstream from protein kinase C in osteoblast-like cells

It has recently been shown that the activation of protein kinase C (PKC) induces protein tyrosine phosphorylation in osteoblast-like MC3T3-E1 cells. We previously reported that the activation of PKC stimulates phosphatidylcholine-hydrolyzing phospholipase D in these cells. In this study, we examined whether protein tyrosine kinase is involved in the PKC-induced activation of phospholipase D in MC3T3-E1 cells. Genistein, an inhibitor of protein tyrosine kinases, which by itself had little effect on choline formation, significantly suppressed the formation of choline induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, in a dose-dependent manner. Tyrphostin, an inhibitor of protein tyrosine kinases chemically distinct from genistein, also dose-dependently suppressed the TPA-induced formation of choline. Sodium orthovandate, an inhibitor of protein tyrosine phosphatases, significantly enhanced the TPA-induced formation of choline in a dose-dependent manner. These results strongly suggest that protein tyrosine kinase regulates phospholipase D activity at a point downstream from PKC in osteoblast-like cells.     

Basic fibroblast growth factor stimulates phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like cells

We examined the effect of basic fibroblast growth factor (bFGF) on the activation of phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. bFGF stimulated both the formations of choline (EC₅₀ was 30 ng/ml) and inositol phosphates (EC₅₀ was 10 ng/ml). Calphostin C, an inhibitor of protein kinase C (PKC), had little effect on the bFGF-induced formation of choline. bFGF stimulated the formation of choline also in PKC down regulated cells. Genistein and methyl 2,5-dihydroxycinnamate, inhibitors of protein tyrosine kinases, significantly suppressed the bFGF-induced formation of choline. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, enhanced the bFGF-induced formation of choline. In vitro kinase assay for FGF receptors revealed that FGF receptor 1 and 2 were autophosphorylated after FGF stimulation. bFGF dose-dependently stimulated DNA synthesis of these cells. These results strongly suggest that bFGF activates phosphatidylcholine-hydrolyzing phospholipase D through the activation of tyrosine kinase, but independently of PKC activated by phosphoinositide hydrolysis in osteoblast-like cells. © 1996 Wiley-Liss, Inc.     

Regulation of phospholipase D by phosphorylation-dependent mechanisms

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4,5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Cα as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.     

鼠重组磷脂酶D2腺病毒的构建及功能的初步研究

将磷脂酰胆碱专一性磷脂酶D2基因及其功能缺陷点突变基因 (K75 8R)从真核表达载体pCGN中克隆至带有绿色荧光标记蛋白的穿梭质粒pAdTrack CMV中 ;再与腺病毒骨架载体一起在大肠杆菌BJ5183中进行同源重组 ,成功构建磷脂酶D2重组腺病毒。该病毒颗粒感染人胚肾 2 93细胞 ,高效表达磷脂酶D2及其功能缺陷蛋白。这种表达对M3乙酰胆碱受体介导的细胞内磷脂酶D激活无影响。但磷脂酶D2功能缺陷蛋白对蛋白激酶C介导的胞内磷脂酶D激活有显著抑制作用 ;相反 ,磷脂酶D2蛋白有显著增强作用。结果表明     

Ceramide does not inhibit protein kinase C β-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) βI and βII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC β-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.     

Dual regulation of phospholipase D1 by protein kinase C alpha in vivo

The regulation of phospholipase D1 (PLD1), which has been shown to be activated by protein kinase C (PKC) alpha, was investigated in the human melanoma cell lines. In G361 cell line, which lacks PKCalpha, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD activation was potentiated by introducing PKCalpha by the adenovirus vector. The kinase-negative PKCalpha elevated TPA-induced PLD activity less significantly than the wild type. A PKC specific inhibitor GF109203X lowered PLD activation in the cells expressing PKCalpha, but did not prevent PLD potentiation induced by the kinase-negative PKCalpha. Expression of PKCbetaII and the kinase-negative PKCbetaII enhanced TPA-stimulated PLD activity moderately in MeWo cell line, in which PKCbetaII is absent. Furthermore, the TPA treatment increased the association of PKCalpha, PKCbetaII, and their kinase-negative mutants with PLD1 in melanoma cells. These results indicate that PLD1 is dually regulated through phosphorylation as well as through the protein-protein interaction by PKCalpha, and probably by PKCbetaII, in vivo.     

The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.     

Phosphorylation-dependent phospholipase D activity of matrix vesicles

A progressive hydrolysis of phospholipids was observed during the mineralization process mediated by extracellular matrix vesicles. Increasing levels of different hydrolysis products revealed phospholipase A and D activities. The importance of these enzymes for the mineralization process lies in a high rate of hydrolysis of neutral phospholipids and lower rate of degradation of anionic phospholipids, which may favor mineral formation in vesicular membrane and membrane breakdown necessary for the release of mineral deposits into extracellular matrix. In this report, we focus on the phosphorylation-dependent phospholipase D activity during mineral formation initiated by chicken embryo matrix vesicles.     

蛋白激酶和D—鞘氨醇对人肝癌细胞磷脂酶D活力的调节

为了研究蛋白激酶Ｃ（ＰＫＣ）和酪氨酸激酶（ＴＰＫ）对７７２１人肝癌细胞中磷脂酰胆碱（ＰＣ０专一性磷脂酶Ｄ（ＰＬＤ）的调节,测定了各种ＰＫＣ和ＴＰＫ抑制剂和ＰＫＣ抗体对该细胞中ＰＬＤ活力的影响。结果发现：４种ＰＫＣ抑制剂Ｃｈｅｌｅｒｙｔｈｒｉｎｅ,Ｈ－７,ＣａｌｐｈｏｓｔｉｎＣ和星形孢菌素（Ｓｔａｕｒｏｓｐｏｒｉｎｅ）,以及２种ＴＰＫ抑制剂Ｔｙｒｐｈｏｓｔｉｎ４６和木质异黄酮（Ｇｅｎｉｓｔｅｉｎ）ｆ     

Endothelin Stimulates Phospholipase D in Striatal Astrocytes

Abstract: In primary cultures of mouse striatal astrocytes prelabeled with [³H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [³H]phosphatidic acid and [³H]diacylglycerol. In the presence of ethanol, a production of [³H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC₅₀ = 2–5 n M ). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a G_i/G_o protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [³H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally depended on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.