Activation of phospholipase D1 by Cdc42 requires the Rho insert region

Members of the Rho subfamily of GTP-binding proteins are implicated in the regulation of phospholipase D (PLD). In the present study, we demonstrate a physical association between a Rho family member, Cdc42, and PLD1. Binding of Cdc42 to PLD1 and subsequent activation are GTP-dependent. Although binding of Cdc42 to PLD1 does not require geranylgeranylation, activation of PLD1 is dependent on this lipid modification of Cdc42. Specific point mutations in the switch I region of Cdc42 abolish binding to and, therefore, activation of PLD1 by Cdc42. Deletion of the Rho insert region, which consists of residues 120-139, from Cdc42 does not interfere with binding to PLD1 but inhibits Cdc42 stimulated PLD1 activity. Interestingly, deletion of the insert region from Cdc42 also inhibits activation of PLD1 by Arf and protein kinase C. With the lack of specific inhibitors of PLD activity, the insert deletion mutant of Cdc42 (designated (DeltaL8)Cdc42) is a novel reagent for in vitro studies of PLD1 regulation, as well as for in vivo studies of Cdc42-mediated signaling pathways leading to PLD1 activation. Because the insert region is required for the transforming activity of Cdc42, regulation of PLD1 by this region on Cdc42 is of major interest.     

Different regulation of phospholipase D activity in glioma C6 cells by sphingosine, propranolol, imipramine and phorbol ester

In has been found that sphingosine, propranolol, imipramine and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) have a stimulatory effect on phospholipase D activity in glioma C6 cells. The cells were prelabelled with [1-(14)C]palmitic acid and phospholipase D-mediated synthesis of [(14)C]phosphatidylethanol was measured. The enhancing effect of TPA was almost completely blocked by a specific protein kinase C inhibitor, GF 109203X. In contrast, GF 109203X failed to inhibit the sphingosine, imipramine and propranolol stimulatory effects, indicating that their stimulation was independent of protein kinase C. The effect of TPA on phospholipase D was also blocked by imipramine and propranolol, whereas sphingosine additively potentiated TPA-mediated phospholipase D activity, both at shorter and longer (2-60 min) times of incubation. These results suggest that in glioma C6 cells, sphingosine is not only involved in a different phospholipase D activation than the TPA regulatory system, but also that it operates in a different compartment of the cell.     

Differential activation by fMet-Leu-Phe and phorbol ester of a plasma membrane phosphatidylcholine-specific phospholipase D in human neutrophil

Signal transduction involving phosphatidylcholine hydrolysis has been investigated in human neutrophils (PMN) after in situ generation of [3H]alkylacyl-sn-glycero-3-phosphocholine ([3H]alkylacyl-GPC) by cell incubation with [3H]alkylacetyl-GPC. When PMN were stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe(fMLP) or phorbol myristate acetate (PMA) in the presence of cytochalasin B, both 1-O-alkyl-2-acyl-sn-glycero-3-phosphate (PA) and 1-O-alkyl-2-acyl-sn-glycerol (AAG) were generated. On addition of the agonists in the presence of ethanol, phosphatidylethanol (PEt) [corrected] was formed with a concomitant decrease in PA and AAG. These results indicate the presence of a phospholipase D (PLD) acting on phosphatidylcholine in human PMN. The kinetics of hydrolysis were quite different according to the stimulus. Whereas fMLP induced a maximum rise in PA and AAG at 30-45 s, these products began to appear only after 1 min upon cell incubation with PMA. Similar amounts of products were formed at 1 min with fMLP and only at 5 min with PMA. Although similar time courses of PA generation were obtained in the absence of cytochalasin B, AAG were no longer involved and therefore cannot account for intracellular second messenger under physiological conditions. Subcellular distribution studies demonstrated the exclusive location of PA and PEt [corrected] in the plasma membrane. The possible involvement of PA in respiratory burst activation is discussed.     

Localization and regulation of phospholipase D2 by ARF6

Phospholipase D (PLD) and ADP-ribosylation factor 6 (ARF6) have been implicated in vesicular trafficking and rearrangement of the actin cytoskeleton. We have explored the co-localization of rat PLD1b and rat PLD2 with wild type and mutant forms of ARF6 in HeLa cells and studied their activation by ARF6 and the role of the actin cytoskeleton. GFP-tagged PLD1 had a similar pattern to multivesicular and late endosomes and the trans-Golgi apparatus, but not to other organelles. When wild type or dominant negative ARF6 and PLD1 or PLD2 were co-expressed, they had a similar localization in cytosolic particles and at the cell periphery. In contrast, dominant active ARF6 caused cell shrinkage and had a similar localization with PLD1 and PLD2 in dense structures, containing the trans-Golgi apparatus and actin. Disruption of the actin cytoskeleton with cytochalasin D did not induce the formation of these structures. To determine, if ARF6 selectively activated PLD1 or PLD2, wild type and mutant forms of the ARF isoform were transfected together with PLD1 or PLD2. Wild type ARF6 did not affect either PLD isozyme, but dominant active ARF6 selectively activated PLD2 and dominant negative ARF6 selectively inhibited PLD2. In contrast, dominant active ARF1 or Rac1 stimulated both PLD isozymes but the ARF1 effect on PLD2 was very small. Cytochalasin D did not affect the activation of PLD by phorbol ester. The localizations of PLD and ARF6 were also analyzed by fractionation after methyl-beta-cyclodextrin extraction to deplete cholesterol. The results showed that all PLD isoforms and ARF6 mutants existed in the membrane fraction, but only wild type ARF6 was dependent on the presence of cholesterol. These experiments showed that wild type ARF6 had a similar location with PLD isoforms on cell staining, but it did not colocalize with PLD isoforms in fractionation experiments. It is proposed that activated ARF6 translocates to the cholesterol independent microdomain and then activates PLD2 there. It is further concluded that PLD2 is selectively activated by ARF6 in vivo and that disruption of the actin cytoskeleton does not affect this activation.     

Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases.

When activated, ERM (ezrin, radixin, moesin) proteins are recruited to the plasma membrane, with concomitant carboxy-terminal threonine phosphorylation, where they crosslink actin filaments to the plasma membrane to form microvilli (reviewed in [1] [2] [3] [4] [5]). Here, we report that, when NIH3T3 or HeLa cells were transfected with a constitutively active mutant of the small GTPase RhoA (V14RhoA), microvilli were induced and the level of carboxy-terminal threonine-phosphorylated ERM proteins (CPERM) [6] [7] increased approximately 30-fold. This increase was not observed following transfection of constitutively active forms of two other Rho-family GTPases, Rac1 and Cdc42, or of a direct effector of Rho, Rho-kinase (also known as ROKalpha or ROCK-II) [8] [9] [10]. The V14RhoA-induced phosphorylation of ERM proteins was not suppressed by Y-27632, a specific inhibitor of ROCK kinases including Rho-kinase [11]. Overexpression of another direct effector of Rho, phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) type Ialpha [12] [13] [14], but not a kinase-inactive mutant [15], increased approximately sixfold the level of CPERM, and induced microvilli. Together with the previous finding that the PI4P5K product phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates ERM proteins in vitro [16], our data suggest that PIP(2), and not ROCK kinases, is involved in the RhoA-dependent activation of ERM proteins in vivo. The active state of ERM proteins is maintained through threonine phosphorylation by as yet undetermined kinases, leading to microvillus formation.     

Activation of phospholipase D by ras proteins is independent of protein kinase C

Growth factors activate phospholipases, causing the generation of diverse lipid metabolites with second messenger function. Among them, the phosphatidylcholine-preferring phospholipase D (PLD) has attracted great interest, since in addition to the transient activation by growth factors stimulation, it is constitutively activated in some of the src- and ras-transformed cells investigated. To establish further the functional relationship of ras oncogenes with PLD, we have investigated its mechanism of regulation. Growth factors such as PDGF or FGF activate the PC-PLD enzyme by a common, PKC-dependent mechanism. By contrast, ras oncogenes activate the PC-PLD enzyme by a PKC-independent mechanism. These results suggest the existence of at least two mechanisms for PLD activation, and ras oncogenes contribute to one of them. © 1996 Wiley-Liss, Inc.     

Stimulation of beta-amyloid precursor protein alpha-processing by phorbol ester involves calcium and calpain activation

Normal processing of Alzheimer's beta-amyloid precursor protein (APP) is markedly stimulated by phorbol esters, but the underlying mechanisms have yet to be fully understood. In this study, we observed that: (a) Phorbol 12,13-dibutyrate (PDBu)-stimulated APP secretion in cultured SH-SY5Y neuroblastoma and fibroblast cells was blocked by EGTA and calpain inhibitors in a concentration-dependent manner, but not by other protease inhibitors. (b) Secretion of fibronectin, another secretory protein tested for comparison, was enhanced by PDBu, but insensitive to calpain inhibitors. (c) PDBu stimulated intracellular calpain activity as measured by the hydrolysis of a fluorogenic calpain substrate. (d) PDBu also induced rapid proteolysis of two endogenous substrates of calpains, i.e., tau and microtubule-associated protein-2 (MAP-2) and the proteolysis was blocked by EGTA and calpain inhibitors. Taken together, these results suggest that stimulation of APP alpha-processing by PDBu is through a mechanism that involves the activation of Ca(2+) and, most notably, calpain. The implications of the findings are discussed in relation to the regulatory mechanism of APP alpha-processing.     

Enhancement of hormone action by a phorbol ester and anti-tubulin alkaloids involves different mechanisms

The tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhanced 1-isoproterenol and prostaglandin E1 stimulated cyclic AMP formation in clones of mouse myeloid leukemic cells. The enhancement was found up to 3h after TPA treatment and had disappeared after 24h, indicating its reversibility. The effect of TPA was not inhibited by removal of extracellular Ca2+ or pre-treatment with the calcium ionophore A23187. This enhancement by TPA seems to involve a different pathway than enhancement of response to the same hormones after treatment with the anti-tubulin alkaloids colchicine or vinblastine, since a myeloid leukemic cell mutant clone that was non-responsive to the anti-tubulin alkaloids responded to TPA. Furthermore, combined treatment of colchicine-sensitive cells with TPA and colchicine showed an additive stimulating effect. The enhancement of cell response to hormones by TPA was found in myeloid leukemic cell clones that either were or were not induced to differentiate after treatment with TPA. This suggests that enhancement of the effect of these and possibly other hormones by TPA may be an initial step of TPA action, but that this enhancement is not sufficient to induce the wide repertoire of TPA effects including induction of differentiation.     

Activation of N-acylethanolamine-releasing phospholipase D by polyamines

N-acylethanolamines including anandamide (an endogenous ligand of cannabinoid receptors) are biosynthesized from N-acyl-phosphatidylethanolamine (PE) by a phosphodiesterase of the phospholipase D type. The enzyme partially purified from the particulate fraction of rat heart hydrolyzed N-palmitoyl-PE to N-palmitoylethanolamine with a specific activity of 50 nmol/min per mg protein at 37 degrees C in the presence of 10 mM CaCl2. We found that the enzyme was highly activated in dose-dependent manner by polyamines like spermine, spermidine, and putrescine. Spermine was the most potent with an EC50 value around 0.1 mM, and increased the specific enzyme activity 27 fold up to 53 nmol/min per mg protein. However, a synergistic effect of spermine and the known activator (Ca2+ or Triton X-100) was not observed. The spermine-stimulated enzyme was also active with N-arachidonoyl-PE (a precursor of anandamide). Thus, polyamines may function as endogenous activators to control the biosynthesis of anandamide and other N-acylethanolamines.     

PKCalpha is involved in phorbol ester TPA-mediated stabilization of p14ARF

We generated A21-13 cells expressing p14(ARF) in the presence of doxycycline in order to examine the stability of p14(ARF) protein. The effects of proteasome inhibitor MG132 on p14(ARF) protein stabilization were detectable using our experimental procedure. Introduction of mutant p53 did not affect MG132-mediated p14(ARF) protein stabilization. We found that phorbol ester TPA (12-o-tetradecanoyl-phorbol 13-acetate) stabilized p14(ARF) protein and that p53 status had no effect on TPA-mediated stabilization. TPA-mediated stabilization was abolished by staurosporine but not by lovastatin or U0126. We further investigated which isoforms of PKC were involved in TPA-mediated p14(ARF) stabilization using short-interference RNA. Knockdown of PKCalpha, but not PKCdelta, attenuated TPA-mediated p14(ARF) stabilization. These findings suggest that PKCalpha is involved in TPA-mediated stabilization of p14(ARF) protein, and this effect of TPA was not affected by the Ras/MAPK pathway or p53 status. Our results are indicative of a novel role of PKC in p14(ARF) protein stability.     

Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.     

Parathyroid hormone induction of creatine kinase activity and DNA synthesis is mimicked by phospholipase C, diacylglycerol and phorbol ester

Parathyroid hormone (PTH), which increases cAMP levels, also induces an increase in the activity of the brain isozyme of creatine kinase and in DNA synthesis in osteoblast-enriched bone cell cultures by a cAMP-independent mechanism. The following results lead us to the conclusion that PTH induction of brain isozyme of creatine kinase activity and DNA synthesis occurs by activation of membranal phospholipid metabolism leading to increased protein kinase C activity and Ca2+ mobilization, a mechanism demonstrated for several growth factors and other hormones. (1) Binding of membranal phospholipids by agents such as gentamycin or antiphospholipid antibodies abolishes the stimulation by PTH of creatine kinase activity and DNA synthesis but not of cAMP production. (2) Treatment of cell cultures with exogenous phospholipase C increases brain isozyme of creatine kinase activity and DNA synthesis, but not cAMP production; these stimulations are also blocked by serum containing anti-phospholipid antibodies. PTH has no additional effect on stimulation of creatine kinase activity by phospholipase C (and only a slight effect on DNA synthesis). (3) A synthetic diacylglycerol (1-oleyl-2-acetyl glycerol) or phorbol ester (phorbol 12-myristate 13-acetate) or Ca2+ ionophore, A23187 induces creatine kinase activity and DNA synthesis in the cultures. However, this effect is not blocked by antiphospholipid sera and PTH has no additional effect. (4) Inhibition of protein kinase C activity by drugs reported to inhibit the enzyme (retinoic acid, quercetin) abolishes the stimulation of brain isozyme of creatine kinase activity and of DNA synthesis by PTH.     

Activation of phospholipase D1 by ADP-ribosylated RhoA

Clostridium botulinum exoenzyme C3 exclusively ADP-ribosylates RhoA, B, and C to inactivate them, resulting in disaggregation of the actin filaments in intact cells. The ADP-ribose resides at Asn-41 in the effector binding region, leading to the notion that ADP-ribosylation inactivates Rho by blocking coupling of Rho to its downstream effectors. In a recombinant system, however, ADP-ribosylated Rho bound to effector proteins such as phospholipase D-1 (PLD1), Rho-kinase (ROK), and rhotekin. The ADP-ribose rather mediated binding of Rho-GDP to PLD1. ADP-ribosylation of Rho-GDP followed by GTP-gamma-S loading resulted in binding but not in PLD activation. On the other hand, ADP-ribosylation of Rho previously activated by binding to GTP-gamma-S resulted in full PLD activation. This finding indicates that ADP-ribosylation seems to prevent GTP-induced change to the active conformation of switch I, the prerequisite of Rho-PLD interaction. In contrast to recombinant systems, ADP-ribosylation in intact cells results in functional inactivation of Rho, indicating other mechanisms of inactivation than blocking effector coupling.     

Activation of phospholipase D by osmotic cell swelling

In response to osmotic cell swelling, Intestine 407 cells react with a rapid and transient activation of phospholipase D (PLD). To investigate the role of PLD during the regulatory volume decrease, cells were treated with 1-butanol resulting in a depletion of PLD substrates. Activation of volume-regulated anion channels, but not the cell swelling-induced release of taurine, was largely inhibited in the presence of low concentrations of 1-butanol. In addition, hypotonicity-induced exocytosis, ATP release and subsequent endocytosis were found to be largely abrogated. The results support a model of cell volume regulation in which PLD plays an essential role in the cell swelling-induced vesicle cycling and in the activation of volume-sensitive anion channels.     

Activation of protein kinase C by a tumor-promoting phorbol ester in pancreatic islets

Rat pancreatic islet homogenates display protein kinase C activity. This phospholipid-dependent and calcium-sensitive enzyme is activated by diacylglycerol or the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of TPA, the Ka for Ca2+ is close to 5 microM. TPA does not affect phosphoinositide turnover but stimulates [32P]- and [3H]choline-labelling of phosphatidylcholine in intact islets. Exogenous phospholipase C stimulates insulin release, in a sustained and glucose-independent fashion. The secretory response to phospholipase C persists in media deprived of CaCl2. It is proposed that protein kinase C participates in the coupling of stimulus recognition to insulin release evoked by TPA, phospholipase C and, possibly, those secretatogues causing phosphoinositide breakdown in pancreatic islets.     

Elevated phospholipase D activity in H-Ras- but not K-Ras-transformed cells by the synergistic action of RalA and ARF6

Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of H-Ras but not K-Ras. H-Ras and K-Ras have been reported to localize to different membrane microdomains, with H-Ras localizing to caveolin-enriched light membrane fractions. We reported previously that PLD activity elevated in response to mitogenic stimulation is restricted to the caveolin-enriched light membrane fractions. PLD activity in H-Ras-transformed cells is dependent upon RalA, and consistent with a lack of elevated PLD activity in K-Ras-transformed cells, RalA was not activated in K-Ras-transformed cells. Although H-Ras-induced PLD activity is dependent upon RalA, an activated mutant of RalA is not sufficient to elevate PLD activity. We reported previously that RalA interacts with PLD activating ADP ribosylation factor (ARF) proteins. In cells transformed by H-Ras, we found increased coprecipitation of ARF6 with RalA. Moreover, ARF6 colocalized with RalA in light membrane fractions. Interestingly, ARF6 protein levels were elevated in H-Ras- but not K-Ras-transformed cells. A dominant-negative mutant of ARF6 inhibited PLD activity in H-Ras-transformed NIH 3T3 cells. Activated mutants of either ARF6 or RalA were not sufficient to elevate PLD activity in NIH 3T3 cells; however, expression of both activated RalA and activated ARF6 in NIH 3T3 cells led to increased PLD activity. These data suggest a model whereby H-Ras stimulates the activation of both RalA and ARF6, which together lead to the elevation of PLD activity.     

Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin (Standaert, M. L., Farese, R. V., Cooper, R. D., and Pollet, R. J. (1988) J. Biol. Chem. 263, 8696-8705). In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle. Exposure to PLC-Cp activated glycogen phosphorylase and potentiated twitch tension in response to electrical stimulation, providing evidence that PLC-Cp increases cytoplasmic Ca2+ concentration. Dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, completely blocked both the activation of phosphorylase and the stimulation of glucose transport by PLC-Cp. These findings provide evidence that an increase in cytoplasmic Ca2+ concentration is involved in the activation of glucose transport in skeletal muscle by PLC-Cp.     

Activation of cathepsin D by glycine ethyl ester.

Cathepsin D purified from bovine spleen is activated by glycine ethyl ester. A maximum of 70% activation was observed at a glycine ethyl ester concentration of 0.1 M and at pH 4.5. The activation effect appears to be reversible.