Dual requirement for rho and protein kinase C in direct activation of phospholipase D1 through G protein-coupled receptor signaling

G protein-coupled and tyrosine kinase receptor activation of phospholipase D1 (PLD1) play key roles in agonist-stimulated cellular responses such as regulated exocytosis, actin stress fiber formation, and alterations in cell morphology and motility. Protein Kinase C, ADP-ribosylation factor (ARF), and Rho family members activate PLD1 in vitro; however, the actions of the stimulators on PLD1 in vivo have been proposed to take place through indirect pathways. We have used the yeast split-hybrid system to generate PLD1 alleles that fail to bind to or to be activated by RhoA but that retain wild-type responses to ARF and PKC. These alleles then were employed in combination with alleles unresponsive to PKC or to both stimulators to examine the activation of PLD1 by G protein-coupled receptors. Our results demonstrate that direct stimulation of PLD1 in vivo by RhoA (and by PKC) is critical for significant PLD1 activation but that PLD1 subcellular localization and regulated phosphorylation occur independently of these stimulatory pathways.     

Evidence for protein kinase C independent activation of phospholipase D by phorbol esters in lymphocytes

Recently it was reported that tumor-promoting phorbol esters stimulate the production of phosphatidylethanol (PEt) in lymphocytes through the activation of phospholipase D (PLD). However, it remains unclear whether this activation is mediated through protein kinase (PKC). The study reported here shows that tumor promoters 12-0-tetradecanoylphorbol-13-acetate (TPA), phorbol dibutyrate (PDBU), 12-deoxyphorbol-13-phenylacetate (DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPPA) and mezerin activated PLD, as measured by the formation of PEt, whereas Concanavalin A (ConA) had no effect. Inhibitors of PKC, sphingosine (2 x 10(-6) M - 5 x 10(-6) M), H-7, HA1004 (5 x 10(-7) - 5 x 10(-6) M) and K252a (1 x 10(-7) - 1 x 10(-6) M) failed to block the PEt synthesis induced by TPA. In fact, sphingosine increased it. Other PKC activators, 1-oleoyl-2-acetylglycerol (OAG) and dioctanoylglycerol (DiC8) had no effect on lymphocyte PLD activity. Analysis of the phospholipid contents after stimulation by TPA showed that only phosphatidylcholine (PC) was significantly decreased. Interestingly, TPA activated PLD in intact cells but not in lysates or subcellular fractions. These observations suggest that stimulation of PLD-catalyzed PEt synthesis by TPA is not solely mediated through PKC activation.     

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.     

AHNAK-mediated activation of phospholipase C-gamma1 through protein kinase C

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by the central repeated units (CRUs) of the AHNAK protein in the presence of arachidonic acid. Here we demonstrate that four central repeated units (4 CRUs) of AHNAK act as a scaffolding motif networking PLC-gamma and PKC-alpha. Specifically, 4 CRUs of AHNAK bind and activate PKC-alpha, which in turn stimulates the release of arachidonic acid near where PLC-gamma1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-gamma and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-gamma activity. Stimulation of NIH3T3 cells expressing 4 CRUs of AHNAK with phorbol 12-myristate 13-acetate resulted in the increased generation of total inositol phosphates (IP(T)) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IP(T) was completely blocked in NIH3T3 cells depleted of PLC-gamma1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-beta isozyme resulting in the generation of a monophasic IP(T) within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IP(T) in NIH3T3 cells expressing 4 CRUs of AHNAK. The secondary activation of PLC is likely because of the scaffolding activity of AHNAK, which is consistent with the role of 4 CRUs as a molecular linker between PLC-gamma and PKC-alpha.     

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.     

Amplification of diacylglycerol activation of protein kinase C by cholesterol

The combined effects of cholesterol, a major cell membrane component, and the lipid second messenger diacylglycerol on the activity of protein kinase C (PK-C) and the structure of phosphatidylcholine/phosphatidylserine bilayers were investigated using specific PK-C assays and ²H NMR. Whereas the classical activation of PK-C was observed as an effect of diacylglycerol, in the absence of this second messenger, cholesterol did not affect PK-C activity. A novel effect of amplified PK-C activation was observed in the presence of both cholesterol and diacylglycerol concentrations within the physiological range of each of these components. ²H NMR results suggest that this phenomenon is due to cholesterol- and diacylglycerol-induced increased propensity of the lipids to adopt nonbilayer phases, effectively destabilizing the bilayer structure. The magnitude of the effect was a function of cholesterol concentration, implying that laterally separated cell membrane domains with distinct cholesterol concentrations have the capacity to differ in their sensitivity to extracellular stimuli.     

Antigen-induced phospholipase D activation in rat mast cells is independent of protein kinase C

In the present study, we investigated the involvement of protein kinase C (PKC) in antigen (Ag, DNP-Ascaris suum)-induced phospholipase D (PLD) activation of rat peritoneal mast cells. Phorbor myristate acetate (PMA) as well as Ag activated PLD as inferred by phosphatidylethanol (PEt) production. PKC inhibitors, staurosporine and H-7, however, failed to suppress PMA-stimulated PLD activation, suggesting that PLD activation by PMA is independent of PKC. By contrast, Ag-stimulated PLD activity was significantly reduced by staurosporine and slightly by H-7. Surprisingly, the inhibitors inhibited Ag-stimulated phospholipase C (PLC), correlated to the inhibition of PLD. These observations lead us to conclude that in Ag-stimulated mast cells 1,2-diacylglycerol (DG) formed by PLC directly or indirectly stimulates PLD, independently of PKC.     

Further studies on the specificity of diacylglycerol for protein kinase C activation

Specificity of 1,2-diacylglycerol for the activation of protein kinase C was investigated with various synthetic products. 1-Stearoyl-2-arachidonylglycerol, a major species of diacylglycerol derived from the receptor-mediated hydrolysis of inositol phospholipids, was most active, but many other diacylglycerols having naturally occurring fatty acids were almost equally active in this role. Hormone-sensitive lipase could produce potentially active diacylglycerols during lipolysis. The lack of the specificity may be reconciled with the possibility that the stearoyl-arachidonyl species is the diacylglycerol with which protein kinase C indeed comes in contact in the membrane when the receptor is stimulated, and that diacylglycerols from other sources are produced in distinct compartments and are not intercalated into the phospholipid bilayer.     

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.     

Dual regulation of sphingosine 1-phosphate-induced phospholipase D activity through RhoA and protein kinase C-alpha in C2C12 myoblasts

Previous studies showed that in C2C12 cells, phospholipase D (PLD) and its known regulators, RhoA and protein kinase Calpha (PKCalpha), were downstream effectors in sphingosine 1-phosphate (SPP) signalling. Moreover, the role of PKC for SPP-mediated PLD activation and the requirement of PKCalpha for RhoA translocation were reported. The present results demonstrated that inactivation of RhoA, by overexpression of RhoGDP dissociation inhibitor (RhoGDI) as well as treatment with C3 exotoxin, attenuated SPP-stimulated PLD activity, supporting the involvement of RhoA in the stimulation of PLD activity by the bioactive lipid in C2C12 myoblasts. In addition, the effect of PKCalpha inhibitor G?6976 on the SPP-induced PLD activation in myoblasts, where RhoA function was inactivated, was consistent with a dual regulation of the enzyme through RhoA and PKCalpha. Interestingly, the subcellular distribution of PLD isoforms, RhoA and PKCalpha, in SPP-stimulated cells supported the view that the functional relationship between the two PLD regulators, demonstrated to occur in SPP signalling, represents a novel mechanism of regulation of specifically localized PLD.     

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.     

Fatty acid activation of protein kinase C: dependence on diacylglycerol

The kinetics of activation of protein kinase C by oleic acid have been reinvestigated, using highly purified preparations of the rat brain and bovine spleen enzymes. Activation of both enzymes by oleic acid is enhanced dramatically by diolein, contrary to previous reports. In the presence of 9.7 microM diolein, the concentrations of oleic acid required for half-maximal activation are 5 microM and 9 microM for the rat brain and bovine spleen enzymes respectively, indicating that the system is much more sensitive to activation by fatty acids than previously recognized. Both enzymes also exhibit a pronounced lag in the activation at low concentrations of oleic acid. The kinetics of activation are very similar to those reported by Hannun et al. (J. Biol. Chem 260, 10039-10043), who characterized the activation of the rat brain enzyme by mixed micelles containing Triton X-100, phosphatidylserine and diolein.     

Membrane translocation of protein kinase Ctheta during T lymphocyte activation requires phospholipase C-gamma-generated diacylglycerol

Protein kinase C (PKC) is the only PKC isoform recruited to the immunological synapse after T cell receptor stimulation, suggesting that its activation mechanism differs from that of the other isoforms. Previous studies have suggested that this selective PKC recruitment may operate via a Vav-regulated, cytoskeletal-dependent mechanism, independent of the classical phospholipase C/diacylglycerol pathway. Here, we demonstrate that, together with tyrosine phosphorylation of PKC in the regulatory domain, binding of phospholipase C-dependent diacylglycerol is required for PKC recruitment to the T cell synapse. In addition, we demonstrate that diacylglycerol kinase alpha-dependent diacylglycerol phosphorylation provides the negative signal required for PKC inactivation, ensuring fine control of the T cell activation response.     

Phenobarbital competes with diacylglycerol for protein kinase C

Phenobarbital inhibits protein kinase C of rat brain by competitively displacing the effector of the enzyme, diacylglycerol. The drug appears to occupy the triple hydrogen bonding site which bonds diacylglycerol - and also phorbol esters - to the enzyme. It remains to be seen if the effect is responsible for the pharmaceutical activity of the drug; even so, it provides an example of a restructuring of lipid-protein hydrogen bonding, in the hydrogen belt of the membrane, in a manner postulated as a mechanism of anesthesia.     

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.     

An essential role for phospholipase D in the activation of protein kinase C and degranulation in mast cells

Activation of phospholipase D (PLD) and protein kinase C (PKC) as well as calcium mobilization are essential signals for degranulation of mast cells. However, the exact role of PLD in degranulation remains undefined. In this study we have tested the hypothesis that the PLD product, phosphatidic acid, and diacylglycerides generated therefrom might promote activation of PKC. Studies were conducted in two rodent mast cell lines that were stimulated with Ag via FcepsilonRI and a pharmacologic agent, thapsigargin. Diversion of production of phosphatidic acid to phosphatidylbutanol (the transphosphatidylation reaction) by addition of l-butanol suppressed both the translocation of diacylglyceride-dependent isoforms of PKC to the membrane and degranulation. Tertiary-butanol, which is not a substrate for the transphosphatidylation, had a minimal effect on PKC translocation and degranulation, and 1-butanol itself had no effect on PKC translocation when PKC was stimulated directly with phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Also, in cells transfected with small inhibitory RNAs directed against PLD1 and PLD2, activation of PLD, generation of diacylglycerides, translocation of PKC, and degranulation were all suppressed. Phorbol ester, which did not stimulate degranulation by itself, restored degranulation when used in combination with thapsigargin whether PLD function was disrupted with 1-butanol or the small inhibitory RNAs. However, degranulation was not restored when cells were costimulated with Ag and phorbol ester. These results suggested that the production of phosphatidic acid by PLD facilitates activation of PKC and, in turn, degranulation, although additional PLD-dependent processes appear to be critical for Ag-mediated degranulation.     

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C.

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.     

IFN-gamma induces a phospholipase D dependent triphasic activation of protein kinase C in endothelial cells.

The IFN-gamma linked PKC activation in endothelial cells was analysed. It was shown that IFN-gamma activates PKC in three transient and separate cycles within the first 60 minutes after IFN-gamma stimulation. Before each PKC activation there was an increase in DAG level. IP3, phosphocholine and choline productions were measured to determine the origin of DAG. Neither of the PLC products, IP3 or phosphocholine, were released after IFN-gamma stimulation. On the other hand the PLD products choline and PA were released before all the three activation cycles of PKC.     

Phosphorylation and activation of phospholipase D1 by protein kinase C in vivo: determination of multiple phosphorylation sites.

Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCalpha in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCalpha in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory "loop" region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.