Depressed phosphatidic acid-induced contractile activity of failing cardiomyocytes

The effects of phosphatidic acid (PA), a known inotropic agent, on Ca(2+) transients and contractile activity of cardiomyocytes in congestive heart failure (CHF) due to myocardial infarction were examined. In control cells, PA induced a significant increase (25%) in active cell shortening and Ca(2+) transients. The phospholipase C (PLC) inhibitor, 2-nitro-4-carboxyphenyl N,N-diphenylcarbonate, blocked the positive inotropic action induced by PA, indicating that PA induces an increase in contractile activity and Ca(2+) transients through stimulation of PLC. Conversely, in failing cardiomyocytes there was a loss of PA-induced increase in active cell shortening and Ca(2+) transients. PA did not alter resting cell length. Both diastolic and systolic [Ca(2+)] were significantly elevated in the failing cardiomyocytes. In vitro assessment of the cardiac sarcolemmal (SL) PLC activity revealed that the impaired failing cardiomyocyte response to PA was associated with a diminished stimulation of SL PLC activity by PA. Our results identify an important defect in the PA-PLC signaling pathway in failing cardiomyocytes, which may have significant implications for the depressed contractile function during CHF.     

Defective sarcolemmal phospholipase C signaling in diabetic cardiomyopathy

Phospholipase C (PLC) activity is known to influence cardiac function. This study was undertaken to examine the status of PLC 3 in the cardiac cell plasma membrane (sarcolemma, SL) in an experimental model of chronic diabetes. SL membrane was isolated from diabetic rat hearts at 8 weeks after a single i.v. injection of streptozotocin (65 mg/kg body weight). The total SL PLC was decreased in diabetes and was associated with a decrease in SL PLC 3 activity, which immunofluorescence in frozen diabetic left ventricular tissue sections revealed to be due to a decrease in PLC 3 protein abundance. In contrast, the SL abundance of Gq was significantly increased during diabetes. These changes were associated with a loss of contractile function (±dP/dt). A 2-week insulin treatment of 6-week diabetic animals partially normalized all of these parameters. These findings suggest a defect in PLC 3-mediated signaling processes may contribute to the cardiac dysfunction seen during diabetes. (Mol Cell Biochem 261: 193–199, 2004)     

Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway

Aluminum (Al³⁺) has been recognized as a main toxic factor in crop production in acid lands. Phosphatidic acid (PA) is emerging as an important lipid signaling molecule and has been implicated in various stress-signaling pathways in plants. In this paper, we focus on how PA generation is affected by Al³⁺ using Coffea arabica suspension cells. We pre-labeled cells with [³²P]orthophosphate (³²Pi) and assayed for ³²P-PA formation in response to Al³⁺. Treating cells for 15 min with either AlCl₃ or Al(NO₃)₃ inhibited the formation of PA. In order to test how Al³⁺ affected PA signaling, we used the peptide mastoparan-7 (mas-7), which is known as a very potent stimulator of PA formation. The Al³⁺ inhibited mas-7 induction of PA response, both before and after Al³⁺ incubation. The PA involved in signaling is generated by two distinct phospholipid signaling pathways, via phospholipase D (PLD; EC: 3.1.4.4) or via Phospholipase C (PLC; EC: 3.1.4.3), and diacylglycerol kinase (DGK; EC 2.7.1.107). By labeling with ³²Pi for short periods of time, we found that PA formation was inhibited almost 30% when the cells were incubated with AlCl₃ suggesting the involvement of the PLC/DGK pathway. Incubation of cells with PLC inhibitor, U73122, affected PA formation, like AlCl₃ did. PLD in vivo activation by mas-7 was reduced by Al³⁺. These results suggest that PA formation was prevented through the inhibition of the PLC activity, and it provides the first evidence for the role of Al toxicity on PA production.     

The role of phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade

Phosphatidic acid (PA) is an important second messenger produced by the activation of numerous cell surface receptors. Recent data have suggested that PA regulates multiple cellular processes. This review addresses primarily the role of PA in the regulation of the Erk1/2 cascade pathway. A model for the regulation of Erk1/2 phosphorylation by cell surface receptors is presented. According to this model, agonists stimulate the binding of GTP to Ras and the activation of phospholipase D to generate phosphatidic acid. PA promotes the binding of cRaf-1 kinase to the membrane, where it interacts with Ras.GTP and other regulatory components of the pathway. Ras-Raf complexes remain bound to the surface of endosomes, where scaffolding complexes involving Ras, cRaf-1, MEK and Erk are formed. Complete activation and coupling of the cascade requires endocytosis, a process that is also modulated by PA.     

Defective sarcolemmal phospholipase C signaling in diabetic cardiomyopathy

Phospholipase C (PLC) activity is known to influence cardiac function. This study was undertaken to examine the status of PLC beta3 in the cardiac cell plasma membrane (sarcolemma, SL) in an experimental model of chronic diabetes. SL membrane was isolated from diabetic rat hearts at 8 weeks after a single i.v. injection of streptozotocin (65 mg/kg body weight). The total SL PLC was decreased in diabetes and was associated with a decrease in SL PLC beta3 activity, which immunofluorescence in frozen diabetic left ventricular tissue sections revealed to be due to a decrease in PLC beta3 protein abundance. In contrast, the SL abundance of Gqalpha was significantly increased during diabetes. These changes were associated with a loss of contractile function (+/- dP/dt). A 2-week insulin treatment of 6-week diabetic animals partially normalized all of these parameters. These findings suggest a defect in PLC beta3-mediated signaling processes may contribute to the cardiac dysfunction seen during diabetes.     

Emerging findings from studies of phospholipase D in model organisms (and a short update on phosphatidic acid effectors)

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Historically, much PLD work has been conducted in mammalian settings although genes encoding enzymes of this family have been identified in all eukaryotic organisms. Recently, important insights on PLD function are emerging from work in yeast, but much less is known about PLD in other organisms. In this review we will summarize what is known about phospholipase D in several model organisms, including C. elegans, D. discoideum, D. rerio and D. melanogaster. In the cases where knockouts are available (C. elegans, Dictyostelium and Drosophila) the PLD gene(s) appear not to be essential for viability, but several studies are beginning to identify pathways where this activity has a role. Given that the proteins in model organisms are very similar to their mammalian counterparts, we expect that future studies in model organisms will complement and extend ongoing work in mammalian settings. At the end of this review we will also provide a short update on phosphatidic acid targets, a topic last reviewed in 2006.     

A novel protein kinase target for the lipid second messenger phosphatidic acid

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.     

A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: application in human myocardium

Phosphatidic acid phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol, the second messenger responsible for activation of protein kinase C. Despite the crucial role of PAP lipid signaling, there are no data on PAP signaling function in the human heart. Here we present a nonradioactive assay for the investigation of PAP activity in human myocardium using a fluorescent derivative of PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate (BODIPY-PA), as substrate in an in vitro PAP-catalyzed reaction. Unreacted BODIPY-PA was resolved from the PAP products by a binary gradient HPLC system and BODIPY-diacylglycerol was detected by fluorimetry. The reaction proceeded at a linear rate for up to 60 min and increased linearly with increasing amounts of cardiac protein in a range of 0.25 to 8.0 μg. This assay proved to be sensitive for accurate quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human atrial tissue and right ventricular endomyocardial biopsies. Total PAP activity was approximately fourfold higher in ventricular myocardium than in atrial tissue. There was negligible PAP-1 activity in atrial myocardium compared with ventricular myocardium, indicating regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart.     

Protecting the heart through MK2 modulation,toward a role in diabetic cardiomyopathy and lipid metabolism

Various signaling pathways have been identified in the heart as important players during development, physiological adaptation or pathological processes. This includes the MAPK families, particularly p38MAPK, which is involved in several key cellular processes, including differentiation, proliferation, apoptosis, inflammation, metabolism and survival. Disrupted p38MAPK signaling has been associated with several diseases, including cardiovascular diseases (CVD) as well as diabetes and its related complications. Despite efforts to translate this knowledge into therapeutic avenues, p38 inhibitors have failed in clinical trials due to adverse effects. Inhibition of MK2, a downstream target of p38, appears to be a promising alternative strategy. Targeting MK2 activity may avoid the adverse effects linked to p38 inhibition, while maintaining its beneficial effects. MK2 was first considered as a therapeutic target in inflammatory diseases such as rheumatoid polyarthritis. A growing body of evidence now supports a key role of MK2 signaling in the pathogenesis of CVD, particularly ischemia/reperfusion injury, hypertrophy, and hypertension and that its inhibition or inactivation is associated with improved heart and vascular functions. More recently, MK2 was shown to be a potential player in diabetes and related complications, particularly in liver and heart, and perturbations in calcium handling and lipid metabolism. In this review, we will discuss recent advances in our knowledge of the role of MK2 in p38MAPK-mediated signaling and the benefits of its loss of function in CVD and diabetes, with an emphasis on the roles of MK2 in calcium handling and lipid metabolism. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

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.     

Plant PA signaling via diacylglycerol kinase

Accumulating evidence suggests that phosphatidic acid (PA) plays a pivotal role in the plant's response to environmental signals. Besides phospholipase D (PLD) activity, PA can also be generated by diacylglycerol kinase (DGK). To establish which metabolic route is activated, a differential ³²P-radiolabelling protocol can be used. Based on this, and more recently on reverse-genetic approaches, DGK has taken center stage, next to PLD, as a generator of PA in biotic and abiotic stress responses. The DAG substrate is generally thought to be derived from PI-PLC activity. The model plant system Arabidopsis thaliana has 7 DGK isozymes, two of which, AtDGK1 and AtDGK2, resemble mammalian DGK?, containing a conserved kinase domain, a transmembrane domain and two C1 domains. The other ones have a much simpler structure, lacking the C1 domains, not matched in animals. Several protein targets have now been discovered that bind PA. Whether the PA molecules engaged in these interactions come from PLD or DGK remains to be elucidated.     

Oxidant stress enhances Lyso-PAF-AcT activity by modifying phospholipase D and phosphatidic acid in aortic endothelial cells

Oxidant stress, as a consequence of selenium (Se) deficiency, alters production of vasoactive compounds including platelet-activating factor (PAF). Recent studies report that enhanced PAF production during Se deficiency is a consequence of increased lyso-PAF:acetyl-coenzyme A acetyltransferase (Lyso-PAF-AcT) activity. To elucidate the mechanism behind increased Lyso-PAF-AcT activity during oxidant stress, phospholipase D (PLD) activity and phosphatidic acid (PA) production were examined. Increased PLD activity and PA production were exhibited in bovine aortic endothelial cells using a Se-deficient model of oxidant stress. The direct effects of PLD and PA on Lyso-PAF-AcT activity were assessed using selective inhibitors and repletion experiments. Following the inhibition of PLD and addition of exogenous PA, Lyso-PAF-AcT activity significantly decreased and increased, respectively. Therefore, Se deficiency enhances Lyso-PAF-AcT activity in part by modifying PLD and PA. This suggests a novel link between Se status and PAF production, providing potential upstream therapeutic targets for PAF regulation under conditions of oxidant stress.     

Modulation of phospholipase D activity in vitro

Most phospholipases D (PLDs) occurring in microorganisms, plants and animals belong to a superfamily which is characterized by several conserved regions of amino acid sequence including the two HKD motifs necessary for catalytic activity. Most eukaryotic PLDs possess additional regulatory structures such as the Phox and Pleckstrin homology domains in mammalian PLDs and the C2 domain in most plant PLDs. Owing to recombinant expression techniques, an increasing number of PLDs from different organisms has been obtained in purified form, allowing the investigation of specific and unspecific interactions of the enzymes with regulatory components in vitro. The present paper gives an overview on different factors which can modulate PLD activity and compares their influence on the enzymes from different sources. While no biological regulator can be recognized for extracellular bacterial PLDs, the most prominent specific activator of eukaryotic PLDs is phosphatidylinositol-4,5-bisphosphate (PIP₂). In a sophisticated interplay PIP₂ seems to cooperate with several regulatory proteins in mammalian PLDs, whereas in plant PLDs it mainly acts in concert with Ca²⁺ ions. Moreover, curvature, charges and heterogeneities of membrane surfaces are assessed as unspecific modulators. A possible physiological role of the transphosphatidylation reaction catalyzed by PLDs in competition with phospholipid hydrolysis is discussed.     

Spectrophotometric determination of phosphatidic acid via iron(III) complexation for assaying phospholipase D activity

The ability of negatively charged phosphatidates to form complexes with Fe³⁺ ions was used to design a simple spectrophotometric assay for the quantitative determination of phosphatidic acid (PA). In the reaction with the purple iron(III)-salicylate, PA extracts Fe³⁺ ions and decreases the absorbance at 490 nm. Lower competition with salicylate for Fe³⁺ ions was observed with single negatively charged phosphatidates such as phosphatidylglycerol (PG), whereas neutral phosphatidates such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) showed no influence on the absorbance of the iron(III) complex. The detection limit of the method on a microplate scale was 10 μM PA. Based on these results, an assay for determining the activity of phospholipase D (PLD) toward natural phospholipids such as PC, PE, and PG was developed. In contrast to other spectroscopic PLD assays, this method is able to determine PLD activity toward different lipids or even lipid mixtures.     

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.     

Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases

Phosphatidic acid (PA) has been increasingly recognized as an important signaling lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization. Recent studies indicate that the signaling PA generated from phospholipase D (PLD) and diacylglycerol kinase (DGK) plays critical roles in regulating the activity of some members of Ras superfamily of small guanosine triphosphatases (GTPases), such as Ras, Rac and Arf. Change of PA levels regulates the activity of small GTPases by modulating membrane localization and activity of small GTPase regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In addition, PA also targets some small GTPases to membranes by direct binding. This review summarizes the roles of PLD and DGK in regulating the activity of several Ras superfamily members and cellular processes they control. Some future directions and the implication of PA regulation of Ras small GTPases in pathology are also discussed.     

Phosphatidic acid signaling to mTOR: Signals for the survival of human cancer cells

During the past decade elevated phospholipase D (PLD) activity has been reported in virtually all cancers where it has been examined. PLD catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger phosphatidic acid (PA). While many targets of PA signaling have been identified, the most critical target of PA in cancer cells is likely to be mTOR — the mammalian target of rapamycin. mTOR has been widely implicated in signals that suppress apoptotic programs in cancer cells — frequently referred to as survival signals. mTOR exists as two multi-component complexes known as mTORC1 and mTORC2. Recent data has revealed that PA is required for the stability of both mTORC1 and mTORC2 complexes — and therefore also required for the kinase activity of both mTORC1 and mTORC2. PA interacts with mTOR in a manner that is competitive with rapamycin, and as a consequence, elevated PLD activity confers rapamycin resistance — a point that has been largely overlooked in clinical trials involving rapamycin-based strategies. The earliest genetic changes occurring in an emerging tumor are generally ones that suppress default apoptotic programs that likely represent the first line of defense of cancer. Targeting survival signals in human cancers represents a rational anti-cancer therapeutic strategy. Therefore, understanding the signals that regulate PA levels and how PA impacts upon mTOR could be important for developing strategies to de-repress the survival signals that suppress apoptosis. This review summarizes the role of PA in regulating the mTOR-mediated signals that promote cancer cell survival.     

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg²⁺-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase -dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.     

Downregulation of adipose triglyceride lipase in the heart aggravates diabetic cardiomyopathy in db/db mice

Adipose triglyceride lipase (ATGL) was recently identified as a rate-limiting triglyceride (TG) lipase and its activity is stimulated by comparative gene identification-58 (CGI-58). Mutations in the ATGL or CGI-58 genes are associated with neutral lipid storage diseases characterized by the accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, is characterized by TG accumulation in coronary atherosclerotic lesions and in the myocardium. Recent reports showed that myocardial TG accumulation is significantly higher in patients with diabetes and is associated with impaired left ventricular diastolic function. Therefore, we investigated the roles of ATGL and CGI-58 in the development of myocardial steatosis in the diabetic state. Histological examination with oil red O staining showed marked lipid deposition in the hearts of diabetic fatty db/db mice. Cardiac triglyceride and diglyceride contents were greater in db/db mice than in db/+ control mice. Next, we determined the expression of genes and proteins that affect lipid metabolism, and found that ATGL and CGI-58 expression levels were decreased in the hearts of db/db mice. We also found increased expression of genes regulating triglyceride synthesis (sterol regulatory element-binding protein 1c, monoacylglycerol acyltransferases, and diacylglycerol acyltransferases) in db/db mice. Regarding key modulators of apoptosis, PKC activity, and oxidative stress, we found that Bcl-2 levels were lower and that phosphorylated PKC and 8-hydroxy-2′-deoxyguanosine levels were higher in db/db hearts. These results suggest that reduced ATGL and CGI-58 expression and increased TG synthesis may exacerbate myocardial steatosis and oxidative stress, thereby promoting cardiac apoptosis in diabetic mice.     

Phosphatidic Acid-Dependent Phosphorylation of a 29-kDa Protein by Protein Kinase Cα in Bovine Brain Cytosol

Abstract: Activation of phospholipase D (PLD) is involved in receptor-mediated signal transduction responses. Signaling from PLD to a downstream molecule(s) appears to be mediated by the PLD product phosphatidic acid (PA). A target molecule(s) of PA, however, has not yet been identified. The present study sought to define such a target molecule(s) of PA. In bovine brain cytosol, proteins with apparent molecular weights of 29,000 (p29) and 32,000 (p32) were prominently phosphorylated in the presence of PA, but not in its absence, indicating that there is a PA-regulated protein kinase (PARK) in bovine brain that phosphorylates p29 and p32. One of these substrates, p29, was purified to near homogeneity. Its partial amino acid sequence was determined and found to be identical to that of a known brain-specific 25-kDa protein (p25). The purified p29 was also readily recognized by and immunoprecipitated with an anti-p25 antibody. These results suggest that p29 is very similar to or identical with p25. Using the purified p29 as a substrate, PARK was purified to near homogeneity. The purified PARK had an apparent molecular weight of 80,000, was strongly recognized by an anti-protein kinase C (PKC)α antibody, and was activated by phosphatidylserine (PS) as well as PA. The PA- and PS-stimulated PARK activity was extremely augmented by the presence of 1 µM free Ca²⁺. In the presence of 1 mM EGTA, phorbol 12-myristate 13-acetate activated PARK synergistically with PA or PS. Similar results were obtained with the purified recombinant PKCα. From these results, it is suggested that the PARK activity purified might be attributed to PKCα. In p25-depleted bovine brain cytosol, which was prepared by treatment of bovine brain cytosol with the anti-p25 antibody, PA-dependent phosphorylation of p29, but not p32, was almost completely eliminated. When PKCα in bovine brain cytosol was depleted by its precipitation with the anti-PKCα antibody, neither p29 nor p32 in this PKCα-depleted cytosol was phosphorylated in the presence of PA. These results indicate that in bovine brain cytosol PA activates PKCα, which, in turn, phosphorylates p29, which may be identical with p25.