Accumulation of phosphatidylalcohol in cultured cells: use of subcellular fractionation to investigate phospholipase D activity during signal transduction

Phosphatidylalcohol accumulates as a product of a phospholipase D (PLD)-catalysed transphosphatidylation reaction in cells incubated in the presence of a primary alcohol. In the presence of ethanol the phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated the accumulation of [3H]phosphatidylethanol (PEth) in HeLa cells prelabelled with [3H]palmitic acid. Radioactivity associated with PEth increased linearly during a 30 min incubation, indicating that a sustained activation of PLD is caused by PMA in these cells. This was accompanied by the membrane association of protein kinase C-alpha (PKC-alpha), the PKC isoform that recent studies indicate is involved in the activation of PLD. In similar experiments, the neuropeptide bradykinin stimulated an accumulation of PEth in 3T3 Li cells. The radioactivity associated with PEth increased to a maximal level at 30 s and plateaued after this time, suggesting that bradykinin induces only a transient activation of PLD in these cells. This is consistent with the effects of bradykinin on PKC-alpha, which underwent a rapid and transient association with cell membranes. The subcellular localization of PEth was examined using the technique of subcellular fractionation on Percoll density gradients to isolate organelle-enriched fractions from HeLa and 3T3 Li cells. An accumulation of [3H]PEth was measured in the plasma-membrane (PM)-enriched fractions of both HeLa and 3T3 Li cells after incubation with PMA and bradykinin respectively. This was accompanied by a time-dependent accumulation of [3H]PEth in the combined mitochondrial and endoplasmic reticulum (MER)-enriched fractions of both cell lines. PMA was also found to cause translocation of PKC-alpha to both the PM- and MER-enriched fractions in HeLa cells. However, bradykinin stimulated the translocation of PKC-alpha to the PM-enriched fractions only of 3T3 Li cells. The results show that PLD activation leads to the accumulation of PEth in both the PM and MER fractions. We therefore propose that either bradykinin activates a PM-associated PLD and the PLD reaction product is rapidly translocated to other membrane systems or it activates an MER-associated PLD by a mechanism that does not involve PKC-alpha.

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Association of protein kinase-C-alpha with cytoplasmic vesicles in melanoma cells.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.2.8609374 ◽

1996 ◽

Vol 44 (2) ◽

pp. 177-182 ◽

Cited By ~ 5

Author(s):

J Timar ◽

B Liu ◽

R Bazaz ◽

K V Honn

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Fluid Phase ◽

Subcellular Fractionation ◽

Melanoma Cells ◽

Kinase C ◽

Cytoplasmic Vesicles ◽

Protein Kinase C Alpha ◽

Pkc Alpha

In B16a melanoma cells, protein kinase-C-alpha (PKC alpha) is immunomorphologically associated with cytoplasmic vesicles in addition to the previously observed locations (plasma membrane, cytoskeleton, nucleus), as detected with monoclonal antibody (MAb) MC3a. Subcellular fractionation indicated that the authentic 80-KD protein as well as PKC activity can be detected in several particulate fractions except for L2, which contains dense lysosomes. The highest PKC activity is associated with the cytosol-ultralight vesicles and the L1 fraction (containing plasma membrane, endosomes, and the Golgi apparatus). Both of these fractions contained the fluid-phase endocytosis marker peroxidase, indicating that PKC alpha, in addition to other subcellular structures, is most probably associated with endosomal membranes in B16a melanoma cells.

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Selective down-regulation of protein kinase c-ε by carcinogens does not prevent stimulation of phospholipase D by phorbol ester and platelet-derived growth factor

Biochemical Journal ◽

10.1042/bj3000751 ◽

1994 ◽

Vol 300 (3) ◽

pp. 751-756 ◽

Cited By ~ 22

Author(s):

Z Kiss ◽

W H Anderson

Keyword(s):

Protein Kinase C ◽

Growth Factor ◽

Protein Kinase ◽

Phospholipase D ◽

Platelet Derived Growth Factor ◽

Kinase C ◽

Pkc Epsilon ◽

Pkc Zeta ◽

Fibroblast Line ◽

Pkc Alpha

It is well established that activators of protein kinase C (PKC) also enhance the activity of phospholipase D (PLD), and that this regulatory mechanism is altered in transformed cells. Here we used the C3H/10T1/2 mouse embryo fibroblast line, a cellular model for the study of carcinogenesis, to examine possible effects of carcinogens on the PKC isoenzyme pattern and on the regulation of PLD by the PKC activators phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF). Treatment of these fibroblasts with 0.5 microgram/ml 7,12-dimethyl-benz[a]anthracene or benzo[a]pyrene for 24 h greatly decreased (> 80%) the amount of immunoreactive PKC-epsilon. Of the remaining three isoenzymes identified, carcinogens alone had no effect on the cellular status of PKC-alpha and PKC-delta, although they appeared to promote slightly PMA-induced membrane translocation of the cytosolic forms of these isoenzymes in exponentially growing cells. Carcinogens and/or PMA had no effects on the cellular content or distribution of PKC-zeta. Chronic (24 h) treatments with carcinogens resulted in increased or decreased release of [14C]ethanolamine or [14C]choline from the appropriate prelabelled phospholipids, respectively. However, carcinogens failed to block the stimulatory effects of PMA and PDGF on the hydrolysis of phosphatidylethanolamine and phosphatidylcholine or on the synthesis of phosphatidylethanol mediated by PLD. These data indicate that in fibroblasts PKC-epsilon is not a major regulator of PLD activity.

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Cholinergic activation of phospholipase D in lacrimal gland acini is independent of protein kinase C and calcium

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.3.c713 ◽

1995 ◽

Vol 268 (3) ◽

pp. C713-C720 ◽

Cited By ~ 32

Author(s):

D. Zoukhri ◽

D. A. Dartt

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase D ◽

Lacrimal Gland ◽

Phorbol Esters ◽

Adrenergic Agonist ◽

Kinase C ◽

Cellular Lipids ◽

Transphosphatidylation Reaction ◽

Cholinergic Activation

To determine if rat lacrimal gland acini contain phospholipase D (PLD) activity, we took advantage of PLD's unique ability, in the presence of ethanol, to catalyze a transphosphatidylation reaction to produce phosphatidylethanol (PEth). Lacrimal gland acini were labeled for 3 h with [14C]stearic acid, preincubated for 20 min in the presence of 2% ethanol, and incubated for 20 min with or without agonists. Total cellular lipids were then extracted and analyzed by thin-layer chromatography, and the radioactivity was determined by liquid scintillation counting. Carbachol (1 mM), a cholinergic agonist, stimulated the production of both [14C]PEth and [14C]phosphatidic acid ([14C]PA) twofold. This effect was completely blocked by the muscarinic antagonist atropine (10 microM). [14C]PEth accumulation was also stimulated twofold by the active phorbol esters, 4 beta-phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-dibutyrate at 1 microM. Ionomycin (1 microM), a Ca2+ ionophore, also stimulated the production of [14C]PEth twofold. In contrast to carbachol, neither phorbol esters nor ionomycin stimulated [14C]PA production. Neither [14C]PEth nor [14C]PA production was altered by epinephrine (1 mM), a nonselective adrenergic agonist, or phenylephrine (0.1 and 1 mM), a specific alpha 1-adrenergic agonist. We concluded that PLD activity, modulated by muscarinic receptors, protein kinase C, and Ca2+, but not by adrenergic receptors, is present in rat lacrimal gland acini. We also concluded that cholinergic activation of PLD appears to be independent of PKC and Ca2+.

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PLD pathway involved in carbachol-induced Cl− secretion: possible role of TNF-α

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.4.c789 ◽

2001 ◽

Vol 280 (4) ◽

pp. C789-C795 ◽

Cited By ~ 14

Author(s):

Judith C. J. Oprins ◽

Claudia van der Burg ◽

Helen P. Meijer ◽

Teun Munnik ◽

Jack A. Groot

Keyword(s):

Primary Alcohol ◽

Electrophysiological Response ◽

Kinase C ◽

Protein Kinase C Activity ◽

Tnf Α ◽

Muscarinic Stimulation ◽

Transphosphatidylation Reaction ◽

Factor Α ◽

Necrosis Factor

In a previous study, it was found that exposure to tumor necrosis factor-α (TNF-α) potentiated the electrophysiological response to carbachol in a time-dependent and cycloheximide-sensitive manner. It was deduced that the potentiation could be due to protein kinase C activity because of increased 1,2-diacylglycerol. It was also observed that propranolol could decrease the electrophysiological response to carbachol (Oprins JC, Meijer HP, and Groot JA. Am J Physiol Cell Physiol 278: C463–C472, 2000). The aim of the present study was to investigate whether the phospholipase D (PLD) pathway plays a role in the carbachol response and the potentiating effect of TNF-α. The transphosphatidylation reaction in the presence of the primary alcohol 1-butanol [leading to stable phosphatidylbutanol (Pbut) formation] was used to measure activity of PLD. The phosphatidic acid (PA) levels were also measured. Muscarinic stimulation resulted in an increased formation of Pbut and PA. TNF-α decreased levels of PA.

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ANG II stimulates phospholipase D through PKCζ activation in VSMC: implications in adhesion, spreading, and hypertrophy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00769.2005 ◽

2006 ◽

Vol 290 (1) ◽

pp. H46-H54 ◽

Cited By ~ 17

Author(s):

Jean-Hugues Parmentier ◽

Zoran Pavicevic ◽

Kafait U. Malik

Keyword(s):

Phospholipase D ◽

Antisense Oligonucleotides ◽

Primary Alcohol ◽

Tertiary Alcohol ◽

Ang Ii ◽

Kinase C ◽

Inactive Form ◽

Maximal Activation ◽

The Relationship

ANG II stimulates phospholipase D (PLD) activity and growth of vascular smooth muscle cells (VSMC). The atypical protein kinase C-ζ (PKCζ) plays a central role in the regulation of cell survival and proliferation. This study was conducted to determine the relationship between ANG II-induced activation of PKCζ and PLD and their implication in VSMC adhesion, spreading, and hypertrophy. ANG II stimulated PKCζ activity with maximal activation at 30 s followed by a decline in its activity to 45% above basal at 5 min. Inhibition of PKCζ activity with a myristoylated pseudosubstrate peptide or overexpression of a kinase-inactive form of PKCζ decreased ANG II-induced PLD activity. Moreover, depletion of PKCζ with selective antisense oligonucleotides also decreased ANG II-induced PLD activity. Interaction between PLD2 and PKCζ in VSMC was detected by coimmunoprecipitation. ANG II-induced PLD activity was inhibited by the primary alcohol n-butanol but not the tertiary alcohol t-butanol. The functional significance of PKCζ and PLD2 in VSMC adhesion, spreading, and hypertrophy was investigated. Inhibition of PKCζ and PLD2 activity or expression attenuated VSMC adhesion to collagen I and ANG II-induced cell spreading and hypertrophy. These results demonstrate that ANG II-induced PLD activation is regulated by PKCζ and suggest a crucial role of PKCζ-dependent PLD2 in VSMC functions such as adhesion, spreading, and hypertrophy, which are associated with the pathogenesis of atherosclerosis and malignant hypertension.

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Epidermal-growth-factor-induced production of phosphatidylalcohols by HeLa cells and A431 cells through activation of phospholipase D

Biochemical Journal ◽

10.1042/bj2870051 ◽

1992 ◽

Vol 287 (1) ◽

pp. 51-57 ◽

Cited By ~ 31

Author(s):

M Kaszkin ◽

L Seidler ◽

R Kast ◽

V Kinzel

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Phosphatidic Acid ◽

Phospholipase D ◽

Hela Cells ◽

Total Phospholipid ◽

Primary Alcohol ◽

A431 Cells ◽

Intact Cells ◽

Epidermal Growth

In response to epidermal growth factor (EGF), HeLa cells and A431 cells rapidly accumulate substantial amounts of phosphatidic acid (up to 0.16 and 0.2 micrograms/10(6) cells respectively), which represents approx. 0.17% of total phospholipid. Phosphatidic acid may be a potential product of diacylglycerol kinase and/or of phospholipase D. To evaluate the contribution of phospholipase D, the phosphatidyl-transfer reaction to a primary alcohol (mostly butan-1-ol; 0.2%) was measured; this reaction is known to be mediated exclusively by phospholipase D in intact cells. In HeLa and in A431 cells prelabelled with [1-14C]oleic acid, EGF (10 and 100 nM respectively) caused a 3-fold increase in radioactive phosphatidylbutanol within 5 min at the expense of labelled phosphatidic acid. Dose-response relationships showed 10 nM- and 100 nM-EGF to be maximally effective in HeLa cells and A431 cells respectively. Mass determinations showed that the phosphatidylbutanol formed within 5 min represented only part of the phosphatidic acid. Depletion of protein kinase C by pretreatment of A431 cells for 17 h with the phorbol ester phorbol 12-myristate 13-acetate (0.1 microM) did not impair EGF-induced formation of phosphatidylbutanol, thus indicating that the reaction was independent of this enzyme. Since phosphatidic acid is suggested to exert second-messenger functions as well as to induce biophysical changes in cellular membranes, its formation, including that via the phospholipase D pathway, may represent an important link between extracellular signals and intracellular targets.

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PKD: a new protein kinase C–dependent pathway in platelets

Blood ◽

10.1182/blood-2002-08-2384 ◽

2003 ◽

Vol 101 (4) ◽

pp. 1392-1399 ◽

Cited By ~ 19

Author(s):

Margaret J. Stafford ◽

Steve P. Watson ◽

Catherine J. Pears

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tyrosine Kinases ◽

Catalytic Domain ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Kinase C ◽

Transient Activation ◽

Dependent Pathway ◽

Sustained Activation

Protein kinase D (PKD, also known as PKCμ) is closely related to the protein kinase C superfamily but is differentially regulated and has a distinct catalytic domain that shares homology with Ca2+-dependent protein kinases. PKD is highly expressed in hematopoietic cells and undergoes rapid and sustained activation upon stimulation of immune receptors. PKD is regulated through phosphorylation by protein kinase C (PKC). In the present study, we show that PKD is expressed in human platelets and that it is rapidly activated by receptors coupled to heterotrimeric G-proteins or tyrosine kinases. Activation of PKD is mediated downstream of PKC. Strong agonists such as convulxin, which acts on GPVI, and thrombin cause sustained activation of PKC and PKD, whereas the thromboxane mimetic U46619 gives rise to transient activation of PKC and PKD. Activation of PKD by submaximal concentrations of phospholipase C–coupled receptor agonists is potentiated by Gi-coupled receptors (eg, adenosine diphosphate and epinephrine). This study shows that PKD is rapidly activated by a wide variety of platelet agonists through a PKC-dependent pathway. Activation of PKD enables phosphorylation of a distinct set of substrates to those targeted by PKC in platelets.

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