Regulation of phospholipase D by protein kinase C (original) (raw)
Related papers
Archives of Biochemistry and Biophysics, 1999
In fibroblasts, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) stimulates phospholipase D (PLD)-mediated hydrolysis of both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) by PKC-␣-mediated nonphosphorylating and phosphorylating mechanisms. Here we have used NIH 3T3 fibroblasts overexpressing holo PKCand its regulatory, catalytic, and zinc finger domain fragments to determine if this isozyme also regulates PLD activity. Overexpression of holo PKCinhibited the stimulatory effects of PMA (5-100 nM) on both PtdCho and PtdEtn hydrolysis. Overexpression of PKC-also was found to inhibit platelet-derived growth factor-induced PLD activity. Expression of the catalytic unit of PKC-had no effect on PMA-induced PLD activity. In contrast, expression of both the regulatory domain fragment and the zinc finger domain of PKCresulted in significant inhibition of PMAstimulated PtdCho and PtdEtn hydrolysis. Interestingly, although PKC-␣ also mediates the stimulatory effect of PMA on the synthesis of PtdCho by a phosphorylation mechanism, overexpression of holo PKCor its regulatory domain fragments did not affect PMA-induced PtdCho synthesis. These results indicate that the PKC-system can act as a negative regulator of PLD activity and that this inhibition is mediated by its regulatory domain.
Biochemical and Biophysical Research Communications, 1990
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-O-tetradecanoylphorbol-13-acetate (TPA), phorbol dibutyrate (PDBU), 12deoxyphorbol-13-phenylacetate (DOPP), 12deoxyphorbol-13-phenylacetate-20acetate (DOPPA) and mezerin activated PLD, as measured by the formation of PEt, whereas Concanvalin A 4 ConA) had no effect. Inhibitors of PKC, sphingosine (2x106M-5x10sM), H-7, HA1004 (5x10 3-5x10 M) and K252a (1~10'~-lxlOBM) failed to block the PEt synthesis induced by TPA. In fact, sphingosine increased it. Other PKC activators, 1-oleoyi-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 phosphatidylchdine (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. @I990 Academic Press, Inc. Phospholipase D (EC 3.1.4.4) (PLD) catalyzes the hydrolysis of nondiglyceride esters at the phosphodiester bond in a variety of phospholipids to produce phosphatidic acid (PA) and an alcohol (1). PLD also is an active transphosphatidylase with a large variety of alcohol acceptors. For example, ethanol is exchanged for a polar group in phospholipids to produce PEt (2). Recently, several researchers observed a significant increase in PEt production in a number of cell types treated with either phorbol esters or a chemotactic peptkie in the presence of 0.5% ethanol (3-6). Such an increase has been reliably attributed to an increase of PLD activity (56). The importance of phorbol esters in tumor promotion and cell activation is well known (7); many studies link phorbol esters to PKC (8-9). However, the role of PKC in PLD activation by phorbol esters remains to be established (10). Work from Mueller's laboratory (3) first showed that TPA induced PLD activity in quiescent bovine lymph node lymphocytes. ConA, a mitogenicledin, stimulates quiescent lymphocytes to proliferate. Wn the work reported here, we investigated the effect of ConA on PLD activity and found that although TPA activated PLD, ConA did not. In addition, PKC seemed to play no role in the activation of PLD by TPA in lymphocytes. l To whom correspondence should be addressed.
FEBS Letters, 2003
The regulation of phospholipase D1 (PLD1) by protein kinase C (PKC) isoforms was analyzed in human melanoma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced PLD1 activation was suppressed by the introduction of PKCN N as well as its kinase-negative mutant in MeWo cells, which contain PKCK K but lack PKCL L. PLD activity was not a¡ected by PKCN N in G361 cells, which have PKCL L but are de¢cient in PKCK K. In MeWo cells introduced by PKCK K and PLD1, the association of these proteins was observed, which was enhanced by the TPA treatment. In cells overexpressing PKCN N in addition to PKCK K and PLD1, TPA treatment increased the association of PKCN N and PLD1, while it attenuated the association of PKCK K and PLD1. These results indicate that PKCN N inhibits TPA-induced PLD1 activation mediated by PKCK K through the association with PLD1.
Phospholipase-D activation can be negatively regulated through the action of protein kinase C
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1994
Transient activation of COS-1 cell phospholipase-D (PLD) in response to the protein kinase C (PKC) agonist tetradecanoyl phorbol acetate (TPA) was demonstrated by monitoring the ethanol-dependent accumulation of phosphatidylethanol (PtdEth). Transfection of COS-1 cells with PKC-a (wild-type and constitutively activated mutants) produced no detectable PtdEth on incubation of transfected cells in the presence of ethanol. However, the response of transfected cells to subsequent TPA stimulation was inhibited, consistent with a role for the PKC-a in the suppression of PLD activity.
Regulation of human PLD1 and PLD2 by calcium and protein kinase C
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2000
Numerous studies show that PLD is activated in cells by calcium and by protein kinase C (PKC). We found that human PLD1 and PLD2 expressed in Sf9 cells can be activated by calcium-mobilizing agonists and by co-expression with PKCK. The calcium-mobilizing agonists A23187 and CryIC toxin triggered large increases in phosphatidylethanol (PtdEth) production in Sf9 cells over-expressing PLD1 and PLD2, but not in vector controls. PLD activation by these agonists was largely dependent on extracellular calcium. Membrane assays demonstrated significant PLD1 and PLD2 activity in the absence of divalent cations, which could be enhanced by low levels of calcium either in the presence or absence of magnesium. PLD1 but not PLD2 activity was slightly enhanced by magnesium. Treatment of Sf9 cells expressing PLD1 and PLD2 with PMA resulted in little PtdEth production. However, a significant and comparable formation of PtdEth occurred when PLD1 or PLD2 were co-expressed with PKCK, but not PKCN, and was further augmented by PMA. In contrast to PLD1, co-expressing PLD2 with PKCK or PKCN further enhanced A23187-induced PtdEth production. Immunoprecipitation experiments demonstrated that PLD1 and PLD2 associated with the PKC isoforms in Sf9 cells. Furthermore, in membrane reconstitution assays, both PLD1 and PLD2 could be stimulated by calmodulin and PKCK-enriched cytosol. The results indicate that PLD2 as well as PLD1 is subject to agonist-induced activation in intact cells and can be regulated by calcium and PKC. ß 0167-4889 / 00 / $^see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -4 8 8 9 ( 0 0 ) 0 0 0 4 9 -5
Biochemistry, 1999
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 PMAinduced 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 PKCR 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 PKCR 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.
Journal of Biological Chemistry, 1997
The role of phosphatidylcholine (PC) hydrolysis in activation of the mitogen-activated protein kinase (MAPK) pathway by platelet-derived growth factor (PDGF) was studied in Rat-1 fibroblasts. PDGF induced the transient formation of phosphatidic acid, choline, diacylglycerol (DG), and phosphocholine, the respective products of phospholipase D (PLD) and phospholipase C (PC-PLC) activity, with peak levels at 5-10 min. PLDcatalyzed transphosphatidylation (with n-butyl alcohol) diminished DG formation at 5 min but not at later stages of PDGF stimulation. Phorbol ester-induced down-regulation of protein kinase C (PKC) completely blocked PLD activation but not the formation of DG and phosphocholine at 10 min of PDGF stimulation. Collectively, these data indicate that PDGF activates both PLD and PC-PLC. In contrast, epidermal growth factor did not activate PC-PLC in these cells, and it activated PLD only weakly. DG formation by itself, through Bacillus cereus PC-PLC treatment of cells, was sufficient to mimic PDGF in activation of MAPK independent of phorbol ester-sensitive PKC. Since PKC down-regulation blocked PDGF-induced PLD but not MAPK activation, we conclude that PLD is not involved in MAPK signaling. In contrast, MAPK activation by exogenous (bacterial) PLD was not affected by PKC down-regulation, indicating that signals evoked by exogenous PLD differ from endogenous PLD. D609 (2-10 g/ml), an inhibitor of PC-PLC, blocked PDGF-but not epidermal growth factor-induced MAPK activation. However, D609 should be used with caution since it also affects PLD activity. The results suggest that PC-PLC rather than PLD plays a critical role in the PDGF-activated MAPK pathway.