scholarly journals Differential effects of platelet-derived growth factor, serum and bombesin on phospholipase D-mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts

1992 ◽  
Vol 285 (1) ◽  
pp. 229-233 ◽  
Author(s):  
Z Kiss
Keyword(s):  
Growth Factor ◽  
Phospholipase D ◽  
Calf Serum ◽  
Platelet Derived Growth Factor ◽  
Maximal Effect ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Fold Stimulation

In previous studies, activators of protein kinase C, sphingosine, ATP and various oncogenes were each found to enhance phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here I examined possible stimulation of PtdEtn hydrolysis by various growth-stimulatory agents, including serum, bombesin, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and insulin. Treatment of NIH 3T3 fibroblasts, prelabelled with [14C]Etn or [32P]PtdEtn, with PDGF-BB resulted in enhanced formation of [14C]Etn or [32P]phosphatidic acid from the respective labelled cellular pools of PtdEtn. A maximal effect (approximately 3-fold stimulation) on PtdEtn hydrolysis was obtained with 50 ng of PDGF/ml after 5 min of treatment. Phosphatidylcholine (PtdCho) was also hydrolysed, although less extensively than PtdEtn, in PDGF-stimulated cells. PDGF-stimulate hydrolysis of both PtdEtn and PtdCho was prevented by prolonged (30 h) treatment of cells with 400 nM-phorbol 12-myristate 13-acetate (PMA). Similar to PDGF, fetal calf serum (1-10%) also stimulated PtdEtn hydrolysis. However, in contrast to PDGF, the effect of serum on PtdEtn hydrolysis (i) was not diminished by pretreatment with PMA, and (ii) was synergistic with that of PMA after a 1 h incubation. Compared with PDGF and serum, bombesin had less effect on PtdEtn hydrolysis, while FGF and insulin had no effects at all. In contrast to PDGF or serum, bombesin inhibited the effect of PMA on PtdEtn hydrolysis.

scholarly journals The zinc chelator 1,10-phenanthroline enhances the stimulatory effects of protein kinase C activators and staurosporine, but not sphingosine and H2O2, on phospholipase D activity in NIH 3T3 fibroblasts

1994 ◽  
Vol 298 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Z Kiss
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Intermediate Step ◽  
Phospholipid Hydrolysis ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Zinc Chelator ◽  
Nih 3T3 ◽  
Hydrolysis Of

Protein kinase C (PKC), an enzyme which is believed to mediate the stimulatory effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on phospholipase D (PLD) activity, has a zinc-dependent structure required for phorbol ester binding. Accordingly, zinc or zinc chelators would be expected to promote or inhibit, respectively, the stimulatory effects of PMA on PLD-mediated phospholipid hydrolysis. Instead, treatment of [14C]choline- and [14C]ethanolamine-labelled NIH 3T3 fibroblasts with the high-affinity zinc chelator 1,10-phenanthroline (0.2-1 mM) for 20-30 min was found to enhance the stimulatory effects of PMA on PLD-mediated hydrolysis of phosphatidylcholine and phosphatidylethanolamine. In [14C]palmitic acid-labelled fibroblasts, in the presence of ethanol, phenanthroline also enhanced the stimulatory effect of PMA on the synthesis of phosphatidylethanol, a marker of PLD activity. Addition of zinc (250 microM) to phenanthroline-treated fibroblasts reversed the stimulatory effects of the chelator. The potentiating effects of phenanthroline were also partially reversed by cadmium, whereas iron, lead, copper, magnesium and calcium were without effects. Of the other activators of PLD tested, phenanthroline also enhanced the stimulatory effects of platelet-derived growth factor and staurosporine, but not that of sphingosine and H2O2, on the hydrolysis of both phospholipids. These results suggest that regulation of PLD by PKC activators and staurosporine involves a common intermediate step, which is inhibited by a chelatable cellular pool of zinc.

scholarly journals Regulation of phospholipase D by sphingosine involves both protein kinase C-dependent and -independent mechanisms in NIH 3T3 fibroblasts

1992 ◽  
Vol 288 (3) ◽  
pp. 853-858 ◽  
Author(s):  
Z Kiss ◽  
E Deli
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Activity Data ◽  
Pkc Inhibitor ◽  
Phospholipid Hydrolysis ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Nih 3T3 ◽  
Hydrolysis Of

Previously, the protein kinase C (PKC) inhibitor sphingosine was found to stimulate phospholipase D (PLD)-mediated hydrolysis of both phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts [Kiss & Anderson (1990) J. Biol. Chem. 265, 7345-7350]. Here we examined the possible relationship between the opposite effects of sphingosine on PKC-mediated protein phosphorylation and PLD activation. After treatments for 3-5 min, sphingosine (25 microM) and the PKC activators phorbol 12-myristate 13-acetate (PMA) (100 nM), bryostatin (100 nM) or platelet-derived growth factor (50 ng/ml) synergistically stimulated the hydrolysis of both PtdEtn and PtdCho in NIH 3T3 fibroblasts prelabelled with [14C]ethanolamine or [14C]choline. Inhibition of PMA-induced phospholipid hydrolysis could also be elicited by sphingosine, but this process required prolonged (60 min) treatments of fibroblasts with 40-60 microM-sphingosine. Similarly to sphingosine, the protein phosphatase inhibitor okadaic acid also had either potentiating or inhibitory effects on PMA-stimulated PLD activity, depending on the length of incubation time and the concentration of PMA. Consistent with the presence of an inhibitory component in the overall action of PKC, the PKC inhibitor staurosporine and down-regulation of PKC activity by prolonged (24 h) treatment with PMA similarly enhanced PLD activity. Data suggest that (a) sphingosine may enhance PMA-mediated phospholipid hydrolysis by neutralizing the action of an inhibitory PKC isoform, and that (b) the stimulatory PKC isoform is less sensitive to the inhibitory action of sphingosine.

Role of nuclear protein kinase C in the mitogenic response to platelet-derived growth factor

1990 ◽  
Vol 96 (1) ◽  
pp. 107-114
Author(s):  
A.P. Fields ◽  
G. Tyler ◽  
A.S. Kraft ◽  
W.S. May
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Nuclear Envelope ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Mitogenic Response ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Nih 3T3

We have assessed the involvement of nuclear envelope protein phosphorylation in the mitogenic response to platelet-derived growth factor (PDGF) in NIH/3T3 fibroblasts. We find that stimulation of quiescent NIH/3T3 cells with PDGF or with the mitogenic protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) or bryostatin 1 (bryo) leads to rapid, dose-dependent phosphorylation of several nuclear envelope polypeptides. The predominant nuclear envelope targets for mitogen-induced phosphorylation are immunologically identified as the nuclear envelope lamins. All three lamin species (A, B and C) are phosphorylated in response to PMA or bryo, while lamins A and C are preferentially phosphorylated in response to PDGF. Phosphopeptide mapping and phosphoamino acid analysis indicate that similar serine sites on the lamins are phosphorylated in response to PDGF, PMA and bryo. Both mitogenicity and lamina phosphorylation induced by these mitogens can be inhibited by the selective PKC inhibitor staurosporine at 2 nM. Treatment of quiescent NIH/3T3 cells with PDGF, PMA or bryo leads to rapid translocation of PKC to the nuclear envelope. These data indicate that rapid nuclear events, including translocation of cytosolic PKC to the nuclear membrane and lamina phosphorylation, may play a role in the transduction of the mitogenic signals of PDGF from the cytoplasm to the nucleus in NIH/3T3 fibroblasts.

The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency

1991 ◽  
Vol 11 (4) ◽  
pp. 2040-2048
Author(s):  
F Fazioli ◽  
U H Kim ◽  
S G Rhee ◽  
C J Molloy ◽  
O Segatto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Phospholipase C ◽  
Signaling Pathway ◽  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Mitogenic Signaling ◽  
Gtpase Activating Protein ◽  
3T3 Fibroblasts ◽  
Nih 3T3

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.

scholarly journals Phorbol ester and bryostatin differentially regulate the hydrolysis of phosphatidylethanolamine in Ha-ras- and raf-oncogene-transformed NIH 3T3 cells

1991 ◽  
Vol 276 (2) ◽  
pp. 505-509 ◽  
Author(s):  
Z Kiss ◽  
U R Rapp ◽  
G R Pettit ◽  
W B Anderson
Keyword(s):  
Phospholipase D ◽  
Second Messengers ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Intact Cells ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

Previously it was reported that transformation of NIH 3T3 fibroblast by the Ha-ras, v-src, v-fms, and A-raf oncogenes decreased the stimulatory effects of phorbol 12-myristate 13-acetate (PMA; ‘TPA’), an activator of protein kinase C (PKC), on the phosphorylation of an endogenous 80 kDa substrate and on 86Rb uptake [Wolfman, Wingrove, Blackshear & Macara (1987) J. Biol. Chem. 262, 16546-16552], as well as on sphingomyelin synthesis [Kiss, Rapp & Anderson (1988) FEBS Lett. 240, 221-226]. Here, we investigated how transformation affects the PMA-stimulated hydrolysis of phosphatidylethanolamine (PtdEtn), a recently characterized mechanism which may contribute to the generation of the second messengers phosphatidic acid and 1,2-diacylglycerol. The effects of PMA were compared with those of bryostatin, a non-tumour-promoter activator of PKC. Transformation of NIH 3T3 cells with Ha-ras, v-raf, or A-raf enhanced the stimulatory effect of PMA on the phospholipase D-mediated hydrolysis of PtdEtn. On the other hand, the effects of bryostatin on PtdEtn hydrolysis were only slightly increased, if at all, in cells transformed with these oncogenes. In crude membrane preparations isolated from these transformed cells, PMA, but not bryostatin, enhanced the combined stimulatory effects of ATP and the GTP analogue guanosine 5′-[gamma-thio]triphosphate on phospholipase D-mediated PtdEtn hydrolysis. The PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine inhibited the stimulatory effect of PMA only in intact cells. These results indicate that transformation of cells by certain oncogenes differentially affects phospholipase D-mediated hydrolysis of PtdEtn induced by PMA and bryostatin, suggesting that the action of PMA might involve two different mechanisms.

scholarly journals The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

1991 ◽  
Vol 11 (4) ◽  
pp. 2040-2048 ◽  
Author(s):  
F Fazioli ◽  
U H Kim ◽  
S G Rhee ◽  
C J Molloy ◽  
O Segatto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Phospholipase C ◽  
Signaling Pathway ◽  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Mitogenic Signaling ◽  
Gtpase Activating Protein ◽  
3T3 Fibroblasts ◽  
Nih 3T3

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.

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