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.

Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells arises from increased choline kinase activity, not from phosphatidylcholine breakdown

1989 ◽  
Vol 9 (1) ◽  
pp. 325-328
Author(s):  
I G Macara
Keyword(s):  
Phospholipase C ◽  
Kinase Activity ◽  
Transformed Cells ◽  
Choline Kinase ◽  
3T3 Cells ◽  
Cellular Concentration ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

The cellular concentration of phosphocholine has been reported to be significantly elevated in Ha-ras-transformed NIH 3T3 cells, but not in v-sis transformants (J. C. Lacal, J. Moscat, and S. A. Aaronson, Nature [London] 330:269-271, 1987). It was suggested that the phosphocholine arises from constitutive hydrolysis of phosphatidylcholine by phospholipase C, an activity that would also account for the elevated 1,2-diacylglycerol found in ras-transformed cells. I have demonstrated that the increased phosphocholine arises through the induction of choline kinase activity. No increased breakdown of phosphatidylcholine was observed in ras-transformed cells. The elevation in diacylglycerol is therefore unlikely to be a consequence of phosphatidylinositol or phosphatidylcholine turnover.

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.

scholarly journals Elevated Phospholipase D Activity in H-Ras- but Not K-Ras-Transformed Cells by the Synergistic Action of RalA and ARF6

2003 ◽  
Vol 23 (2) ◽  
pp. 645-654 ◽  
Author(s):  
Lizhong Xu ◽  
Paul Frankel ◽  
Desmond Jackson ◽  
Thuy Rotunda ◽  
Rita L. Boshans ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Dominant Negative ◽  
Membrane Microdomains ◽  
Transformed Cells ◽  
Dominant Negative Mutant ◽  
3T3 Cells ◽  
Protein Levels ◽  
Nih 3T3 ◽  
Adp Ribosylation Factor ◽  
Nih 3T3 Cells

ABSTRACT Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of H-Ras but not K-Ras. H-Ras and K-Ras have been reported to localize to different membrane microdomains, with H-Ras localizing to caveolin-enriched light membrane fractions. We reported previously that PLD activity elevated in response to mitogenic stimulation is restricted to the caveolin-enriched light membrane fractions. PLD activity in H-Ras-transformed cells is dependent upon RalA, and consistent with a lack of elevated PLD activity in K-Ras-transformed cells, RalA was not activated in K-Ras-transformed cells. Although H-Ras-induced PLD activity is dependent upon RalA, an activated mutant of RalA is not sufficient to elevate PLD activity. We reported previously that RalA interacts with PLD activating ADP ribosylation factor (ARF) proteins. In cells transformed by H-Ras, we found increased coprecipitation of ARF6 with RalA. Moreover, ARF6 colocalized with RalA in light membrane fractions. Interestingly, ARF6 protein levels were elevated in H-Ras- but not K-Ras-transformed cells. A dominant-negative mutant of ARF6 inhibited PLD activity in H-Ras-transformed NIH 3T3 cells. Activated mutants of either ARF6 or RalA were not sufficient to elevate PLD activity in NIH 3T3 cells; however, expression of both activated RalA and activated ARF6 in NIH 3T3 cells led to increased PLD activity. These data suggest a model whereby H-Ras stimulates the activation of both RalA and ARF6, which together lead to the elevation of PLD activity.

scholarly journals Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells arises from increased choline kinase activity, not from phosphatidylcholine breakdown.

1989 ◽  
Vol 9 (1) ◽  
pp. 325-328 ◽  
Author(s):  
I G Macara
Keyword(s):  
Phospholipase C ◽  
Kinase Activity ◽  
Transformed Cells ◽  
Choline Kinase ◽  
3T3 Cells ◽  
Cellular Concentration ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

The cellular concentration of phosphocholine has been reported to be significantly elevated in Ha-ras-transformed NIH 3T3 cells, but not in v-sis transformants (J. C. Lacal, J. Moscat, and S. A. Aaronson, Nature [London] 330:269-271, 1987). It was suggested that the phosphocholine arises from constitutive hydrolysis of phosphatidylcholine by phospholipase C, an activity that would also account for the elevated 1,2-diacylglycerol found in ras-transformed cells. I have demonstrated that the increased phosphocholine arises through the induction of choline kinase activity. No increased breakdown of phosphatidylcholine was observed in ras-transformed cells. The elevation in diacylglycerol is therefore unlikely to be a consequence of phosphatidylinositol or phosphatidylcholine turnover.

scholarly journals Both the Catalytic and Regulatory Domains of Protein Kinase C Chimeras Modulate the Proliferative Properties of NIH 3T3 Cells

1997 ◽  
Vol 272 (45) ◽  
pp. 28793-28799 ◽  
Author(s):  
Péter Ács ◽  
Qiming J. Wang ◽  
Krisztina Bögi ◽  
Adriana M. Marquez ◽  
Patricia S. Lorenzo ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
3T3 Cells ◽  
Regulatory Domains ◽  
Kinase C ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes

1987 ◽  
Vol 7 (7) ◽  
pp. 2378-2387 ◽  
Author(s):  
C W Rettenmier ◽  
M F Roussel ◽  
R A Ashmun ◽  
P Ralph ◽  
K Price ◽  
...  
Keyword(s):  
Growth Factor ◽  
Disulfide Bonds ◽  
Mononuclear Phagocyte ◽  
Transformed Cells ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Membrane Bound ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.

scholarly journals Isolation of cellular genes differentially expressed in mouse NIH 3T3 cells and a simian virus 40-transformed derivative: growth-specific expression of VL30 genes.

1985 ◽  
Vol 5 (10) ◽  
pp. 2590-2598 ◽  
Author(s):  
K Singh ◽  
S Saragosti ◽  
M Botchan
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Mouse Cell ◽  
Simian Virus ◽  
Differentially Expressed ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Transformed Cell ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We constructed and screened a cDNA library made from simian virus 40 (SV40)-transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression.

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.

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