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Phorbol ester and bryostatin differentially regulate the hydrolysis of phosphatidylethanolamine in Ha-ras- and
1Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Abstract:
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.
Insights
Oncogene transformation of NIH 3T3 cells enhances phorbol 12-myristate 13-acetate (PMA) stimulated phosphatidylethanolamine (PtdEtn) hydrolysis. This suggests PMA utilizes distinct mechanisms, differing from bryostatin, in transformed cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Oncogene transformation alters cellular signaling pathways.
- Phorbol 12-myristate 13-acetate (PMA) activates protein kinase C (PKC) and influences various cellular processes.
- Phosphatidylethanolamine (PtdEtn) hydrolysis generates second messengers like diacylglycerol.
Purpose of the Study:
- To investigate the impact of oncogene transformation on PMA-stimulated PtdEtn hydrolysis.
- To compare the effects of PMA with bryostatin, another PKC activator, in transformed cells.
- To elucidate the mechanisms underlying PMA's action in oncogene-transformed cells.
Main Methods:
- NIH 3T3 fibroblasts were transformed with Ha-ras, v-raf, or A-raf oncogenes.
- PtdEtn hydrolysis was measured in response to PMA and bryostatin stimulation.
- Phospholipase D activity was assessed in crude membrane preparations.
- PKC inhibition was evaluated using 1-(5-isoquinolinesulphonyl)-2-methylpiperazine.
Main Results:
- Oncogene transformation enhanced PMA-stimulated phospholipase D-mediated PtdEtn hydrolysis.
- Bryostatin's effects on PtdEtn hydrolysis were minimally affected by transformation.
- PMA, but not bryostatin, enhanced PtdEtn hydrolysis in membrane preparations stimulated with ATP and GTP analogue.
- PKC inhibitor blocked PMA's effect only in intact cells.
Conclusions:
- Oncogene transformation differentially affects PMA- and bryostatin-induced PtdEtn hydrolysis.
- The findings suggest that PMA's action in transformed cells involves at least two distinct mechanisms.
- This differential response highlights the complex interplay between oncogenic transformation and signal transduction pathways.