DECREASED ACTIVITIES OF PHOSPHATIDATE PHOSPHOHYDROLASE AND PHOSPHOLIPASE-D IN RAS AND TYROSINE KINASE (FPS) TRANSFORMED FIBROBLASTS

The activity of N-ethylmaleimide-insensitive phosphatidate phosphohydr olase (PAP-2) was characterized in control, ras-transformed, and tyros ine kinase-(fps) transformed rat fibroblasts. PAP-2 was assayed in two different ways: 1) within its natural membrane using liposomes of pho sphatidate and 2) in the presence of sufficient Triton X-100 to solubi lize PAP-2, and to form mixed micelles with the phosphatidate. Harvest ing the fibroblasts in medium containing orthovanadate and Zn2+ gave u p to 3-fold higher PAP-2 activities when measured in the absence, but not in the presence, of Triton X-100. PAP-2-specific activities from b oth assays increased in the control fibroblasts as the cells reached c onfluence. Both specific activities were lower in the oncogenically tr ansformed fibroblasts than in controls at all cell densities tested. T he specific activities of PAP-2 did not increase with time in culture in transformed cells which continued to divide. The relative increase in activity of phospholipase D after stimulation with serum or phorbol myristate acetate was lower in the transformed fibroblasts compared t o control cells. This indicates a coordinated decrease in. the phospho lipase D/phosphatidate phosphohydrolase pathway at the level of both e nzymes in ras and fps transformed fibroblasts. The ratio of the produc tion of diacylglycerol relative to phosphatidate, after stimulation wi th serum, or phorbol ester, was lower in both transformed fibroblasts relative to the controls. This is compatible with the decreased specif ic activity of PAP-2 and indicates functional significance for the dif ferences in PAP-2 activity in regulating the balance between the two m itogenic lipids, phosphatidate and diacylglycerol. Control of PAP-2 ac tivity could be an important factor in regulating appropriate signals for cell division.