Effects of normal and mutant ras genes on inositol lipid metabolism in Dictyostelium.

Dictyostelium cells transformed with multiple copies of a mutant Dictyostelium ras gene (ras-Thr12 that gave a Gly to Thr substitution at position 12 of the ras protein, showed 2 to 3 times greater incorporation of 32P into PtdInsP and PtdInsP2 (without changing the specific radioactivity) compared to the untransformed strain or a strain transformed with multiple copies of the normal ras-Gly12 gene. The ratio of labelled PtdInsP2/PtdInsP, however, was not affected by the ras-Thr12 gene. Stimulation with the chemoattractant, cyclic AMP, caused a rapid but transient decrease in the levels of labelled PtdInsP and PtdInsP2 in the normal and ras-Gly12-transformed strains but ras-Thr12-transformed strains failed to respond. In untransformed cells a small, very rapid rise in the level of labelled PtdInsP and PtdInsP2 was seen immediately after stimulation of the cells with cyclic AMP (before the transient decrease) and this rise was greatly accentuated in cells transformed with multiple copies of the normal ras-Gly12 gene. Agents that induce prolonged activation of phosphoinositidase C such as AlF4- or GTPYS gave a lowered steady-state level of incorporation of 32P into PtdInsP and PtdInsP2 in all strains. The results indicate that the enzyme in the inositol phosphate pathway that is affected by the ras gene is not phosphoinositidase C, but is an enzyme before PtdInsP kinase, possibly PtdIns kinase.