Wood C A, Europe-Finner G N, Newell P C
Department of Biochemistry, University of Oxford, U.K.
Cell Signal. 1991;3(5):473-81. doi: 10.1016/0898-6568(91)90077-8.
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.
用突变型盘基网柄菌ras基因（ras-Thr12，其导致ras蛋白第12位的甘氨酸被苏氨酸取代）的多个拷贝转化的盘基网柄菌细胞，与未转化的菌株或用正常ras-Gly12基因的多个拷贝转化的菌株相比，显示出32P掺入磷脂酰肌醇磷酸（PtdInsP）和磷脂酰肌醇二磷酸（PtdInsP2）的量增加了2至3倍（而不改变比放射性）。然而，标记的PtdInsP2/PtdInsP的比率不受ras-Thr12基因的影响。用趋化剂环磷酸腺苷（cAMP）刺激后，正常和ras-Gly12转化菌株中标记的PtdInsP和PtdInsP2水平迅速但短暂下降，但ras-Thr12转化菌株没有反应。在未转化的细胞中，在用cAMP刺激细胞后（在短暂下降之前），立即观察到标记的PtdInsP和PtdInsP2水平有一个小的、非常迅速的上升，并且在用正常ras-Gly12基因的多个拷贝转化的细胞中这种上升大大加剧。诱导磷脂酶C长期激活的试剂，如AlF4-或鸟苷5'-O-(3-硫代三磷酸)（GTPγS），使所有菌株中32P掺入PtdInsP和PtdInsP2的稳态水平降低。结果表明，受ras基因影响的肌醇磷酸途径中的酶不是磷脂酶C，而是在PtdInsP激酶之前的一种酶，可能是磷脂酰肌醇激酶。
