Inokuchi S, Imboden J B
Department of Medicine, University of California, San Francisco.
J Biol Chem. 1990 Apr 15;265(11):5983-9.
Stimulation of the human T cell line, Jurkat, by the addition of monoclonal antibodies reactive with the T cell antigen receptor complex (CD3/Ti) leads to sustained increases in levels of inositol 1,4,5-trisphosphate. To investigate the possibility that the production of polyphosphoinositides is regulated during CD3/Ti stimulation, we studied Jurkat cells whose inositol phospholipids had been labeled to steady state with [3H]inositol, as well as Jurkat cells during nonequilibrium labeling with [32P]orthophosphate. The addition of CD3 monoclonal antibodies led to a 4-5-fold increase in [3H]inositol trisphosphate that was sustained for greater than 20 min. Within 60 s of CD3/Ti stimulation, [3H] phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) decreased by 65 and 35%, respectively. This change in [3H]PtdIns(4,5)P2 persisted for greater than 20 min. The decrease in [3H]PtdIns4P, however, was transient, and, after 5 min, the levels of [3H]PtdIns4P were comparable in stimulated and unstimulated cells. To examine the rate of flux through inositol phospholipids, we measured the CD3/Ti-stimulated changes in the ratio, 32P cpm/3H cpm, in each inositol phospholipid. CD3/Ti stimulation led to accelerated fluxes through PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) that were maintained for greater than 20 min. After the initial 30 s, however, there was no detectable effect of anti-CD3 on flux through Ptsins4p. This observation suggested that, during CD3/Ti stimulation, production of PtdIns(4,5)P2 from PtdIns might occur via a small pool of PtdIns4P with a very high turnover. The existence of such a pool was established by determining that, in stimulated cells, the 32P-specific activity of the 1-position phosphate of PtdIns(4,5)P2 was 8-10-fold that of PtdIns4P. We conclude that, during the initial 60 s of CD3/Ti stimulation, there is a substantial depletion of cellular PtdIns(4,5)P2 and PtdIns4P. Thereafter, a CD3/Ti-regulated pathway generates PtdIns(4,5)P2 from PtdIns through a small, but highly labile, pool of PtdIns4P.
通过添加与T细胞抗原受体复合物（CD3/Ti）反应的单克隆抗体刺激人T细胞系Jurkat，会导致肌醇1,4,5 -三磷酸水平持续升高。为了研究在CD3/Ti刺激过程中多磷酸肌醇的产生是否受到调节，我们研究了用[3H]肌醇将肌醇磷脂标记至稳态的Jurkat细胞，以及在用[32P]正磷酸盐进行非平衡标记期间的Jurkat细胞。添加CD3单克隆抗体导致[3H]肌醇三磷酸增加4 - 5倍，并持续超过20分钟。在CD3/Ti刺激的60秒内，[3H]磷脂酰肌醇4,5 -二磷酸（PtdIns(4,5)P2）和[3H]磷脂酰肌醇4 -磷酸（PtdIns4P）分别下降了65%和35%。[3H]PtdIns(4,5)P2的这种变化持续超过20分钟。然而，[3H]PtdIns4P的下降是短暂的，5分钟后，刺激细胞和未刺激细胞中的[3H]PtdIns4P水平相当。为了检测肌醇磷脂的通量速率，我们测量了CD3/Ti刺激后每种肌醇磷脂中32P cpm/3H cpm比值的变化。CD3/Ti刺激导致通过PtdIns(4,5)P2和磷脂酰肌醇（PtdIns）的通量加速，并持续超过20分钟。然而，在最初的30秒后，抗CD3对通过Ptsins4p的通量没有可检测到的影响。这一观察结果表明，在CD3/Ti刺激过程中，从PtdIns产生PtdIns(4,5)P2可能是通过一小部分周转非常快的PtdIns4P池进行的。通过确定在刺激细胞中PtdIns(4,5)P2的1 -位磷酸的32P比活性是PtdIns4P的8 - 10倍，证实了这样一个池的存在。我们得出结论，在CD3/Ti刺激的最初60秒内，细胞中的PtdIns(4,5)P2和PtdIns4P大量消耗。此后，一条由CD3/Ti调节的途径通过一小部分但高度不稳定的PtdIns4P池从PtdIns产生PtdIns(4,5)P2。
