Park D J, Min H K, Rhee S G
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Jan 25;267(3):1496-501.
The mechanisms by which phorbol 12-myristate 13-acetate (PMA) and cAMP attenuate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) induced by ligation of the T-cell antigen receptor complex (TCR) was studied in the human Jurkat T-cell line. It has previously been shown that stimulation of Jurkat cells with antibodies to CD3, components of the TCR, elicits a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1, the predominant PLC isozyme in Jurkat cells, at multiple tyrosine residues and that such tyrosine phosphorylation leads to activation of PLC-gamma 1. Prior incubation of Jurkat cells with PMA or forskolin, which increases intracellular cAMP concentrations, prevented tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of PtdIns 4,5-P2 induced by ligation of CD3. Dose-response curves of PMA and of forskolin for the inhibition of PLC-gamma 1 tyrosine phosphorylation and of PtdIns 4,5-P2 hydrolysis were similar. These results suggest that the inhibition of PtdIns 4,5-P2 hydrolysis by PMA and cAMP is attributable to reduced tyrosine phosphorylation of PLC-gamma 1. Treatment of Jurkat cells with PMA or forskolin stimulated the phosphorylation of PLC-gamma 1 at serine 1248. PMA treatment also elicited the phosphorylation of PLC-gamma 1 at an unidentified serine site. Phosphopeptide map analysis indicated that the sites of PLC-gamma 1 phosphorylated in Jurkat cells treated with PMA and forskolin are the same as those phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), respectively. Stimulation of Jurkat cells with antibodies to CD3 also elicited phosphorylation of PLC-gamma 1 at serine 1248 and at the unidentified serine site phosphorylated in PLC-gamma 1 from PMA-treated cells. Thus, phosphorylation of PLC-gamma 1 by PKC or PKA at serine 1248 may modulate the interaction of PLC-gamma 1 with the protein tyrosine kinase or the protein tyrosine phosphatase; this altered interaction may, at least in part, be responsible for the decreased tyrosine phosphorylation of PLC-gamma 1 seen in PMA- and forskolin-treated Jurkat cells. Furthermore, in the absence of PMA, activation of PKC by diacylglycerol provides a negative feedback signal responsible for reducing the phosphotyrosine contents of PLC-gamma 1.
在人Jurkat T细胞系中研究了佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA）和环磷酸腺苷（cAMP）减弱T细胞抗原受体复合物（TCR）连接诱导的磷脂酰肌醇4,5 -二磷酸（PtdIns 4,5 - P2）水解的机制。先前已经表明，用抗CD3抗体（TCR的组分）刺激Jurkat细胞会引发磷脂酶C（PLC）-γ1（Jurkat细胞中主要的PLC同工酶）在多个酪氨酸残基处快速且短暂的磷酸化，并且这种酪氨酸磷酸化会导致PLC -γ1的激活。用PMA或福斯可林（可增加细胞内cAMP浓度）预先孵育Jurkat细胞，可防止PLC -γ1的酪氨酸磷酸化以及CD3连接诱导的PtdIns 4,5 - P2水解。PMA和福斯可林对PLC -γ1酪氨酸磷酸化和PtdIns 4,5 - P2水解抑制作用的剂量反应曲线相似。这些结果表明，PMA和cAMP对PtdIns 4,5 - P水解的抑制作用归因于PLC -γ1酪氨酸磷酸化的减少。用PMA或福斯可林处理Jurkat细胞会刺激PLC -γ1在丝氨酸1248处的磷酸化。PMA处理还会引发PLC -γ1在一个未明确的丝氨酸位点的磷酸化。磷酸肽图谱分析表明，用PMA和福斯可林处理的Jurkat细胞中PLC -γ1磷酸化的位点分别与蛋白激酶C（PKC）和cAMP依赖性蛋白激酶（PKA）在体外磷酸化的位点相同。用抗CD3抗体刺激Jurkat细胞也会引发PLC -γ1在丝氨酸1248以及PMA处理细胞的PLC -γ1中磷酸化的未明确丝氨酸位点的磷酸化。因此，PKC或PKA在丝氨酸1248处对PLC -γ1的磷酸化可能会调节PLC -γ1与蛋白酪氨酸激酶或蛋白酪氨酸磷酸酶的相互作用；这种改变的相互作用可能至少部分地导致了在PMA和福斯可林处理的Jurkat细胞中所见的PLC -γ1酪氨酸磷酸化的减少。此外，在没有PMA的情况下，二酰基甘油对PKC的激活提供了一个负反馈信号，负责降低PLC -γ1的磷酸酪氨酸含量。
