Ecto-phosphorylation on aortic endothelial cells. Exquisite sensitivity to staurosporine.
Ecto-phosphorylation on aortic endothelial cells. Exquisite sensitivity to staurosporine.
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主动脉内皮细胞的外磷酸化。
DOI：
10.1042/bj2850585
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
J. Boeynaems
中科院分区：
文献类型：
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作者：
S. Pirotton;O. Boutherin;B. Robaye;J. Boeynaems
从牛主动脉内皮细胞（BAEC）与[γ-32 P]ATP孵育的蛋白质的一维和二维凝胶电泳显示的细胞相关的21 kDa底物的优先标记。该条带的标记在30 s内可检测到，增加至30 min，并在ATP洗脱后稳定至少3 h。在细胞与[γ-35S]ATP孵育后，也标记了该蛋白质。如果在标记期之前或之后用低浓度胰蛋白酶（0.01%）处理内皮细胞，则不会发生放射性掺入21 kDa条带。BAEC蛋白磷酸化的[γ-32 P]ATP的模式是完全不同的胎牛血清用于细胞培养。在BAEC与[γ-32 P]ATP孵育期间血清的存在没有定性地改变标记模式，特别是没有增强21 kDa底物的磷酸化;这表明激酶和21 kDa底物都不被吸附的血清蛋白。Staurosporine是一种低特异性的蛋白激酶抑制剂，可降低21 kDa蛋白的标记，IC 50为2 nM。相比之下，在100 nM时，星形孢菌素没有减少ATP通过激活P2 γ受体诱导的肌醇磷酸的积累。这些数据表明存在的主动脉内皮细胞的胞外激酶，其使用细胞外ATP产生的选择性和长寿命的磷酸化的21 kDa的内皮底物。除了P2 γ受体之外，该蛋白质的外磷酸化可能在ATP调节内皮细胞功能中起作用[Boeynaems & Pearson（1990）Trends Pharmacol. Sci. 11，34-37]。外磷酸化对星形孢菌素抑制的敏感性和某些异喹啉磺酰胺化合物的特异性抑制为研究这一假说提供了潜在的药理学工具。
One- and two-dimensional gel electrophoresis of proteins from bovine aortic endothelial cells (BAEC) incubated with [gamma-32P]ATP revealed the preferential labelling of a cell-associated 21 kDa substrate. The labelling of this band was detectable within 30 s, increased up to 30 min and was stable for at least 3 h following the wash-out of the ATP. This protein was also labelled after incubation of the cells with [gamma-35S]ATP. Incorporation of radioactivity into the 21 kDa band did not occur if the endothelial cells were treated with low concentrations of trypsin (0.01%) before or after the labelling period. The pattern of BAEC protein phosphorylation by [gamma-32P]ATP was completely different from that of the fetal calf serum used for the cell culture. The presence of serum during the incubation of BAEC with [gamma-32P]ATP did not modify qualitatively the labelling pattern and, in particular, did not enhance the phosphorylation of the 21 kDa substrate; this suggests that neither the kinase nor the 21 kDa substrate are adsorbed serum proteins. Staurosporine, a protein kinase inhibitor with low specificity, decreased the labelling of the 21 kDa protein with an IC50 of 2 nM. In contrast, at 100 nM, staurosporine did not decrease the accumulation of inositol phosphates induced by ATP via the activation of P2y receptors. These data indicate the presence of aortic endothelial cells of an ecto-kinase which uses extracellular ATP to produce the selective and long-lived phosphorylation of a 21 kDa endothelial substrate. Ecto-phosphorylation of this protein might play a role in the modulation of endothelial cell functions by ATP, in addition to the P2y receptors [Boeynaems & Pearson (1990) Trends Pharmacol. Sci. 11, 34-37]. The exquisite sensitivity of ecto-phosphorylation to inhibition by staurosporine and its specific inhibition by some isoquinolinesulphonamide compounds provide potential pharmacological tools to investigate this hypothesis.