Al-Nedawi K N, Pawłowska Z, Cierniewski C S
Department of Biophysics, Medical University of Lódź, Poland.
Acta Biochim Pol. 1999;46(3):693-702.
The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.
在内皮细胞质膜外表面已证实存在蛋白激酶活性及其磷酸化产物。通过将原代内皮细胞（人脐静脉内皮细胞）和内皮细胞系EA.hy 926与[γ-32P]ATP孵育来检测细胞外磷酸化。将反应产物进行SDS/PAGE、放射自显影和扫描密度测定。在实验条件下，两种细胞中均有5种表观分子量分别为19、23、55、88和110 kDa的蛋白质显著磷酸化。19 kDa蛋白质的磷酸化最快，60秒后达到最大值，然后该蛋白质去磷酸化。负责膜蛋白表面标记的胞外蛋白激酶通过使用以下物质进行表征：(a)蛋白激酶C抑制剂：K-252b、氯化白屈菜红碱和[Ala113]髓鞘碱性蛋白(104 - 118)；(b)蛋白激酶A抑制剂肯普肽8334；以及(c)酪蛋白激酶II抑制剂5,6-二氯-1-β-D-呋喃核糖基苯并咪唑(DRB)。用肿瘤坏死因子α(TNFα)和干扰素γ(IFNγ)刺激内皮细胞与所有膜蛋白细胞外磷酸化降低20 - 80%相关。与膜受体结合的IFNγ迅速磷酸化。仅在IFNγ的情况下，它与出现一条对应于IFNγ自身的17 kDa强磷酸化带相关。这种17 kDa外源底物的磷酸化被胞外激酶抑制剂K-252b阻止。通过测试微囊藻毒素LR（一种抑制PP-1和PP-2a磷蛋白磷酸酶的膜不可渗透试剂）的作用，证明了内皮细胞中存在胞外磷蛋白磷酸酶活性。在微囊藻毒素存在的情况下，19 kDa和ll0 kDa磷蛋白的磷酸化程度显著增加。我们的结果表明内皮细胞上至少存在两种胞外激酶活性，它们可能在细胞因子功能调节中起重要作用。
