Huckle W R, Dy R C, Earp H S
Lineberger Comprehensive Cancer Center, Department of Medicine, School of Medicine, University of North Carolina, Chapel Hill 27599-7295.
Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8837-41. doi: 10.1073/pnas.89.18.8837.
The cellular effects of numerous hormones and neurotransmitters, including the vasoactive agents angiotensin II (AngII) and [Arg8]vasopressin, are mediated in part by protein-serine threonine kinases activated by increase of cytosolic Ca2+ concentration. In this study, we have tested the ability of Ca(2+)-mobilizing agents to activate cellular tyrosine kinases. Treatment of intact GN4 liver epithelial cells with AngII rapidly (less than or equal to 15 sec) increased tyrosine kinase activity measured either in unfractionated cell lysates or in anti-phosphotyrosine immune complexes from detergent-solubilized cells. Increased phosphorylation of the exogenous substrate poly(Glu80Tyr20) (3- to 4-fold over control) by immunoprecipitated kinases closely paralleled the time- and dose-dependence of the appearance of tyrosine phosphoproteins in intact cells. This effect of AngII was mimicked by thapsigargin, a Ca(2+)-elevating tumor promoter. The ability of AngII, but not epidermal growth factor, to increase tyrosine kinase activity was blocked in cells loaded with the Ca2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. Dephosphorylation of immunoprecipitated proteins by tyrosine phosphatase treatment was accompanied by a 60-70% loss in in vitro kinase activity, suggesting that the AngII-sensitive kinase(s) are activated by phosphorylation in intact cells. These findings demonstrate a link between two widely occurring signaling pathways, the tyrosine kinases and the Ca2+ second-messenger system, and suggest the possible involvement of Ca(2+)-activated tyrosine kinases in the endocrine actions of AngII and [Arg8]vasopressin.
包括血管活性因子血管紧张素 II（AngII）和[精氨酸 8]加压素在内的众多激素和神经递质的细胞效应，部分是由胞质 Ca2+浓度升高激活的蛋白丝氨酸苏氨酸激酶介导的。在本研究中，我们测试了 Ca2+动员剂激活细胞酪氨酸激酶的能力。用 AngII 处理完整的 GN4 肝上皮细胞后，在未分级的细胞裂解物或去污剂溶解细胞的抗磷酸酪氨酸免疫复合物中测量的酪氨酸激酶活性迅速（小于或等于 15 秒）增加。免疫沉淀激酶对外源底物聚（Glu80Tyr20）的磷酸化增加（比对照高 3 至 4 倍）与完整细胞中酪氨酸磷酸化蛋白出现的时间和剂量依赖性密切平行。毒胡萝卜素（一种 Ca2+升高的肿瘤促进剂）模拟了 AngII 的这种作用。在加载 Ca2+螯合剂双（O-氨基苯氧基）乙烷-N，N，N'，N'-四乙酸的细胞中，AngII 增加酪氨酸激酶活性的能力被阻断，而表皮生长因子则没有。酪氨酸磷酸酶处理使免疫沉淀蛋白去磷酸化的同时，体外激酶活性损失 60 - 70%，这表明 AngII 敏感的激酶在完整细胞中通过磷酸化被激活。这些发现证明了两个广泛存在的信号通路——酪氨酸激酶和 Ca2+第二信使系统之间的联系，并提示 Ca2+激活的酪氨酸激酶可能参与 AngII 和[精氨酸 8]加压素的内分泌作用。
