Schorb W, Peeler T C, Madigan N N, Conrad K M, Baker K M
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
J Biol Chem. 1994 Jul 29;269(30):19626-32.
Angiotensin II has been demonstrated to act as a growth factor in rat cardiac fibroblasts. However, the signaling events that lead to fibroblast cell growth in response to angiotensin II remain to be elucidated. This study was designed to determine whether angiotensin II stimulated tyrosine phosphorylation of proteins in cardiac fibroblasts. Immunoblot analysis demonstrated rapid tyrosine phosphorylation of distinct substrates of 125, 95, 46-60, and 44 kDa in response to 10 nM angiotensin II. Tyrosine phosphorylation was maximal at 5 min and persisted for at least 180 min. Additional tyrosine-phosphorylated proteins of 185, 145, and 85 kDa were detected in response to 10 ng/ml platelet-derived growth factor BB. A cluster of 75-80-kDa proteins were phosphorylated in response to angiotensin II, phorbol ester, and platelet-derived growth factor. Angiotensin II-induced tyrosine phosphorylation was unaffected by phorbol ester-sensitive protein kinase C down-regulation and could be partially blocked by pertussis toxin pretreatment. Angiotensin II stimulation resulted in increased cytosolic tyrosine kinase activity which was recovered by immunoprecipitation. Immunoblot analysis demonstrated tyrosine phosphorylation of p44MAPK, and, in addition, we demonstrated for the first time tyrosine phosphorylation of p125FAK, p46SHC, and p56SHC in response to angiotensin II. The finding that angiotensin II and platelet-derived growth factor stimulated tyrosine phosphorylation of p46SHC and p56SHC suggested that this protein may serve as a common tyrosine kinase substrate in the mitogenic signaling cascade induced by G-protein-coupled receptors and growth factors and is consistent with the hypothesis that angiotensin II-induced tyrosine phosphorylation is involved in mitogenic signaling pathways in neonatal rat cardiac fibroblasts.
血管紧张素II已被证明在大鼠心脏成纤维细胞中作为一种生长因子起作用。然而，响应血管紧张素II导致成纤维细胞生长的信号转导事件仍有待阐明。本研究旨在确定血管紧张素II是否刺激心脏成纤维细胞中蛋白质的酪氨酸磷酸化。免疫印迹分析表明，响应10 nM血管紧张素II，125、95、46 - 60和44 kDa的不同底物迅速发生酪氨酸磷酸化。酪氨酸磷酸化在5分钟时达到最大值，并持续至少180分钟。响应10 ng/ml血小板衍生生长因子BB，检测到另外185、145和85 kDa的酪氨酸磷酸化蛋白。一组75 - 80 kDa的蛋白响应血管紧张素II、佛波酯和血小板衍生生长因子发生磷酸化。血管紧张素II诱导的酪氨酸磷酸化不受佛波酯敏感的蛋白激酶C下调的影响，并且可被百日咳毒素预处理部分阻断。血管紧张素II刺激导致胞质酪氨酸激酶活性增加，该活性可通过免疫沉淀回收。免疫印迹分析表明p44MAPK发生酪氨酸磷酸化，此外，我们首次证明响应血管紧张素II，p125FAK、p46SHC和p56SHC发生酪氨酸磷酸化。血管紧张素II和血小板衍生生长因子刺激p46SHC和p56SHC的酪氨酸磷酸化这一发现表明，该蛋白可能在由G蛋白偶联受体和生长因子诱导的有丝分裂信号级联反应中作为一种常见的酪氨酸激酶底物，并且与血管紧张素II诱导的酪氨酸磷酸化参与新生大鼠心脏成纤维细胞有丝分裂信号通路的假说一致。
