Huckle W R, Prokop C A, Dy R C, Herman B, Earp S
Cell Biology Program of the Lineberger Cancer Research Center, Chapel Hill, North Carolina.
Mol Cell Biol. 1990 Dec;10(12):6290-8. doi: 10.1128/mcb.10.12.6290-6298.1990.
Cellular responses to epidermal growth factor (EGF) are dependent on the tyrosine-specific protein kinase activity of the cell-surface EGF receptor. Previous studies using WB rat liver epithelial cells have detected at least 10 proteins whose phosphotyrosine (P-Tyr) content is increased by EGF. In this study, we have examined alternate modes of activating tyrosine phosphorylation. Treatment of WB cells with hormones linked to Ca2+ mobilization and protein kinase C (PKC) activation, including angiotensin II, [Arg8]vasopressin, or epinephrine, stimulated rapid (less than or equal to 15-s) and transient increases in the P-Tyr content of several proteins (p120/125, p75/78, and p66). These proteins, detected by anti-P-Tyr immunoblotting, were similar in molecular weight to a subset of EGF-sensitive P-Tyr-containing proteins (P-Tyr-proteins). The increased P-Tyr content was confirmed by [32P]phosphoamino acid analysis of proteins recovered by anti-P-Tyr immunoprecipitation. Elevating intracellular [Ca2+] with the ionophore A23187 or ionomycin or with the tumor promoter thapsigargin mimicked the effects of hormones on tyrosine phosphorylation, whereas treatment with a PKC-activating phorbol ester did not. In addition, responses to angiotensin II were not diminished in PKC-depleted cells. Ca2+ mobilization, measured by fura-2 fluorescence, was coincident with the increase in tyrosine phosphorylation in response to angiotensin II or thapsigargin. Loading cells with the intracellular Ca2+ chelator bis-(o-aminophenoxy)ethane-N ,N ,N' , N'-tetraacetic acid (BAPTA) inhibited the appearance of all P-Tyr-proteins in response to angiotensin II, thapsigargin, or ionophores, as well as two EGF-stimulated P-Tyr-proteins. The majority of EGF-stimulated P-Tyr-proteins were not affected by BAPTA. These studies indicate that angiotensin II can alter protein-tyrosine phosphorylation in a manner that is secondary to, and apparently dependent on, Ca2+ mobilization. Thus, ligands such as EGF and angiotensin II, which act through distinct types of receptors, may activate secondary pathways involving tyrosine phosphorylation. These results also raise the possibility that certain growth-promoting effects of Ca2+ -mobilizing agents such as angiotensin II may be mediated via tyrosine phosphorylation.
细胞对表皮生长因子（EGF）的反应依赖于细胞表面EGF受体的酪氨酸特异性蛋白激酶活性。先前使用WB大鼠肝上皮细胞的研究已检测到至少10种蛋白质，其磷酸酪氨酸（P-Tyr）含量会因EGF而增加。在本研究中，我们研究了激活酪氨酸磷酸化的其他方式。用与Ca2+动员和蛋白激酶C（PKC）激活相关的激素处理WB细胞，包括血管紧张素II、[Arg8]加压素或肾上腺素，会刺激几种蛋白质（p120/125、p75/78和p66）的P-Tyr含量快速（小于或等于15秒）且短暂增加。通过抗P-Tyr免疫印迹检测到的这些蛋白质，其分子量与一部分对EGF敏感的含P-Tyr蛋白质（P-Tyr蛋白质）相似。通过抗P-Tyr免疫沉淀回收的蛋白质的[32P]磷酸氨基酸分析证实了P-Tyr含量的增加。用离子载体A23187或离子霉素或肿瘤启动子毒胡萝卜素提高细胞内[Ca2+]可模拟激素对酪氨酸磷酸化的作用，而用激活PKC的佛波酯处理则没有这种作用。此外，在PKC缺失的细胞中，对血管紧张素II的反应并未减弱。通过fura-2荧光测量的Ca2+动员与对血管紧张素II或毒胡萝卜素的酪氨酸磷酸化增加同时发生。用细胞内Ca2+螯合剂双（邻氨基苯氧基）乙烷-N，N，N'，N'-四乙酸（BAPTA）加载细胞可抑制对血管紧张素II、毒胡萝卜素或离子载体产生反应的所有P-Tyr蛋白质以及两种EGF刺激的P-Tyr蛋白质的出现。大多数EGF刺激的P-Tyr蛋白质不受BAPTA影响。这些研究表明，血管紧张素II可以以一种继发于且明显依赖于Ca2+动员的方式改变蛋白质酪氨酸磷酸化。因此，诸如EGF和血管紧张素II等通过不同类型受体起作用的配体，可能会激活涉及酪氨酸磷酸化的次级途径。这些结果还增加了一种可能性，即诸如血管紧张素II等Ca2+动员剂的某些促生长作用可能通过酪氨酸磷酸化介导。
