Calcium-dependent stimulation of mitogen-activated protein kinase activity in A431 cells by low doses of ionizing radiation.
Calcium-dependent stimulation of mitogen-activated protein kinase activity in A431 cells by low doses of ionizing radiation.
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DOI：
10.2307/3579904
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发表时间：
1998-06
影响因子：
3.4
通讯作者：
Brian D. Kavanagh;P. Dent;Rupert Schmidt-Ullrich;Phylip Chen;Ross B. Mikkelsen
中科院分区：
文献类型：
--
作者：
Brian D. Kavanagh;P. Dent;Rupert Schmidt-Ullrich;Phylip Chen;Ross B. Mikkelsen
2Gy射线电离辐射可激活A431鳞癌细胞中的表皮生长因子受体(EGFR)激酶活性，从而瞬时激活下游效应因子--丝裂原活化蛋白激酶(MAPK)。剂量-反应分析显示，0.5Gy3-5min后4倍激活，4Gy后无额外激活。根据蛋白激酶C下调和蛋白激酶C抑制剂白屈菜红碱的边际效应来定义，激活是独立于蛋白激酶C的。相反，细胞内钙离子螯合剂(BAPTA/AM)、钙离子拮抗剂(TMB-8)和磷脂酶C抑制剂(U73223)均可阻断MAPK的刺激。上游成分Raf-1也通过一种依赖于EGFR和钙离子的机制被激活。BAPTA/AM和TMB-8抑制了Raf-1的激活，通过酪氨酸磷酸化和与RAS的免疫共沉淀来监测，表明钙依赖的步骤发生在Ras和Raf-1相互作用的前后。Ras鸟苷三磷酸交换蛋白(SOS)和与MAPK途径相关的钙激活的酪氨酸激酶(粘着斑激酶和PYK2)都不受辐射刺激。相反，在免疫共沉淀实验中，通过增强SOS与EGFR的结合，EGF激活了SOS。这些结果表明，辐射诱导的EGFR依赖的下游信号的激活不同于EGFR的天然配体诱导的激活。
Ionizing radiation at 2 Gy activates the epidermal growth factor receptor (EGFR) kinase activity in A431 squamous carcinoma cells and as a consequence transiently activates a downstream effector, mitogen-activated protein kinase (MAPK). A dose-response analysis shows fourfold activation 3-5 min after irradiation at 0.5 Gy with no additional activation after doses up to 4 Gy. Activation is independent of protein kinase C as defined by marginal effects of protein kinase C down-regulation and the protein kinase C inhibitor, chelerythrine. In contrast, an intracellular Ca2+ chelator (BAPTA/AM), a Ca2+ antagonist (TMB-8) and a phospholipase C inhibitor (U73223), which inhibits radiation-induced Ca2+ oscillations, all block MAPK stimulation. The upstream component, Raf-1, is also activated through a mechanism that is dependent on EGFR and Ca2+. Activation of Raf-1, monitored by tyrosine phosphorylation and co-immunoprecipitation with Ras, was inhibited by BAPTA/AM and TMB-8, indicating that the Ca2+-dependent step occurs at or before the interaction of Ras and Raf-1. Neither the Ras guanosine triphosphate exchange protein, SOS, nor Ca2+-activated tyrosine kinases linked to the MAPK pathway, focal adhesion kinase and PYK2, were stimulated by radiation. In contrast, EGF activated SOS as shown by the enhanced association of SOS with EGFR in co-immunoprecipitation experiments. These results suggest that activation of EGFR-dependent downstream signaling induced by radiation differs from that induced by the natural ligands of EGFR.