Overexpression of C-terminal Src kinase blocks 14,15-epoxyeicosatrienoic acid-induced tyrosine phosphorylation and mitogenesis
Overexpression of C-terminal Src kinase blocks 14,15-epoxyeicosatrienoic acid-induced tyrosine phosphorylation and mitogenesis
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DOI：
10.1074/jbc.275.18.13789
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发表时间：
2000-05-05
影响因子：
4.8
通讯作者：
Harris, RC
中科院分区：
文献类型：
--
作者：
Chen, JR;Capdevila, J;Harris, RC
14，15-环氧二十碳三烯酸（14，15-epoxyeicosatrienoic acid，14，15-EET）是肾上皮细胞株LLCPKc 14的一种有效的促有丝分裂剂，这种促有丝分裂作用依赖于蛋白酪氨酸激酶级联反应的激活，该级联反应导致促有丝分裂剂活化的蛋白激酶和磷脂酰肌醇3-激酶的激活。由于暗示性的证据表明，14，15-EET也激活Src在这些细胞中，我们稳定转染LLCPKc 14与C-末端Src激酶（CSK），抑制Src家族激酶活性的表达构建体。体外Src激酶活性测定证实，在空载体转染的细胞中，（载体细胞），14，15-EET增加Src激酶活性，而在过表达CSK mRNA和免疫反应蛋白的克隆中，（CSK细胞），14，15-EET诱导的Src活化几乎被完全阻断（94%抑制）。感兴趣的是，表皮生长因子（EGF）和胎牛血清（FBS）也增加载体细胞中的Src活性，但不增加CSK细胞中的Src活性，进一步证实了CSK过表达阻止Src活化的能力。CSK细胞对外源性14，15-EET不能增加[H-3]胸苷掺入。用抗磷酸酪氨酸抗体免疫沉淀和细胞外信号调节激酶（ERK）抗体免疫印迹表明，14，15-EET不能激活CSK细胞的ERK 1和ERK 2;然而，EGF和FBS诱导的ERK活化与在载体细胞中所见的没有不同。在载体细胞中，14，15-EET刺激的ERK酪氨酸磷酸化被1 μ M PP 2（Src激酶的选择性抑制剂）预处理阻断。本研究表明，14，15-EET主要通过Src激酶介导的途径发挥其促有丝分裂作用，Src激酶介导的途径是迄今为止在肾上皮细胞中14，15-EET激活的酪氨酸激酶级联中确定的最上游信号步骤。
We have previously reported that 14,15-epoxyeicosatrienoic acid (14,15-EET) is a potent mitogen for the renal epithelial cell line, LLCPKc14, This mitogenic effect is dependent upon activation of a protein-tyrosine kinase cascade that results in activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Because of suggestive evidence that 14,15-EET also activated Src in these cells, we stably transfected LLCPKc14 with an expression construct of the C-terminal Src kinase (CSK), which inhibits Src family kinase activity. In vitro Src kinase activity assays confirmed that in empty vector-transfected cells (Vector cells), 14,15-EET increased Src kinase activity, while in clones overexpressing CSK mRNA and immunoreactive protein (CSK cells), 14,15-EET-induced activation of Src was almost completely blocked (94% inhibition), Of interest, epidermal growth factor (EGF) and fetal bovine serum (FBS) also increased Src activity in Vector cells, but not in CSK cells, further confirming the ability of CSK overexpression to prevent Src activation. CSK cells failed to increase [H-3]thymidine incorporation in response to exogenous 14,15-EET. In contrast, both EGF and FBS significantly increased [H-3]thymidine incorporation in CSK cells, Immunoprecipitation with anti-phosphotyrosine antibodies and immunoblotting with an antibody against extracellular signal-regulated kinase (ERK) indicated that in CSK cells, 14,15-EET failed to activate ERK1 and ERK2; however, EGF- and FBS-induced activation of ERKs was not different from that seen in Vector cells, In Vector cells, the 14,15-EET-stimulated tyrosine phosphorylation of ERKs was blocked by pretreatment with 1 mu M PP2, a selective inhibitor of Src kinases. The present study demonstrates that 14,15-EET exerts its mitogenic effects predominantly through a Src kinase-mediated pathway, which is the most upstream signaling step determined to date in the 14,15-EET-activated tyrosine kinase cascade in renal epithelial cells.