Enan E, Matsumura F
Department of Environmental Toxicology, University of California, Davis 95616, USA.
Biochem Pharmacol. 1996 Nov 22;52(10):1599-612. doi: 10.1016/s0006-2952(96)00566-7.
We have shown previously that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) under cell-free conditions causes a significant rise in protein tyrosine kinase activity of cytosol from male guinea pig adipose tissue, and that such an effect of TCDD is Ah-receptor dependent. In the present study, we obtained evidence indicating that c-Src protein kinase is the protein kinase activated by TCDD and that this kinase is associated specifically with the Ah-receptor-complex proteins in cytosol from adipose tissue and liver of guinea pig and liver of C57BI/6J mouse, and in NIH 3T3 mouse fibroblast cells. Here, we present evidence that c-Src protein is functionally attached to the Ah-receptor (AhR) and is specifically activated upon ligand binding. This conclusion is based on several lines of evidence: (a) TCDD caused activation of protein tyrosine kinase activity when administered directly to purified Ah-receptor immunoprecipitate; (b) this stimulatory effect of TCDD was abolished when the cytosol was immunodepleted of c-Src protein or Ah-receptor protein by preincubating with anti-c-Src or anti-Ah-receptor antibody, followed by the addition of TCDD to the remaining portions of cytosol; (c) when Ah-receptor immunoprecipitate was incubated with TCDD, and the kinase(s) released to the supernatant was analyzed on autoradiography of two-dimensional (2D) electrophoresis, 32P-labeled c-Src protein was recognized; (d) the same 32P-labeled-phosphoprotein with M(r) = 60 kDa and pl = 6.1 was found in the immunoprecipitate with anti-c-Src antibody on 2D autoradiograms; (e) this same phosphoprotein disappeared when the supernatant of the Ah-receptor immunoprecipitate was immunodepleted of c-Src protein by anti-c-Src antibody; and (f) a structure-activity relationship study with TCDD and three dioxin-congeners revealed a rank order for their potency in activation of c-Src kinase activity to be identical to that of previously determined toxicity indices: i.e., TCDD > 1,2,3,7,8-pentachlorodibenzo-p-dioxin (1,2,3,7,8-PCDD) > 1,2,4,7,8-pentachlorodibenzo-p-dioxin (1,2,4,7,8-PCDD) > 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). Consistent with these results, TCDD-induced c-Src kinase activity was abolished when c-Src immunoprecipitate's suspension was preincubated with 0.1 or 1 microM alpha-naphthoflavone (AhR blocker) for 10 min prior to the addition of TCDD. In addition, pretreatment of 3T3 fibroblast cells with 3-methylcholanthrene abolished TCDD-induced c-Src kinase activity in AhR-immunoprecipitate. We conclude that c-Src protein kinase is associated specifically with the AhR complex along with hsp90 in the cytosol of these cells and that upon ligand binding to the Ah-receptor subunit, c-Src is activated and released from the complex.
我们之前已经表明，在无细胞条件下，2,3,7,8 - 四氯二苯并 - p - 二恶英（TCDD）会使雄性豚鼠脂肪组织胞质溶胶中的蛋白质酪氨酸激酶活性显著升高，并且TCDD的这种作用依赖于芳烃受体（Ah受体）。在本研究中，我们获得的证据表明，c - Src蛋白激酶是被TCDD激活的蛋白激酶，并且该激酶与豚鼠脂肪组织和肝脏以及C57BI/6J小鼠肝脏的胞质溶胶中的Ah受体复合蛋白特异性相关，在NIH 3T3小鼠成纤维细胞中也是如此。在此，我们提供证据表明c - Src蛋白在功能上与Ah受体（AhR）相连，并在配体结合后被特异性激活。这一结论基于以下几条证据：（a）直接将TCDD施用于纯化的Ah受体免疫沉淀物时，会导致蛋白质酪氨酸激酶活性的激活；（b）当通过用抗c - Src或抗Ah受体抗体预孵育使胞质溶胶中的c - Src蛋白或Ah受体蛋白免疫耗竭，然后向剩余的胞质溶胶部分添加TCDD时，TCDD的这种刺激作用就会消失；（c）当Ah受体免疫沉淀物与TCDD一起孵育，并对释放到上清液中的激酶进行二维（2D）电泳放射自显影分析时，可识别出32P标记的c - Src蛋白；（d）在2D放射自显影图上，用抗c - Src抗体的免疫沉淀物中发现了相同的M(r) = 60 kDa且pl = 6.1的32P标记的磷蛋白；（e）当Ah受体免疫沉淀物的上清液用抗c - Src抗体免疫耗竭c - Src蛋白时，这种相同的磷蛋白消失；（f）对TCDD和三种二恶英同系物进行的构效关系研究表明，它们激活c - Src激酶活性的效力顺序与先前确定的毒性指数顺序相同，即：TCDD > 1,2,3,7,8 - 五氯二苯并 - p - 二恶英（1,2,3,7,8 - PCDD）> 1,2,4,7,8 - 五氯二苯并 - p - 二恶英（1,2,4,7,8 - PCDD）> 2,7 - 二氯二苯并 - p - 二恶英（2,7 - DCDD）。与这些结果一致，在添加TCDD之前，将c - Src免疫沉淀物的悬浮液与0.1或1 microM的α - 萘黄酮（AhR阻断剂）预孵育10分钟，TCDD诱导的c - Src激酶活性就会消失。此外，用3 - 甲基胆蒽预处理3T3成纤维细胞可消除AhR免疫沉淀物中TCDD诱导的c - Src激酶活性。我们得出结论，在这些细胞的胞质溶胶中，c - Src蛋白激酶与AhR复合物以及热休克蛋白90（hsp90）特异性相关，并且在配体与Ah受体亚基结合后，c - Src被激活并从复合物中释放出来。
