2,3,7,8-tetrachlorodibenzo-p-dioxin-induced activation of a protein tyrosine kinase, pp60(src), in murine hepatic cytosol using a cell-free system
2,3,7,8-tetrachlorodibenzo-p-dioxin-induced activation of a protein tyrosine kinase, pp60(src), in murine hepatic cytosol using a cell-free system
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DOI：
10.1124/mol.52.4.667
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发表时间：
1997-10-01
影响因子：
3.6
通讯作者：
Matsumura, F
中科院分区：
文献类型：
--
作者：
Blankenship, A;Matsumura, F
在分离的C57小鼠肝胞质溶胶（100，000 x g上清液）中，发现2，3，7，8-四氯二苯并对二恶英（TCDD）在无细胞和无核条件下激活蛋白激酶。TCDD的这一作用具有芳香烃受体（AHR）依赖性、浓度依赖性，并可被酪氨酸激酶抑制剂genistein抑制。产生蛋白质磷酸化的统计学显著增加的TCDD的最低浓度为10 pM。我们还研究了蛋白激酶与胞质AHR复合物物理相关的可能性。激酶复性试验旨在检测十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后再活化的蛋白激酶，结果显示，使用抗AHR抗体（IgG）和蛋白A/G/琼脂糖珠从肝细胞溶质中获得的洗涤免疫沉淀物中存在60 kDa激酶，但当使用非特异性IgG代替抗AHR抗体时，则不存在。相同的60 kDa条带存在于以类似方式从相同的胞质溶胶制备的免疫沉淀物中，但具有抗热休克蛋白90抗体（IgM）。这60-kDa激酶被发现被激活TCDD处理的整个细胞质从未经处理的小鼠。此外，pp 60（src）免疫沉淀细胞质中，已被预先处理的TCDD在无细胞条件下表现出2倍以上的激酶活性比同等制剂处理的溶剂对照。同样，当使用非特异性IgG代替抗pp 60（src）抗体时，无法检测到TCDD的这种作用。增加蛋白磷酸化后，观察到直接TCDD处理的免疫沉淀物获得使用抗体AHR和pp 60（src），分别，但不当非特异性IgG用于免疫沉淀在任何情况下。这一观察结果是一致的想法，在胞质溶胶中，AHR和pp 60（src）共存的一部分，可以特异性免疫共沉淀的多聚体蛋白质复合物。这些结果提供的证据表明，（i）TCDD激活蛋白激酶在小鼠肝胞质溶胶，（ii）一个60 kDa的蛋白激酶与胞质形式的AHR复合物，（iii）配体结合直接激活这种激酶，因为TCDD处理免疫沉淀的AHR复合物的结果增加蛋白激酶活性，和（iv）AHR相关的蛋白激酶似乎是pp 60（src）激酶。目前的研究结果提供了一个潜在的重要机制的线索，TCDD可以发挥快速，多效性的影响，通过AHR相关激酶改变许多蛋白质的功能，通过级联的蛋白质磷酸化。
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was found to activate protein kinases under cell-and nucleus-free conditions in isolated C57 mouse liver cytosol (100,000 x g supernatant). This action of TCDD was found to be aryl hydrocarbon receptor (AHR) dependent, concentration dependent, and inhibited by genistein, a tyrosine kinase inhibitor. The lowest concentration of TCDD to produce a statistically significant increase in protein phosphorylation was 10 pM. We also investigated the possibility that a protein kinase is physically associated with the cytosolic AHR complex. Kinase renaturation tests designed to detect reactivated protein kinases after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of a 60-kDa kinase in the washed immunoprecipitate obtained from liver cytosol using anti-AHR antibody (IgG) and protein A/G/ agarose beads but not when a nonspecific IgG was used instead of anti-AHR antibody. The same 60-kDa band was present in an immunoprecipitate prepared in a similar manner from the same cytosol but with anti-heat shock protein 90 antibody (IgM). This 60-kDa kinase was found to be activated by TCDD treatment of whole cytosol from untreated mice. Moreover, pp60(src) immunoprecipitated from cytosol that had been previously treated with TCDD under cell-free conditions exhibited 2-fold more kinase activity than the equivalent preparation treated with a solvent control. Again, such an effect of TCDD could not be detected when a nonspecific IgG was used in place of an anti-pp60(src) antibody. Increased protein phosphorylation was observed after direct TCDD treatment of immunoprecipitates obtained using antibodies to AHR and pp60(src), respectively, but not when a nonspecific IgG was used for immunoprecipitation in either case. This observation is consistent with the idea that in cytosol, the AHR and pp60(src) coexist as part of a multimeric protein complex that can be specifically coimmunoprecipitated. These results provide evidence that (i) TCDD activates protein kinases in murine hepatic cytosol, (ii) a 60-kDa protein kinase is associated with the cytosolic form of the AHR complex, (iii) ligand binding directly activates this kinase because TCDD treatment of immunoprecipitated AHR complex results in increased protein kinase activity, and (iv) the AHR-associated protein kinase seems to be pp60(src) kinase. The current findings provide a clue to a potentially important mechanism by which TCDD can exert rapid, pleiotropic effects through the AHR-associated kinase to alter functions of many proteins through a cascade of protein phosphorylations.