erbB2 通过与 erbB1 形成异二聚体表达对于电离辐射诱导但不是 EGF 诱导的 Akt 生存途径的激活是必需的。

ErbB2 expression through heterodimerization with erbB1 is necessary for ionizing radiation- but not EGF-induced activation of Akt survival pathway.

机构信息

Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany.

出版信息

Radiother Oncol. 2010 Nov;97(2):338-45. doi: 10.1016/j.radonc.2010.03.008. Epub 2010 Mar 27.

DOI:10.1016/j.radonc.2010.03.008

PMID:20347166

Abstract

PURPOSE

ErbB1-dependent Akt phosphorylation improves post-irradiation cellular survival. In the present study, we investigated the contribution of erbB2 as a heterodimerization partner of erbB1 in activation of Akt survival signaling after irradiation or EGF treatment.

MATERIALS AND METHODS

Pattern of receptor dimerization and protein phosphorylation were investigated by Western and immunoblotting as well as immunoprecipitation techniques. Residual DNA double-strand breaks (DNA-DSB) and clonogenic activity were analyzed by γH2AX and standard clonogenic assay. To knocked erbB2 expression siRNA was used.

RESULTS

In lung carcinoma cell lines A549 and H661, the erbB1-tyrosine kinase (TK) inhibitor erlotinib blocked EGF as well as ionizing radiation (IR)-induced Akt and DNA-PKcs phosphorylation. Targeting Akt and erbB1 induced cellular radiation sensitivity while, the erbB2-TK inhibitor AG825 neither affected phosphorylation of Akt and DNA-PKcs nor induced radiosensitization. ErbB2-siRNA and the anti-erbB2 antibody trastuzumab blocked IR-induced, but not EGF-stimulated Akt phosphorylation and impaired the repair of DNA-DSB. Likewise, IR but not EGF enhanced erbB1/erbB2 heterodimerization and resulted in the release of phosphorylated erbB2 cleavage products p135 and p95. Trastuzumab prevented radiation-induced formation of an active erbB1/erbB2 heterodimer and increased cellular radiation sensitivity. ErbB1- but not erbB2-TK inhibition stabilized erbB2 (p185) through preventing its cleavage.

CONCLUSIONS

The data indicates that ErbB2 through heterodimerization with erbB1 is necessary for the activation of Akt signaling following irradiation but not following EGF treatment.

摘要

目的

ErbB1 依赖性 Akt 磷酸化可改善放射后细胞存活。本研究探讨了 erbB2 作为 erbB1 的异二聚化伙伴在放射或 EGF 处理后激活 Akt 存活信号中的作用。

材料和方法

通过 Western 和免疫印迹以及免疫沉淀技术研究受体二聚化和蛋白磷酸化的模式。通过 γH2AX 和标准克隆形成测定分析残留的 DNA 双链断裂（DNA-DSB）和克隆形成活性。使用针对 erbB2 的 siRNA 敲除 erbB2 表达。

结果

在肺癌细胞系 A549 和 H661 中，erbB1 酪氨酸激酶（TK）抑制剂厄洛替尼阻断了 EGF 以及电离辐射（IR）诱导的 Akt 和 DNA-PKcs 磷酸化。靶向 Akt 和 erbB1 诱导细胞放射敏感性，而 erbB2-TK 抑制剂 AG825 既不影响 Akt 和 DNA-PKcs 的磷酸化，也不诱导放射增敏。erbB2-siRNA 和抗 erbB2 抗体曲妥珠单抗阻断了 IR 诱导的 Akt 磷酸化，但不阻断 EGF 刺激的 Akt 磷酸化，并损害了 DNA-DSB 的修复。同样，IR 而非 EGF 增强了 erbB1/erbB2 异二聚体的形成，并导致磷酸化的 erbB2 裂解产物 p135 和 p95 的释放。曲妥珠单抗阻止了放射诱导的活性 erbB1/erbB2 异二聚体的形成，并增加了细胞的放射敏感性。erbB1-TK 抑制而非 erbB2-TK 抑制通过防止其裂解稳定了 erbB2（p185）。