MITOGEN MEDIATED SIGNAL TRANSDUCTION IN CARCINOGENESIS

致癌过程中丝裂原介导的信号转导

• 批准号: 3752738
• 负责人: S S THORGEIRSSON
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3752738
• 关键词: 3T3 cells; biological signal transduction; carcinogenesis; cell transformation; chimeric proteins; epidermal growth factor; gel electrophoresis; gene expression; growth factor receptors; immunoprecipitation; mutagens; neoplastic transformation; oncogenes; oncoproteins; phosphoproteins; phosphorylation; protein tyrosine kinase; western blottings

Using genetically engineered chimeric molecules between epidermal growth factor receptor (EGFR) and gp185-erbB-2, this study was undertaken to investigate and characterize the molecular basis of the different biological properties of EGFR and erbB-2 kinases. NIH3T3 cells, overexpressing (NIH-EGFR) or the chimeric EGFR/erbB-2 receptor (NIH- EGFR/erbB-2) containing the extracellular ligand-binding domain of EGFR and the intracellular transduction competent-domain of gp185-erbB-2, were triggered with 50 nM EGF for 15 minutes at 37 degrees C. Phosphotyrosine (pTyr) proteins were immunoprecipitated with anti-pTyr antibodies and analyzed using two-dimensional polyacrylamide gel electrophoresis (2D- PAGE) and anti-pTyr immunoblot analysis. Several proteins were specifically phosphorylated by either EGFR or erbB-2 with markedly different stoichiometry. In NIH3T3 cells, erbB-2 is 100-fold more transforming than EGFR. Concomitant with this transforming capacity is the preferential phosphorylation of at least 28 proteins by erbB-2, as compared to EGFR. To identify which substrates might be directly involved in mitogenic signaling, 2D-PAGE maps of proteins phosphorylated on tyrosine by EGFR/erbB-2-TK, a chimeric molecule in which the tyrosine kinase (TK) domain of erbB-2 replaced that of EGFR, and EGFR/ret, a chimera containing the intracellular portion of ret kinase, a TK belonging to a different family of receptor tyrosine kinases (RTKs), were utilized. Both EGFR/erbB-2-TK and EGFR/ret display an erbB-2-like biological phenotype and transform NIH3T3 with high efficiency. It was anticipated that the use of such chimeras could aid in the identification of specific substrates for erbB-2 mediated mitogenic signaling. In all patterns of pTyr protein expression, paxillin and a 23 kDa substrate were invariably phosphorylated to higher stoichiometry whenever potent mitogenic and transforming signals were activated. It is proposed that paxillin and the 23 kDa substrate are important elements in the erbB-2 and ret-activated mitogenic signaling.

在表皮生长之间使用基因工程嵌合分子 因子受体（EGFR）和gp 185-erbB-2，这项研究进行， 研究和表征不同的分子基础 EGFR和erbB-2激酶的生物学特性。 NIH 3 T3细胞， 过表达EGFR（NIH-EGFR）或嵌合EGFR/erbB-2受体（NIH-erbB-2受体）的小鼠， EGFR/erbB-2）的细胞外配体结合结构域 和gp 185-erbB-2的胞内转导活性域， 在37 ℃下用50 nM EGF触发15分钟。 磷酸酪氨酸 用抗-pTyr抗体免疫沉淀（pTyr）蛋白， 使用二维聚丙烯酰胺凝胶电泳（2D- PAGE）和抗pTyr免疫印迹分析。 几种蛋白质被 被EGFR或erbB-2特异性磷酸化，并显着 不同的化学计量 在NIH 3 T3细胞中，erbB-2比正常细胞高100倍。 比EGFR转化。 与这种转变能力相伴随的是 erbB-2对至少28种蛋白质的优先磷酸化， 与EGFR相比。 为了确定哪些底物可能直接 参与有丝分裂信号传导，磷酸化蛋白质的2D-PAGE图谱 EGFR/erbB-2-TK（一种嵌合分子，其中酪氨酸 erbB-2的激酶（TK）结构域取代了EGFR的结构域，而EGFR/ret是一种 含有ret激酶（一种TK）细胞内部分的嵌合体 属于不同的受体酪氨酸激酶（RTK）家族， 利用。 EGFR/erbB-2-TK和EGFR/ret均表现出erbB-2样表达。 生物学表型，高效转化NIH 3 T3。 这是 预计使用这种嵌合体可以帮助识别 erbB-2介导的促有丝分裂信号的特异性底物。 在所有 pTyr蛋白表达、桩蛋白和23 kDa底物的模式是 总是磷酸化到更高的化学计量， 有丝分裂和转化信号被激活。 拟将 桩蛋白和23 kDa底物是erbB-2中的重要元件 和RET激活的促有丝分裂信号传导。