MITOGEN MEDIATED SIGNAL TRANSDUCTION IN CARCINOGENESIS

利用基因工程的嵌合分子在表皮生长之间 因子受体(EGFR)和gp185-erbB-2，本研究旨在 研究和表征不同基因的分子基础 EGFR和erbB-2激酶的生物学特性。NIH3T3细胞， 过度表达(NIH-EGFR)或嵌合EGFR/erbB-2受体(NIH-EGFR) 含有EGFR胞外配体结合域的EGFR/erbB-2) 和gp185-erbB-2的胞内转导功能域，分别为 在37℃下用50 NM EGF触发15分钟 (PTyr)蛋白与抗pTyr抗体免疫共沉淀 采用双向聚丙烯酰胺凝胶电泳法(2D- PAGE)和抗pTyr免疫印迹分析。有几种蛋白质 被EGFR或erbB-2特异性磷酸化，明显 不同的化学计量比。在NIH3T3细胞中，erbB-2的表达是NIH3T3细胞的100倍 转化能力强于EGFR。伴随着这种转变能力的是 Erb B-2、AS对至少28种蛋白质的选择性磷酸化 与EGFR相比。以确定哪些底物可能直接 参与有丝分裂信号，磷酸化蛋白质的2D-PAGE图谱 EGFR/erbB-2-TK嵌合分子对酪氨酸的作用 ErbB-2的TK结构域取代了EGFR的结构域，EGFR/ret，a 嵌合体包含RET激酶的胞内部分，一种TK 属于不同的受体酪氨酸激酶(RTK)家族， 被利用了。EGFR/erbB-2-Tk和EGFR/ret都表现为erbB-2样蛋白 生物学表型和高效转化NIH3T3。确实是 预期这种嵌合体的使用可以帮助识别 ErbB-2介导的有丝分裂信号的特定底物。总而言之， PTyr蛋白的表达模式、巴西林和23 kDa底物 一旦有效，总是被磷酸化到更高的化学计量比 有丝分裂和转化信号被激活。现建议： 巴西林和23 kDa底物是erbB-2的重要成分 和ret激活的有丝分裂信号。

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.