Salh B, Marotta A, Matthewson C, Ahluwalia M, Flint J, Owen D, Pelech S
Department of Medicine, University of British Columbia, Vancouver, Canada.
Anticancer Res. 1999 Jan-Feb;19(1B):731-40.
Mitogenic signaling through the principal growth factor receptor tyrosine kinase (RTK) pathway, i.e. RTK-->Ras-->Raf-->Mek-->MAPK has been implicated in the pathogenesis of human cancer. However, biochemical characterization of this has not been adequately assessed in human cancers.
Using extracts from 23 human breast cancers and control tissue from the same resected specimens, the protein levels, phosphotransferase activities and subcellular locations of the mitogen-activated protein (MAP) kinase isoforms p42 Erk2 and p44 Erk1 were examined, together with their phosphotransferase activities towards myelin basic protein (MBP) and a peptide substrate patterned after the Thr-669 site in the epidermal growth factor receptor (EGFR T669) that is phosphorylated by MAP kinase.
Overexpression of both Erk2 and Erk1 isoforms was evident using specific antibodies. A universal activation of MBP and EGFR T669 peptide phosphotransferase activities was also found (up to 3-fold). MonoQ fractionation resolved the bulk of the EGFR T669 peptide phosphorylation from elution of the MAP kinase protein. Erk1 and Erk2 activities determined by specific immunoprecipitation were increased by up to only 2.5-fold in only 50% of tumors overall. Immunohistochemical studies, using a monoclonal antibody specific for Erk2 demonstrated that the cellular distribution of this MAP kinase was similar in both control and tumor tissues, and Erk2 was largely confined to normal and malignant acini, whilst the intensity of staining was actually reduced in the tumor tissue. Mek1 and especially Mek2 protein expression, as well as MAP kinase kinase activity as determined by phosphorylation of kinase-inactive Erk [GST-K71A] were increased in cancer samples.
a) This confirms that MAP kinase activity is increased in human breast cancer. However, the frequency and magnitude of this change is dependent upon the chosen methodology (i.e. crude lysate assays versus specific immunoprecipitation). b) A MAP-kinase-independent source of increased EGFR T669 phosphotransferase activity in tumor extracts has been demonstrated for the first time in human breast cancer. c) By immunohistochemistry, Erk2 protein was actually found to exhibit lower intensity in tumor samples; the increased expression was most likely due to its increased distribution. d) Increased Mek protein expression and activation have been demonstrated for the first time in human breast tumors.
通过主要生长因子受体酪氨酸激酶（RTK）途径的促有丝分裂信号传导，即RTK→Ras→Raf→Mek→MAPK，已被认为与人类癌症的发病机制有关。然而，在人类癌症中对此的生化特征尚未得到充分评估。
使用来自23例人类乳腺癌的提取物以及同一切除标本的对照组织，检测有丝分裂原激活蛋白（MAP）激酶亚型p42 Erk2和p44 Erk1的蛋白质水平、磷酸转移酶活性和亚细胞定位，以及它们对髓鞘碱性蛋白（MBP）和表皮生长因子受体（EGFR T669）中Thr-669位点磷酸化后的肽底物的磷酸转移酶活性。
使用特异性抗体可明显看出Erk2和Erk1亚型均有过表达。还发现MBP和EGFR T669肽磷酸转移酶活性普遍激活（高达3倍）。MonoQ分级分离从MAP激酶蛋白洗脱中分离出大部分EGFR T669肽磷酸化。通过特异性免疫沉淀测定的Erk1和Erk2活性在总体仅50%的肿瘤中仅增加至2.5倍。免疫组织化学研究使用针对Erk2的单克隆抗体表明，该MAP激酶在对照组织和肿瘤组织中的细胞分布相似，并且Erk2主要局限于正常和恶性腺泡，而肿瘤组织中的染色强度实际上降低。癌症样本中Mek1尤其是Mek2蛋白表达以及通过激酶失活的Erk[GST-K71A]磷酸化测定的MAP激酶激酶活性增加。
a）这证实了人类乳腺癌中MAP激酶活性增加。然而，这种变化的频率和幅度取决于所选择的方法（即粗裂解物测定与特异性免疫沉淀）。b）首次在人类乳腺癌中证明肿瘤提取物中EGFR T669磷酸转移酶活性增加存在不依赖MAP激酶的来源。c）通过免疫组织化学，实际上发现肿瘤样本中Erk2蛋白的强度较低；表达增加很可能是由于其分布增加。d）首次在人类乳腺肿瘤中证明Mek蛋白表达和激活增加。
