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BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.