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Proteins phosphorylated on tyrosine are detectable by antibodies against phosphotyrosine (P-Tyr antibodies) in cells transformed by oncogene-encoded tyrosine kinases. We used P-Tyr antibodies to investigate the existence of abnormal levels of phosphoproteins in human breast cancer. Three human breast cancer cell lines (SK-BR-3, MCF-7 and CG-5) and 37 human breast cancer specimens were examined by Western blot analysis and "in vitro" kinase assay. In the SK-BR-3 cell line three major phosphoproteins of the approximate Mr of 185,000 (p185), 135,000 (p135) and 110,000 (p110) were detected. The former was identified as the HER-2 gene product by specific antibodies against HER-2 encoded protein. In the other cell lines, a product of the approximate Mr of 170,000 (p170), together with a p135 and a p110, were phosphorylated on tyrosine. P185 and p170 were shown to have an associated tyrosine kinase activity. Two proteins, comigrating with p135 and p110, were found to be highly phosphorylated on tyrosine in 50% of the breast cancer samples, but not in samples harvested from 12 human tumors of the gastro-intestinal tract. These data show that 50% of human breast cancer samples display an abnormal level of tyrosine phosphorylated proteins.