Activation of Src in human breast tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530

Elevated levels of Src kinase activity have been reported in a number of hu man cancers, including colon and breast cancer, We have analysed four human breast tumor cell lines that exhibit high levels of Src kinase activity, a nd have determined that these cell lines also exhibit a high level of a pho sphotyrosine phosphatase activity that recognizes the Src carboxy-terminal P-Tyr530 negative regulatory site. Total Src kinase activity in these cell lines is elevated as much as 30-fold over activity in normal control cells and specific activity is elevated as much as 5.6-fold. When the breast tumo r cells were grown in the presence of the tyrosine phosphatase inhibitor va nadate, Src kinase activity was reduced in all four breast tumor cell lines , suggesting that Src was being activated by a phosphatase which could reco gnize the Tyr530 negative regulatory site, In fractionated cell extracts fr om the breast tumor cells, we found elevated levels of a membrane associate d tyrosine phosphatase activity that preferentially dephosphorylated a Src family carboxy-terminal phosphopeptide containing the regulatory tyrosine 5 30 site, Src was hypophosphorylated in vivo at tyrosine 530 in at least two of the tumor cell lines, further suggesting that Src was being activated b y a phosphatase in these cells, In preliminary immunoprecipitation and anti body depletion experiments, we were unable to correlate the major portion o f this phosphatase activity with several known phosphatases.