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

Abstract

Elevated levels of Src kinase activity have been reported in a number of human cancers, including colon and breast cancer. We have analysed four human breast tumor cell lines that exhibit high levels of Src kinase activity, and have determined that these cell lines also exhibit a high level of a phosphotyrosine phosphatase activity that recognizes the Src carboxy-terminal PTyr530 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 speci®c activity is elevated as much as 5.6-fold. When the breast tumor cells were grown in the presence of the tyrosine phosphatase inhibitor vanadate, Src kinase activity was reduced in all four breast tumor cell lines, suggesting that Src was being activated by a phosphatase which could recognize the Tyr530 negative regulatory site. In fractionated cell extracts from the breast tumor cells, we found elevated levels of a membrane associated tyrosine phosphatase activity that preferentially dephosphorylated a Src family carboxy-terminal phosphopeptide containing the regulatory tyrosine 530 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 by a phosphatase in these cells. In preliminary immunoprecipitation and antibody depletion experiments, we were unable to correlate the major portion of this phosphatase activity with several known phosphatases.

Selected glimpses into the activation and function of Src kinase

“…This approach is of particular relevance because several di erent PTPs are known to target Src for dephosphorylation and activation, and the expression pattern of these PTPs varies according to cell type. Two cell types that have been identi®ed by our laboratory as possessing elevated Src activity are human melanocytes (O'Connor et al, 1992(O'Connor et al, , 1995 and several human breast cancer cell lines (Egan et al, 1999). In both situations, we have detected elevated c-Src speci®c activity and hypophosphorylation of Tyr(527/530).…”

“…There is a large body of evidence that has demonstrated that Src kinase activity, and sometimes Src protein levels, are elevated in several cancers including colon and breast cancer, with a correlation often observed between increases in Src kinase activity and degree of malignancy (Bolen et al, 1987;Cartwright et al, 1989;Ottenho -Kal et al, 1992;Verbeek et al, 1996;Egan et al, 1999;Biscardi et al, 1999). As mentioned previously, Src kinase activity can be activated through a number of mechanisms including: (i) binding of Src to activated growth factor receptor tyrosine kinases; (ii) dephosphorylation of the negative regulatory Tyr(527/530) residue by a PTP; and (iii) mutation.…”

Expression of protein tyrosine phosphatase alpha (RPTPα) in human breast cancer correlates with low tumor grade, and inhibits tumor cell growth in vitro and in vivo

“…In a pilot analysis of c-Src kinase activity in a subset of tumors analysed, we failed to observe a simple 1 : 1 correlation between RPTPa overexpression and c-Src activation (data not shown). A recent biochemical approach concluded that RPTPa is not the major Src-activating PTP in breast cancer cell lines (Egan et al, 1999).…”

The interplay between Src and integrins in normal and tumor biology

“…Increased Src activity has been found, for example, in human mammary carcinomas, colon cancer and pancreatic cancer (Jacobs and Rubsamen, 1983;Rosen et al, 1986;Ottenhoff-Kalff et al, 1992;Talamonti et al, 1993;Termuhlen et al, 1993;Maa et al, 1995;Verbeek et al, 1996;Mao et al, 1997;Egan et al, 1999;Hakak and Martin, 1999;Harris et al, 1999;Shimakage et al, 2000). The increased Src kinase activity in these tumors has been proposed to be due to tyrosine phosphatase-mediated dephosphorylation of the carboxy-terminal negative regulatory element, an increase in Src protein levels and/or altered protein stability, an increase in upstream receptor tyrosine kinase activity, or loss of key regulatory proteins (Maa et al, 1995;Mao et al, 1997;Egan et al, 1999;Hakak and Martin, 1999;Harris et al, 1999;Shimakage et al, 2000;Masaki et al, 2003). A few human colon cancers exhibit Src activating mutations (Irby et al, 1999).…”