Association of SH2 Domain Protein Tyrosine Phosphatases with the Epidermal Growth Factor Receptor in Human Tumor Cells

Abstract

The SH2 domain protein tyrosine phosphatases (PTPases) PTP1C and PTP1D were found associated with epidermal growth factor (EGF) receptor which was purified from A431 cell membranes by several steps of chromatography. Both PTPases also associated with the EGF receptor upon exposure of immunoprecipitated receptor to lysates of MCF7 mammary carcinoma cells. The associated PTPases had little activity toward the bound receptor when it was autophosphorylated in vitro. Receptor dephosphorylation could, however, be initiated by treatment of the receptor-PTPase complex with phosphatidic acid (PA). When autophosphorylated EGF receptor was exposed to lysates of PTP1C or PTP1D overexpressing 293 cells, the association of PTP1C but not of PTP1D was enhanced in the presence of PA. In intact A431 cells, an association of PTP1C and PTP1D with the EGF receptor was detectable by coimmunoprecipitation experiments. PA treatment reduced the phosphorylation state of ligand activated EGF receptors in A431 cells and in 293 cells overexpressing EGF receptors together with PTP1C but not in 293 cells overexpressing EGF receptors alone or together with PTP1D. We conclude that PTP1C but not PTP1D participates in dephosphorylation of activated EGF receptors. A possible role of PA for physiological modulation of EGF receptor signaling is discussed.

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

“…This preparative material was digested with CNBr and the digestion products were separated on a 22% SDS ± PAGE gel. The intensity of the phosphorylated Tyr 530 band from Src protein isolated from BT-483 cells was slightly reduced relative to HFF cells (77%) (Figure 4c and d Preliminary analysis of some known tyrosine phosphatases in breast tumor cell lines Several previous reports have suggested possible roles for speci®c PTPases in the development of breast and other cancers (den Hertog et al, 1993;Elson and Leder, 1995;Galaktionov et al, 1995;Pallen, 1993;Shen et al, 1991;Tomic et al, 1995;Zhai et al, 1993), including SH-PTP1, PTP1B, SH-PTP2, PTPalpha, LAR and Cdc25B. We examined the expression of these phosphatases in the four breast tumor cell lines studied here, which we had shown to have higher levels of Src-speci®c phosphatase activity.…”

“…It is not clear, however if any of these phosphatases are involved in Src activation in breast tumor cells. For example, SH-PTP1 and SH-PTP2 can associate in vitro and in vivo with the EGF receptor (Tomic et al, 1995), and SH-PTP2 has been detected in association with Src in a human colon carcinoma cell line (Peng and Cartwright, 1995), although biological e ects of these associations have not been demonstrated. In addition, SH-PTP1 is capable of dephosphorylating Src in vitro at Tyr530 and there is a positive correlation between the expression of SH-PTP1 and Src kinase activity in vivo in mouse thymocytes (Somani et al, 1997).…”

Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s)

“…A431 epidermoid carcinoma cells (CRL 1555) were obtained from the American Type Culture Collection (Rockville, USA). A43 1 cell culture and isolation of a membrane fraction were performed as described (Tomic et al, 1995).…”

“…Then the cells were stimulated with 1 jg ml-' EGF for 1 min at room temperature or left unstimulated. Cell extracts were prepared as described (Tomic et al, 1995), using 200 jil of lysis buffer per well. About 20 jig or 5 jig of protein per lane of H125 or A431 cell extract, respectively, was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with anti-phosphotyrosine antibodies (RC20-peroxidase conjugate, Transduction Laboratories) and ECL (Amersham) detection.…”

“…Apparently, different PTPases, including those of cytosolic or transmembrane type, have the capacity to dephosphorylate autophosphorylated EGFR (Hashimoto et al, 1992;Lammers et al, 1993). Recently, the SH2-domain PTPase, PTP1C, has been shown to associate with the EGFR and to dephosphorylate it in A431 cells and in 293 cells overexpressing PTP1C and EGFR (Tomic et al, 1995). Receptor dephosphorylation is considered a major mechanism of negative regulation of receptor activity.…”