Figure 6.

A, bicarbonate is required for EGF-dependent reversible PTP1B oxidation in A431 cells. A431 cells were incubated overnight either in low-serum regular DMEM-containing bicarbonate or in HEPES-buffered DMEM (50 mm, pH 7.4). Pretreatment of the cells in bicarbonate-containing DMEM with the NBC inhibitor S0859 (50 μm) was for 1 h prior to stimulation. Bicarbonate (60 mm) was added to cells in HEPES-buffered DMEM and subsequently stimulated. At the indicated times after EGF stimulation, cells were subjected to the cysteinyl-labeling assay using biotinylated iodoacetyl-PEG2-biotin for analysis of reversible PTP1B oxidation. Biotinylated proteins were purified on streptavidin-Sepharose beads, resolved by SDS-PAGE, and visualized using antibodies against PTP1B. PTP1B control levels were determined from total cell lysate by SDS-PAGE and blotting (IB) against PTP1B. Representative Western blotting of three independent experiments is shown. B, model for regulation of PTP1B activity during growth factor signaling. EGFR activation induces a transient burst of H₂O₂, which reacts with bicarbonate to give PTP1B oxidation via peroxymonocarbonate (red) and activation of phosphorylation pathways (green arrow). Prxs compete for the H₂O₂, and the Trx system decreases the availability of H₂O₂ by supporting the Prx cycle and also acts by reactivating oxidized PTP1B. See “Discussion” for further details.