Abstract
Protein-tyrosine phosphatases (PTPs) counteract protein tyrosine phosphorylation and cooperate with receptor-tyrosine kinases in the regulation of cell signaling. PTPs need to undergo oxidative inhibition for activation of cellular cascades of protein-tyrosine kinase phosphorylation following growth factor stimulation. It has remained enigmatic how such oxidation can occur in the presence of potent cellular reducing systems. Here, using in vitro biochemical assays with purified, recombinant protein, along with experiments in the adenocarcinoma cell line A431, we discovered that bicarbonate, which reacts with H2O2 to form the more reactive peroxymonocarbonate, potently facilitates H2O2-mediated PTP1B inactivation in the presence of thioredoxin reductase 1 (TrxR1), thioredoxin 1 (Trx1), and peroxiredoxin 2 (Prx2) together with NADPH. The cellular experiments revealed that intracellular bicarbonate proportionally dictates total protein phosphotyrosine levels obtained after stimulation with epidermal growth factor (EGF) and that bicarbonate levels directly correlate with the extent of PTP1B oxidation. In fact, EGF-induced cellular oxidation of PTP1B was completely dependent on the presence of bicarbonate. These results provide a plausible mechanism for PTP inactivation during cell signaling and explain long-standing observations that growth factor responses and protein phosphorylation cascades are intimately linked to the cellular acid-base balance.
Highlights
Protein-tyrosine phosphatases (PTPs) counteract protein tyrosine phosphorylation and cooperate with receptor-tyrosine kinases in the regulation of cell signaling
Using in vitro biochemical assays with purified, recombinant protein, along with experiments in the adenocarcinoma cell line A431, we discovered that bicarbonate, which reacts with H2O2 to form the more reactive peroxymonocarbonate, potently facilitates H2O2-mediated PTP1B inactivation in the presence of thioredoxin reductase 1 (TrxR1), thioredoxin 1 (Trx1), and peroxiredoxin 2 (Prx2) together with NADPH
We examined whether inclusion of bicarbonate facilitated H2O2-mediated inactivation of PTP1B in the presence of a functional Trx system
Summary
Protein-tyrosine phosphatases (PTPs) counteract protein tyrosine phosphorylation and cooperate with receptor-tyrosine kinases in the regulation of cell signaling. PTPs need to undergo oxidative inhibition for activation of cellular cascades of protein-tyrosine kinase phosphorylation following growth factor stimulation. EGF-induced cellular oxidation of PTP1B was completely dependent on the presence of bicarbonate These results provide a plausible mechanism for PTP inactivation during cell signaling and explain long-standing observations that growth factor responses and protein phosphorylation cascades are intimately linked to the cellular acid– base balance. Ceutical disruption of several NBCs (SLCA4, SLC4A7, and SLC4A9) suppresses tumor growth of breast cancer spheroids and murine xenografts [43, 44], and chemical inhibition of bicarbonate influx using S0859 decreases spheroid growth of HCT116 cells [45] Considering these observations, we asked whether bicarbonate can directly facilitate PTP1B oxidation in the presence of a peroxiredoxin-recycling system and in a cellular setting. Our results reveal that bicarbonate is an obligate component of cellular PTP1B oxidation and EGF responsiveness
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