Protein-Protein Interactions Control PTP1B Oxidation and Activation

Abstract

Protein Tyrosine Phosphatase 1B (PTP1B) catalytic activity is negatively regulated by reversible oxidation. In vivo, PTP1B reversible inactivation by hydrogen peroxide allows phosphorylation of specific tyrosine residues of several receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). Hence PTP1B acts as a tumor suppressor by preventing excessive signaling from growth factors. We re-examined the redox cycle of PTP1B in order to find underlying molecular mechanisms that control its activation. For this purpose, we performed structural analyses of reduced and oxidized PTP1B (PTP1B-OX) and identified peptides that were uniquely exposed to the cytosol in PTP1B-OX (OX-Peptides). We used these OX-Peptides, fished for novel PTP1B interactors and we identified 14-3-3β as a specific binding partner for PTP1B-OX. As expected, the interaction between 14-3-3β and PTP1B was dependent on PTP1B oxidation downstream of EGFR in cells. 14-3-3s are phosphoserine binding proteins and inhibiting PKB activity and PTP1B Ser-50 phosphorylation prevented interaction between 14-3-3β- and PTP1B-OX. Interestingly, preventing PTP1B Ser-50 phosphorylation or inhibiting 14-3-3s using R18 also prevented PTP1B oxidation in cells, suggesting that 14-3-3β binding is necessary for PTP1B to become oxidized. Similarly, the interaction between 14-3-3 β and PTP1B-OX was compromised in cells following exposure to cell-permeable OX-Peptides. Preventing this protein-protein interaction directly perturbed the redox cycle of PTP1B: cell-permeable OX-peptides effectively prevented PTP1B reversible inactivation by reactive oxygen species. We propose a mechanism whereby 14-3-3β stabilizes the inactive form of PTP1B, transiently allowing EGFR signaling to occur. Destabilizing the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for drugs that activate protein tyrosine phosphatases.