H2O2-induced intermolecular disulfide bond formation between receptor protein-tyrosine phosphatases.

Receptor protein-tyrosine phosphatase alpha (RPTPalpha) belongs to the subfamily of receptor-like protein-tyrosine phosphatases that are characterized by two catalytic domains of which only the membrane-proximal one (D1) exhibits appreciable catalytic activity. The C-terminal catalytic domain (D2) regulates RPTPalpha catalytic activity by controlling rotational coupling within RPTPalpha dimers. RPTPalpha-D2 changes conformation and thereby rotational coupling within RPTPalpha dimers in response to changes in the cellular redox state. Here we report a decrease in motility of RPTPalpha from cells treated with H2O2 on non-reducing SDS-polyacrylamide gels to a position that corresponds to RPTPalpha dimers, indicating intermolecular disulfide bond formation. Using mutants of all individual cysteines in RPTPalpha and constructs encoding the individual protein-tyrosine phosphatase domains, we located the intermolecular disulfide bond to the catalytic Cys-723 in D2. Disulfide bond formation and dimer stabilization showed similar levels of concentration and time dependence. However, treatment of lysates with dithiothreitol abolished intermolecular disulfide bonds but not stable dimer formation. Intermolecular disulfide bond formation and rotational coupling were also found using a chimera of the extracellular domain of RPTPalpha fused to the transmembrane and intracellular domain of the leukocyte common antigen-related protein (LAR). These results suggest that H2O2 treatment leads to oxidation of the catalytic Cys in D2, which then rapidly forms a disulfide bond with the D2 catalytic Cys of the dyad-related monomer, rendering an inactive RPTP dimer. Recovery from oxidative stress first leads to the reduction of the disulfide bond followed by a slower refolding of the protein to the active conformation.