O induced intermolecular disulfide bond formation between Receptor Protein-Tyrosine Phosphatases

Receptor Protein-Tyrosine Phosphatase a 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 RPTP a catalytic activity by controlling rotational coupling within RPTP a dimers. RPTP a -D2 changes conformation and thereby rotational coupling within RPTP a dimers in response to changes in the cellular redox state. Here we report a decrease in motility of RPTP a from cells treated with H 2 O 2 on non-reducing SDS-PAGE gels to a position that corresponds to RPTP a dimers, indicating intermolecular disulfide bond formation. Using mutants of all individual cysteines in RPTP a and constructs encoding the individual PTP domains, we located the intermolecular disulfide bond to the catalytic Cys723 in D2. Disulfide bond formation and dimer stabilization showed similar concentration-and time-dependencies. However, treatment of lysates with dithiotreitol 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 RPTP a fused to the transmembrane and intracellular domain of LAR. These results suggest that H 2 O 2 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.

Reactive oxygen species (ROS) induce oxidation of catalytic cysteines, thereby inactivating these PTPs (5)(6)(7)(8). Extracellular stimuli like growth factors and UV result in an increase in intracellular ROS and oxidation of PTPs (5,(9)(10)(11)(12). Inhibition of enzyme activity by oxidative stress is increasingly recognized as an important mechanism of regulation of the PTP family. Therefore, PTPs may serve as sensors of the cellular redox state.
RPTPα belongs to the receptor like PTPs that are characterized by a single transmembrane domain. RPTPα has two catalytic domains of which the N-terminal one (D1) contains almost all of the catalytic activity of the enzyme. RPTPα was found to constitutively form dimers in the cell membrane (13,14) and activity of the dimer is dependent on the relative orientation of the two monomers in the dimer (15).
Studies on the crystal structure of RPTPα-D1 indicate that a helix-loop-helix wedgelike structure to the N-terminal side of D1 occludes the catalytic site of the dyadrelated monomer (16). The RPTP CD45 is also regulated by dimerization (17,18).
Mutations in the wedge-like structure of CD45 and RPTPα abolished dimerization induced inactivation (15,19) proving that the wedge-like structure is essential for the regulation of RPTP activity by rotational coupling of the monomers in the dimer.
Previously, we showed that the catalytically inactive C-terminal PTP domain (D2) of RPTPs has a regulatory role (20,21). Recent studies on oxidative stress and RPTPα indicate that D2 acts as a redox sensor (22). Using an antibody that recognizes oxidized classical PTPs, RPTPα-D2 exhibits a higher susceptibility to oxidation than RPTPα-D1 (12 7

HA-accessibility assay
The conformation of the extracellular domain was detected by the accessibility of the HA-tag at the N-terminal side of the extracellular domain of HA-α and of the EDα-LAR chimeric protein. The procedure is described in detail in (27). In short, after stimulation, the living cells were incubated ice-cold with anti-HA-antibody for 1 h.
After extensive washing, cells were lysed and the antibody-bound fraction of the protein was pulled down from the lysate with protein A-sepharose. This part of the total amount of transfected protein is called the accessible fraction. The rest of the HA-tagged protein, the non-accessible fraction, was immunoprecipitated from the remaining lysate using protein A-sepharose bound anti-HA-antibody. The immunoprecipitating proteins were visualized following SDS-PAGE by immunoblotting as described above.

Rapid formation of intermolecular S-S bridges between RPTPα monomers in response to H 2 O 2
Previously, we showed that H

Role of intermolecular disulfide bonds in stable RPTPα dimer formation
Since Cys723 is required both for intermolecular disulfide bond formation and for stable dimer formation as detected by co-immunoprecipitation of differentially tagged RPTPα monomers, and both emerge with the same kinetics in response to H 2 O 2 treatment (Fig. 1B) (Fig. 7). We found that reduction with DTT abolished the intermolecular disulfide bonds, while stabilized RPTPα dimers were still detected. Pervanadate is a strong oxidizing agent that triply oxidizes the PTP catalytic cysteine into the sulfonic acid form, which cannot form disulfide bonds (28). As shown in Figure 7, treatment of cells with pervanadate did not induce stable dimer formation nor intermolecular disulfide bond formation. To confirm triple oxidation upon pervanadate treatment, we used an antibody generated against triply oxidized PTP-catalytic cysteine, anti-oxPTP (12). As shown in Figure 8   Immunoblots developed by ECL are depicted.   Immunoblots probed with anti-HA antibody and developed by ECL are depicted.