Regulation of receptor protein-tyrosine phosphatase alpha by oxidative stress

Abstract

The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane-distal PTP domain (RPTP-D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C-terminus in RPTPa prohibited intermolecular interactions. Interestingly, stress factors such as H 2 O 2 , UV and heat shock induced reversible, free radical-dependent, intermolecular interactions between RPTPa and RPTPa-SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPa-SpD2, Cys723, was required for the H 2 O 2 effect on RPTPa. H 2 O 2 induced a rapid, reversible, Cys723-dependent conformational change in vivo, as detected by¯uorescence resonance energy transfer, with cyan¯uorescent protein (CFP) and yellow¯uorescent protein (YFP) anking RPTPa-SpD2 in a single chimeric protein. Importantly, H 2 O 2 treatment stabilized RPTPa dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPa-D2, leading to stabilization of RPTPa dimers, and thus to inhibition of RPTPa activity.

H2O2-induced Intermolecular Disulfide Bond Formation between Receptor Protein-tyrosine Phosphatases

“…Indeed, DTT treatment of lysates from cells stimulated with H 2 O 2 immediately reduced the intermolecular disulfide bonds between RPTP␣ monomers, whereas differentially tagged monomers still co-immunoprecipitated. In line with this, recovery from the H 2 O 2 -induced conformational changes of RPTP␣-D2 in cells as detected by FRET was relatively slow (22), indicating that refolding of RPTP␣-D2 is independent of disulfide bond reduction but probably a result of another intrinsic property of the protein.…”

“…Individual Cys 3 Ser mutants were generated by PCR using appropriate oligonucleotides and were verified by sequencing. Expression vectors for the individual RPTP␣-D1 and RPTP␣-D2 domains were described previously (20,22). The fusion protein encoding the RPTP␣ ectodomain, human LAR transmembrane, and intracellular domain fusion protein (ED␣-LAR) was generated by PCR.…”

“…showed that H 2 O 2 treatment of cells transfected with differentially tagged RPTP␣ constructs leads to the formation of stable RPTP␣ dimers as detected by co-immunoprecipitation of the two constructs (22). To study whether dimer stabilization coincides with the formation of intermolecular disulfide bond(s), we co-transfected COS-1 cells with both HA-and Myc-tagged RPTP␣.…”

“…Recent studies on oxidative stress and RPTP␣ indicate that D2 acts as a redox sensor (22). Using an antibody that recognizes oxidized classical PTPs, RPTP␣-D2 exhibits a higher susceptibility to oxidation than RPTP␣-D1 (12).…”

“…Using an antibody that recognizes oxidized classical PTPs, RPTP␣-D2 exhibits a higher susceptibility to oxidation than RPTP␣-D1 (12). Furthermore, H 2 O 2 treatment induces a rapid, reversible, and catalytic site Cys-723-dependent in vivo change in protein conformation, rendering a more stable, catalytically inactive RPTP␣ dimer (22).…”