Abstract
Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTPalpha-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTPalpha-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTPalpha-D1 and RPTPalpha-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPmu-D1, with a similar conformation as RPTPalpha-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H(2)O(2) concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.
Highlights
Phosphorylation of proteins on tyrosine residues has an important role in many cellular processes like proliferation, differentiation, and migration
We investigated differential oxidation of protein-tyrosine phosphatases (PTPs). oxidized catalytic site cysteines in PTPs (oxPTPs) immunoprobing and matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry indicated that the mutations in the Tyr(P) loop and the WPD loop that are responsible for the difference in catalytic activity between receptor PTPs (RPTPs)␣-D1 and RPTP␣-D2 were involved in the difference in oxidizability
RPTP␣-D2, much less active than D1, is more susceptible to H2O2 than RPTP␣-D1 and is already inactivated at low concentrations of H2O2 (31 M; Fig. 1D). These results show that RPTP␣-D2 is more susceptible to oxidation than RPTP␣-D1 as detected by the oxPTP antibody and by PTP activity assays
Summary
Phosphorylation of proteins on tyrosine residues has an important role in many cellular processes like proliferation, differentiation, and migration. The human genome encodes 103 cysteine-based PTP family members, of which 38 are strictly phosphotyrosine-specific [4, 5]. These “classical” PTPs are subdivided into transmembrane, receptor PTPs (RPTPs) (21 genes) and intracellular, nonreceptor PTPs (17 genes). Little is known about the regulation of PTPs. Reversible oxidation of the absolutely conserved catalytic site cysteine that is essential for catalysis [14] is emerging as an important regulatory mechanism [15]. Reynolds et al [27] demonstrated in a mathematical model that epidermal growth factor-induced epidermal growth factor receptor activation, together with ROS-mediated inactivation of PTPs is sufficient to cause lateral signal propagation.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
