Redox Regulation of Receptor Protein-Tyrosine Phosphatases

Abstract

Phosphorylation is of major importance in cell signalling processes like cell migration, cell proliferation and cell differentiation within higher eukaryotic organisms. Therefore, the balance between phosphorylation, mediated by kinases, and dephosphorylation, mediated by phosphatases, must be tightly controlled. Kinases as well as phosphatases are known to play a role in diseases like cancer and diabetes. Future research on PTPs will give important information about the exact roles they fulfill in signal transduction and the implications they have in diseases. The aim of this work is the elucidation of the regulatory mechanism of oxidation induced inhibition of Receptor Protein-Tyrosine Phosphatases (RPTPs). RPTPa activity is inhibited by the formation of stable dimers upon oxidation. The crystal structure of RPTPa shows that a wedge-like structure of one subunit inserts into the catalytic cleft of the other, thereby preventing substrate binding. The cysteine of the membrane distal domain (RPTP-D2) acts as a redox sensor since mutating this residue into a serine prevents the formation of stable dimers and renders the phosphatase active after oxidation. In chapter II an antibody is described that specifically recognizes oxidized cysteines in the PTP-loop. We found that the cysteine of RPTPa-D2 is indeed more susceptible to oxidation than the cysteine of RPTPa-D1. In chapter III we investigated whether differential oxidation is a common theme in RPTPs. The guanidinium group of a nearby serine appears to define the susceptibility of the catalytic site cysteine. Chapter IV shows the crystal structure of oxidized RPTPa-D2 in which a sulphenamide form was identified just as was found in the crystal structure of PTP1B. This form appeared to be reversible and stable up to 24 hours. Whether dimerization induced inhibition could be a general regulatory mechanism for RPTPs was investigated in chapter V. The intracellular domains of RPTPa, RPTPm, LAR and CD45 all formed stable dimers upon oxidation. Besides they all showed profound conformational changes in response to oxidation. Here, we propose the following model. RPTPs exist on the cell membrane as preformed active dimers. In the presence of Reactive Oxygen Species (ROS), the conserved second, membrane distal catalytic domain functions as a redox sensor, transducing the oxidation information to the first, membrane proximal catalytically active domain. The inhibitory wedge of one subunit will subsequently occlude the catalytic cleft of the other, thereby preventing substrate binding, rendering the RPTP inactive. Since the oxidation of the cysteines is reversible, reduction of the cysteine of the second catalytic domain will reform the RPTPs to an active dimeric state. Taken together, we propose that the activity of RPTPs is regulated by an oxidation-induced dimerization mechanism.