Reversible Oxidation of the Membrane Distal Domain of Receptor PTPα Is Mediated by a Cyclic Sulfenamide

Abstract

Protein tyrosine phosphatases (PTPs) are fundamental to the regulation of cellular signalling cascades triggered by protein tyrosine kinases. Most receptor-like PTPs (RPTPs) comprise two tandem PTP domains, with only the membrane proximal domains (D1) having significant phosphatase activity; the membrane distal domains (D2) display little to no catalytic activity. Intriguingly, however, many RPTP D2s share the catalytically essential Cys and Arg residues of D1s. D2 of RPTPR may function as a redox sensor that mediates regulation of D1 via reactive oxygen species. Oxidation of Cys723 of RPTPR D2 (equivalent to PTP catalytic Cys residues) stabilizes RPTPR dimers, induces rotational coupling, and is required for inactivation of D1 phosphatase activity. Here, we investigated the structural consequences of RPTPR D2 oxidation. Exposure of RPTPR D2 to oxidants promotes formation of a cyclic sulfenamide species, a reversibly oxidized state of Cys723, accompanied by conformational changes of the D2 catalytic site. The cyclic sulfenamide is highly resistant to terminal oxidation to sulfinic and sulfonic acids. Conformational changes associated with RPTPR D2 oxidation have implications for RPTPR quaternary structure and allosteric regulation of D1 phosphatase activity.The reversible tyrosine phosphorylation of proteins, reciprocally controlled by tyrosine kinases and phosphatases, is crucial to the regulation of diverse cellular processes, including growth, proliferation, and differentiation (1). Redox reactions, mediated by reactive oxygen species (ROS), 1 control PTP activity in response to a variety of hormones and growth factors (2-6). Generation of ROS accompanies PTK activation (7,8), with oxidation-induced inhibition of PTP activity amplifying PTK-dependent signaling (9-11).PTP-catalyzed protein dephosphorylation proceeds via a nucleophilic displacement reaction utilizing an essential cysteine residue conserved within the PTP signature motif Cys(X) 5 Arg (12-15). The low pK a of the catalytic cysteine necessary for its function (16) renders PTPs susceptible to inactivation by ROS-induced cysteine oxidation. However, the reversibility of redox-mediated inactivation of PTPs requires that a reversibly oxidized state of the cysteine residue be stabilized at the PTP catalytic site and that formation of SO 2 and SO 3 , two irreversibly oxidized species of cysteine, be suppressed (17). Crystallographic studies of the tyrosine specific cytosolic PTP1B revealed that the reversibly oxidized form of the enzyme was not the expected sulfenic acid or disulfide bond, but an oxidation state of cysteine termed a cyclic sulfenamide, a species not previously observed in proteins (18,19). Generation of the cyclic sulfenamide inhibits catalytic activity by blocking the nucleophilic cysteine residue through a covalent bond of its thiol group with the neighboring main chain amide, a linkage that is accompanied by profound conformational transitions of the catalytic site PTP and pTyr loops. The oxidation properties of the catalyt...

Does the Transcription Factor NemR Use a Regulatory Sulfenamide Bond to Sense Bleach?

“…However, in both of these cases, the oxidized cysteine reacts with a backbone amide of the polypeptide chain causing a cyclic sulfenamide formation. In PTPs, sulfenamide formation is thought to protect the active site cysteine from irreversible overoxidation (9), and it is possible that formation of a Cys106-Lys175 sulfenamide in NemR plays a similar role, especially given the high reactivity of RCS with cysteine residues (2). Based on the conservation of Cys106 and Lys175 among NemR homologs from diverse bacterial genera, we propose that formation of a regulatory sulfenamide bond may represent a broadly distributed redox switch mechanism, although the functions of most NemR homologs have not yet been examined.…”

Discovering mechanisms of signaling-mediated cysteine oxidation

“…The initial evidence for this species was crystallographic [16], leaving some doubt as to the relevance of this species in a biological setting. On the other hand, a functional role for the generation of such a species is suggested by the profound changes in structure imparted to the active site of protein tyrosine phosphatases with this modification, changes which may enhance access by reductants and/or modulate interactions with other domains ( [17]; reviewed in [18]). Studies using model chemistry have also better defined the parameters which promote this cyclization reaction in the constrained setting of a protein active site [19].…”

Receptor Protein-tyrosine Phosphatase α Regulates Focal Adhesion Kinase Phosphorylation and ErbB2 Oncoprotein-mediated Mammary Epithelial Cell Motility

“…Uniquely to PTP␣, the membrane-distal D2 domain is also active, but with lower specific activity than D1. Furthermore, D2 appears to play a role in sensing reactive oxygen species (14,15) and, following oxidation, may participate in "inside-out" signaling by altering the rotational coupling of PTP␣ molecules within a receptor dimer (16). There is considerable evidence supporting a role for PTP␣ in activating SRC and other SRC family kinases (13,(17)(18)(19).…”