Abstract
The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.
Highlights
The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR)
We used the catalytic domains of HePTP, STEP, and PTPRR to determine the H2O2-mediated oxidation and subsequent thiolmediated regeneration of KIM-PTP activities
All KIM-PTPs are sensitive to H2O2-mediated oxidation (Fig. 2), with their steady-state activity decreasing with increasing H2O2 concentrations
Summary
The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). The kinase interaction motif (KIM) family of non-receptor protein-tyrosine phosphatases (PTP) includes hematopoietic protein-tyrosine phosphatase (HePTP, PTPN7), striatal-enriched protein-tyrosine phosphatase (STEP, PTPN5), and protein-tyrosine phosphatase receptor type R (PTPRR, PTPSL, PTPBR7) These PTPs negatively regulate cell activation and proliferation through their ability to bind and subsequently dephosphorylate and inactivate the MAP kinases (MAPK) p38 and Erk2 [1,2,3,4]. We used kinetic assays, iodoacetamide labeling, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS) to show that HePTP uses a novel mechanism, namely the formation of an intermolecular disulfide bond, to prevent its irreversible oxidation, which leads to the reversible dimerization of HePTP
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