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. 2017 May 26;292(21):8786-8796.
doi: 10.1074/jbc.M116.774174. Epub 2017 Apr 7.

The KIM-family Protein-Tyrosine Phosphatases Use Distinct Reversible Oxidation Intermediates: Intramolecular or Intermolecular Disulfide Bond Formation

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Free PMC article

The KIM-family Protein-Tyrosine Phosphatases Use Distinct Reversible Oxidation Intermediates: Intramolecular or Intermolecular Disulfide Bond Formation

Luciana E S F Machado et al. J Biol Chem. .
Free PMC article

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.

Keywords: biophysics; enzyme inactivation; nuclear magnetic resonance (NMR); oxidation-reduction (redox); tyrosine-protein phosphatase (tyrosine phosphatase).

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
General redox regulation scheme for PTPs. In the presence of H2O2, the thiolate ion of the PTP catalytic cysteine is reversibly oxidized to sulfenic acid (k1). Due to its instability, sulfenic acid quickly converts into: 1) the thiolate ion (k−1), 2) one of two irreversibly oxidized states, sulfinic acid (k2) or sulfonic acid (not shown), or 3) one of two reversibly oxidized states, a disulfide or a sulfenamide (k3). The latter can both be reduced and reactivated by reducing agents such as DTT (k4).
Figure 2.
Figure 2.
Inhibition and reversibility of the steady-state activity of HePTP, STEP, and PTPRR. 5 μm HePTP (A), STEP (C), and PTPRR (E) were incubated with 0–2000 μm H2O2 for 15 min at room temperature; the incubations were then continued for 10 min either without (closed symbols) or with (open symbols) 10 mm DTT (±S.E., n = 6–12; two-way ANOVA test, ****, p < 0.0001). IC50 values for H2O2-mediated inhibition of HePTP (B), STEP (D), and PTPRR (F) at different incubation times (15, 60, and 120 min) either without (filled bars) and with (hatched bars) 10 mm DTT during the second incubation step (±S.E., n = 6–12; paired t test, *, p < 0.05 or **, p < 0.001).
Figure 3.
Figure 3.
Oxidation and reactivation rate constants for HePTP, STEP, and PTPRR. A–C, time dependence of HePTP (A), STEP (B), and PTPRR (C) inactivation by H2O2. H2O2 concentrations are shown in different colors. The data were fit to a single exponential; insets show the concentration dependence of inactivation used to define the second-order rate constant k1. D and E, time-dependent reactivation of KIM-PTPs by DTT using stopped-flow kinetics: D, HePTP at DTT concentrations of 2.5, 5, 10, 20, 30, and 40 mm (curve for 2.5 mm is shown). E, STEP at identical DTT concentrations (curve for 10 mm DTT shown). The insets yield the second-order rate constants k4. F–H, time-dependent irreversible inactivation of KIM-PTPs by high H2O2 concentrations: HePTP (F), STEP (G), and PTPRR (H). Experiments were performed as described in A–C, with the exception that higher concentrations of H2O2 were used. The insets show the concentration dependence of the inactivation used to define the second-order rate constant k2. ±S.E., n = 3.
Figure 4.
Figure 4.
STEP and PTPRR form intramolecular disulfides, whereas HePTP does not. A, C, and E, inactivation of KIM-PTPs and BC and cysteine variants. 20 μm STEP and STEP(C384S) (BC) (A), PTPRR and PTPRR(C501S) (BC) (C), and HePTP, HePTP(C116S), HePTP(C183S) (BC), and HePTP(C207S) (E) were incubated with 0–2000 μm H2O2 for 15 min at room temperature and the resulting activities at steady-state measured. B, D, and F, steady-state reversibility of KIM-PTPs and their variants. 20 μm STEP (B), PTPRR (D), and HePTP (F) were incubated with 500 μm H2O2 for 15 min at room temperature; the samples were then incubated with varying concentrations of DTT (0–20 mm) for 15 min and their activities were measured to determine the recovered activities (described under “Experimental procedures”). ±S.E., n = 4. Two-way ANOVA test, with ****, p < 0.0001.
Figure 5.
Figure 5.
Identification of the disulfide-linked peptides in oxidized HePTP, STEP, and PTPRR using LC-MS. A–C, STEP, PTPRR, and HePTP treated with H2O2 (top) or treated with H2O2 and subsequently incubated with 10 mm DTT and 55 mm IAM (bottom). Mass errors at MH4+ were 6.0, 5.2, and −0.2 ppm for STEP, PTPRR, and HePTP, respectively.
Figure 6.
Figure 6.
Size exclusion chromatography and non-reducing SDS-PAGE of oxidized HePTP. A, HePTP (34 kDa) was incubated in the absence (black) or presence of 500 μm (red) H2O2 for 10 min (120 units/ml of catalase was used to quench the reaction); blue line corresponds to 44-kDa standard. B, HePTP was incubated in the presence of 0, 100, and 500 μm H2O2 for 10 min (60 units/ml of catalase was used to quench the reaction). 10 mm IAM or 10 mm DTT were used to either stabilize the free cysteines or reduce the disulfide bonds, respectively.
Figure 7.
Figure 7.
2D [2H,15N]TROSY NMR data of HePTP in presence of H2O2. Left, [2H,15N]TROSY spectra of 2H,15N-labeled 97 μm HePTP (black) in the presence of 100 (dark cyan), 300 (red), and 500 μm (purple) H2O2. Right, 2H,15N-labeled 97 μm HePTP in the presence of 100 μm (dark cyan), 300 μm (light pink), and 500 μm (light purple) H2O2 after addition of 10 mm DTT.
Figure 8.
Figure 8.
Reversible oxidation mechanisms of PTPs.

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