S-Nitrosation of Arabidopsis thaliana Protein Tyrosine Phosphatase 1 Prevents Its Irreversible Oxidation by Hydrogen Peroxide

Abstract

Tyrosine-specific protein tyrosine phosphatases (Tyr-specific PTPases) are key signaling enzymes catalyzing the removal of the phosphate group from phosphorylated tyrosine residues on target proteins. This post-translational modification notably allows the regulation of mitogen-activated protein kinase (MAPK) cascades during defense reactions. Arabidopsis thaliana protein tyrosine phosphatase 1 (AtPTP1), the only Tyr-specific PTPase present in this plant, acts as a repressor of H₂O₂ production and regulates the activity of MPK3/MPK6 MAPKs by direct dephosphorylation. Here, we report that recombinant histidine (His)-AtPTP1 protein activity is directly inhibited by H₂O₂ and nitric oxide (NO) exogenous treatments. The effects of NO are exerted by S-nitrosation, i.e., the formation of a covalent bond between NO and a reduced cysteine residue. This post-translational modification targets the catalytic cysteine C265 and could protect the AtPTP1 protein from its irreversible oxidation by H₂O₂. This mechanism of protection could be a conserved mechanism in plant PTPases.

Keywords: Arabidopsis thaliana; H2O2; S-nitrosation; mitogen-activated protein kinases; nitric oxide; oxidation; post-translational modifications; protein tyrosine phosphatase 1.

Figure 1

Sequence alignment of eight plant Protein Tyrosine Phosphatase 1 (PTP1) proteins, including Arabidopsis thaliana Protein Tyrosine Phosphatase 1 (AtPTP1), and the human PTP1B protein. Perfectly conserved amino acid residues are indicated by stars. Cys residues are indicated in gray. The typical conserved PTPase signature motif is highlighted by a red frame. The sequences compared are as follows: Arabidopsis thaliana AtPTP1, O82656; Zea mays ZmPTP1, NP_001149088; Oryza sativa OsPTP1, Q2QX07; Pisum sativum PsPTP1, O82710; Medicago truncatula MtPTP1, A0A072VQ92; Glycine max GmPTP1, O82687; Populus nigra × (Populus deltoides × P. nigra) PdPTP1, Lu et al., 2020; Nicotiana tabacum NtPTP1, A0A1S4A2F4; and Homo sapiens HsPTP1B, P18031.

Figure 2

Identification by mass spectrometry of the Cys residues of histidine (His)-AtPTP1 targeted by S-nitrosation. (A) Primary sequence of AtPTP1 protein. The seven peptides identified by mass spectrometry are boxed, the Cys residues are shaded and the active site signature motif of PTPases is indicated in bold. (B) Relative percentage of biotinylated peptide to the total abundance of this peptide in the sample following treatment of His-AtPTP1 by 100 μM glutathione (GSH) or S-nitrosoglutathione (GSNO). Values are the results of three mass spectrometry analyses. Significant difference is denoted by an asterisk: ^**p ≤ .01.

Figure 3

Effect of GSH or GSNO treatment on His-AtPTP1 activity in the absence or presence of DTT. (A) Experimental design. (B) Measurement of the enzymatic rate of His-AtPTP1 following treatment with 100 or 500 μM of GSNO (grey columns) or GSH (black columns) in the absence or presence of 20 mM DTT. Following the treatment, GSH or GSNO was removed through exclusion chromatography. Results are expressed relative to the enzyme activity measured without GSH/GSNO or DTT treatments. Values are means of four measurements ±SEM. The results shown are from one representative experiment among three independent experiments. Significant differences are denoted by different letters (p ≤ .05).

Figure 4

Effect of H₂O₂ treatment on His-AtPTP1 activity in the absence or presence of DTT. (A) Experimental design. (B) The enzymatic activity of His-AtPTP1 was measured following treatment with 50, 100, 250, and 500 μM or 1, 2, or 5 mM of H₂O₂ in the absence or presence of 20 mM DTT. Results are expressed relative to the enzyme activity measured without H₂O₂ or DTT treatments. Values are means of four measurements ±SEM. The results shown are from one representative experiment among three independent experiments. Significant differences are denoted by different letters (p ≤ .05).

Figure 5

Effect of H₂O₂ treatment on His-AtPTP1 activity in the absence or presence of DTT following pre-treatment with GSH or GSNO. (A) Experimental design. (B) Measurement of the enzymatic rate of His-AtPTP1 following treatment with 50 and 250 μM or 1 mM of H₂O₂ in the absence of 20 mM DTT after pre-treatment with 100 or 500 μM of GSH or GSNO. (C) Measurement of the enzymatic rate of His-AtPTP1 following treatment with 50 μM, 250 μM or 1 mM of H₂O₂ in the presence of 20 mM DTT after pre-treatment with 100 or 500 μM of GSH or GSNO. Values are means of four measurements ±SEM. The results shown are from one representative experiment among three independent experiments. Significant differences are denoted by different letters (p ≤ .05).