Abstract
The formation of a reversible disulfide bond between the catalytic cysteine and a spatially neighboring cysteine (backdoor) in protein tyrosine phosphatases (PTPs) serves as a critical regulatory mechanism for maintaining the activity of protein tyrosine phosphatases. The failure of such protection results in the formation of irreversibly oxidized cysteines into sulfonic acid in a highly oxidative cellular environment in the presence of free radicals. Hence, it is important to develop methods to interconvert PTPs into reduced and oxidized forms to understand their catalytic function in vitro. Protein tyrosine phosphatase 4A type 1 (PTP4A1), a dual-specificity phosphatase, is catalytically active in the reduced form. Unexpectedly, also its oxidized form performs a key biological function in systemic sclerosis (SSc) by forming a kinase–phosphatase complex with Src kinases. Thus, we developed simple and efficient protocols for producing oxidized and reduced PTP4A1 to elucidate their biological function, which can be extended to study other protein tyrosine phosphatases and other recombinantly produced proteins.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hunter T, Karin M (1992) The regulation of transcription by phosphorylation. Cell 70:375–387
Humphrey SJ, James DE, Mann M (2015) Protein phosphorylation: a major switch mechanism for metabolic regulation. Trends Endocrinol Metab TEM 26:676–687
Barford D, Das AK, Egloff MP (1998) The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27:133–164
Alonso A, Sasin J, Bottini N, Friedberg I, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T (2004) Protein tyrosine phosphatases in the human genome. Cell 117:699–711
Tonks NK (2006) Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 7:833–846
Netto LES, Machado LESF (2022) Preferential redox regulation of cysteine-based protein tyrosine phosphatases: structural and biochemical diversity. FEBS J 289:5480–5504
Caselli A, Marzocchini R, Camici G, Manao G, Moneti G, Pieraccini G, Ramponi G (1998) The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H2O2. J Biol Chem 273:32554–32560
Lee S-R, Yang K-S, Kwon J, Lee C, Jeong W, Rhee SG (2002) Reversible inactivation of the tumor suppressor PTEN by H2O2. J Biol Chem 277:20336–20342
Savitsky PA, Finkel T (2002) Redox regulation of Cdc25C. J Biol Chem 277:20535–20540
Zhang R, Kumar GS, Hansen U, Zoccheddu M, Sacchetti C, Holmes ZJ, Lee MC, Beckmann D, Wen Y, Mikulski Z, Yang S, Santelli E, Page R, Boin F, Peti W, Bottini N (2022) Oxidative stress promotes fibrosis in systemic sclerosis through stabilization of a kinase-phosphatase complex. JCI Insight 7:e155761
Denton CP, Khanna D (2017) Systemic sclerosis. Lancet Lond Engl 390:1685–1699
Gabrielli A, Svegliati S, Moroncini G, Amico D (2012) New insights into the role of oxidative stress in scleroderma fibrosis. Open Rheumatol J 6:87–95
Piera-Velazquez S, Makul A, Jiménez SA (2015) Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: regulation by transforming growth factor β. Arthritis Rheumatol Hoboken NJ 67:2749–2758
Karisch R, Fernandez M, Taylor P, Virtanen C, St-Germain JR, Jin LL, Harris IS, Mori J, Mak TW, Senis YA, Östman A, Moran MF, Neel BG (2011) Global proteomic assessment of the classical protein-tyrosine phosphatome and “Redoxome”. Cell 146:826–840
Sacchetti C, Bai Y, Stanford SM, Di Benedetto P, Cipriani P, Santelli E, Piera-Velazquez S, Chernitskiy V, Kiosses WB, Ceponis A, Kaestner KH, Boin F, Jimenez SA, Giacomelli R, Zhang Z-Y, Bottini N (2017) PTP4A1 promotes TGFβ signaling and fibrosis in systemic sclerosis. Nat Commun 8:1060
Gulerez I, Funato Y, Wu H, Yang M, Kozlov G, Miki H, Gehring K (2016) Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis. EMBO Rep 17:1890–1900
Kozlov G, Cheng J, Ziomek E, Banville D, Gehring K, Ekiel I (2004) Structural insights into molecular function of the metastasis-associated phosphatase PRL-3. J Biol Chem 279:11882–11889
Sun J-P, Wang W-Q, Yang H, Liu S, Liang F, Fedorov AA, Almo SC, Zhang Z-Y (2005) Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion. Biochemistry 44:12009–12021
Peti W, Page R (2007) Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51:1–10
Kapust RB, Tözsér J, Fox JD, Anderson DE, Cherry S, Copeland TD, Waugh DS (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14:993–1000
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kumar, G.S. (2024). Preparation of Oxidized and Reduced PTP4A1 for Structural and Functional Studies. In: Thévenin, D., P. Müller, J. (eds) Protein Tyrosine Phosphatases. Methods in Molecular Biology, vol 2743. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3569-8_14
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3569-8_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3568-1
Online ISBN: 978-1-0716-3569-8
eBook Packages: Springer Protocols