Elsevier

Methods

Volume 35, Issue 1, January 2005, Pages 28-36
Methods

Development of a modified in-gel assay to identify protein tyrosine phosphatases that are oxidized and inactivated in vivo

https://doi.org/10.1016/j.ymeth.2004.07.005Get rights and content

Abstract

Protein tyrosine phosphatases (PTPs) comprise a superfamily of enzymes that control a diverse array of signal transduction pathways. However, the function and regulation of many of these enzymes remain undefined. Previous studies have shown that the optimal tyrosine phosphorylation response to various exogenous stimuli requires the production of reactive oxygen species (ROS). It has been proposed that ROS might transiently inactivate inhibitory PTPs, thus facilitating tyrosine phosphorylation-dependent signaling. Interestingly, the unique chemistry of the invariant, active site Cys residue located in the signature motif renders it highly susceptible to oxidation, leading to the inactivation of PTPs. We have developed a novel strategy to identify those PTPs that are oxidized and therefore, inactivated in response to extracellular stimuli. Iodoacetic acid (IAA) was used to alkylate selectively the thiolate anion of the active site Cys in the reduced PTPs. In contrast, any PTPs in which the active site Cys had been oxidized in response to the stimulus were resistant to alkylation. Following this key step to differentiate between the two pools of PTPs, the oxidized phosphatases were reduced back to the active state during the process of a standard in-gel PTP activity assay. This novel technique revealed, for the first time, that multiple cellular PTPs were indeed oxidized and inactivated in response to exogenous hydrogen peroxide. We have used this technique extensively to show that the ligand-stimulated production of intracellular hydrogen peroxide reversibly regulates the activity of specific PTPs in vivo. By defining the precise PTP targets of intracellular oxidants, the mechanistic details of signal transduction can be delineated. Due to the potential use of this method in finding the molecular targets of intracellular oxidants in diverse signaling pathways, we describe here the theoretical background and the detailed protocols of the modified in-gel PTP assay.

Introduction

Reversible protein tyrosyl phosphorylation is tightly regulated by the coordinated actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Although much attention has been focused on the functions of PTKs, it is now clear that PTPs play a key role in the regulation of signal transduction events [1]. The application of new technologies, such as gene knockout in mice [2], [3] and the substrate trapping approach in cell culture [4], promoted our understanding of the functions of PTPs. Recent studies have revealed that many in vivo substrates of PTPs are either the PTKs themselves [5], [6], [7], [8], [9], [10] or the downstream signaling modulators of PTKs, for instance, tyrosine phosphorylated adaptor proteins [7], [11], [12]. These findings highlight the important regulatory role of PTPs in fundamental physiological functions such as cell growth, proliferation, differentiation, survival, metabolism, and motility.

Being such a critical regulator of cell signaling, the enzymatic activity and substrate specificity of PTPs must be tightly controlled in vivo. The zip-code model, which suggests that the substrate specificity of PTPs is determined by intracellular targeting through non-catalytic domains of these enzymes, has been thought to be a primary regulatory mechanism [13]. However recent studies, guided by crystal structures, have revealed intrinsic specificity in substrate recognition by the catalytic domain of PTPs [10]. It has been proposed that ligand binding to the receptor-like PTPs also provides a means to regulate the enzymatic activity of these phosphatases. Recent progress in the study of the regulatory mechanisms of PTPs, however, has revealed a new tier of control, in response to stimulus-induced production of Reactive Oxygen Species (ROS).

The enzymes in the superfamily of PTPs, including classical PTPs which dephosphorylate phosphotyrosine residues, and dual specificity phosphatases (DSPs) which recognize phosphorylated Ser/Thr and Tyr residues, are characterized by the presence of a signature motif [I/V]HCXXGXXR[S/T] [14]. A unique feature of the invariant Cys residue within the signature motif is its unusually low pKa (between pH 4.5 and pH 5.5, compared to the typical pKa for Cys  pH 8.5), due to the local environment surrounding the active site of PTPs [15], [16]. Due to the low pKa, the invariant Cys residue is present as a thiolate anion at physiological pH, thus enhancing it’s ability to serve as a nucleophile a to attack the phosphate group of the substrate, but also rendering the active site Cys highly susceptible to oxidation. Once oxidized, the nucleophilic properties are lost, leading to the inactivation of PTPs. Such a redox switch of the invariant Cys residue provides a means to regulate the enzymatic activity of PTPs in response to the production of intracellular ROS, which have been shown to function as a second messenger in diverse signaling pathways. We have applied this principle to formulate a novel analysis, which allows investigation of the redox-mediated regulation of PTPs in vivo.

The theory of the modified in-gel phosphatase assay is shown in Fig. 1. Cells are treated with a stimulus that triggers the production of intracellular ROS. Despite the presence of a pool of PTPs that encounter ROS and become oxidized, the majority of cellular PTPs remain in the reduced form. The task at hand is to distinguish the reversibly oxidized PTPs from the background of reduced PTPs in a cellular context. To achieve this goal, we harvest cells under anaerobic conditions to prevent spontaneous oxidation and lyse cells in buffer supplemented with a free thiol-reactive alkylating reagent, such as iodoacetic acid (IAA) (Fig. 1). Those reduced PTPs, once alkylated on the invariant, active site Cys residue, are permanently inactivated (Fig. 1). In contrast, due to the presence of the sulfenic acid, the invariant Cys of oxidized PTPs is resistant to alkylation. This is the key step to differentiating the two pools of PTPs in cell lysates. Following alkylation, the PTPs in lysates are analyzed by a standard in-gel phosphatase activity assay [17]. A SDS-gel is cast containing a radioactively labeled substrate. An aliquot of cell lysate is subjected to SDS–PAGE, followed by sequential denaturation and renaturation of proteins in the presence of reducing reagents, which reactivates those PTPs which the invariant Cys residue has been oxidized to the sulfenic acid in response to a stimulus. In contrast, those PTPs that have not encountered ROS in cells, and have been alkylated by IAA during cell lysis, remain inactive. The final step of incubation in the renaturation buffer promotes reactivation of the oxidized PTPs, which dephosphorylate substrate in the region of the gel immediately surrounding the PTP proteins. The gel is then stained with Coomassie blue to stop the dephosphorylation reaction. This step also provides a record of protein loading. After drying, the gel is exposed to film and the activity of PTPs is visualized by autoradiography (Fig. 1).

Section snippets

Purified PTP1B

A clone encoding amino acids 1–321 (37 kDa) of wild type human PTP1B was placed in a pET19b vector (Novagen) for protein expression. Briefly, the 37 kDa PTP1B was expressed in Escherichia coli strain BL21 and purified by chromatography sequentially on Fast Flow S-Sepharose and Mono Q Sepharose, as described previously [18].

Buffers used in this study

  • Buffer A: 20 mM Tris–HCl, pH 8, 150 mM NaCl, 2.5 mM EDTA, and 1 mg/ml lysozyme.

  • Buffer B: 50 mM imidazole, pH 7.2, 10 mM DTT.

  • Buffer C: 50 mM imidazole, pH 7.2, 10 mM DTT, 5 mM EDTA, and

Principle

Iodoacetic acid (IAA), which reacts with the free Cys thiols, can be used to differentiate oxidized PTPs from reduced PTPs in cell lysates (Fig. 1). To test the effectiveness of IAA in alkylating the active site Cys residue, thereby inactivating PTPs, the purified, 37 kDa form of PTP1B was used. The IAA-reacted PTP1B was then subjected to the in-gel phosphatase activity assay.

IAA reaction

In order to prevent the spontaneous oxidation of phosphatase through the air oxygen dissolved in solution, degassed

Principle

The successful application of the modified in-gel phosphatase assay relies on the quantitative labelling by IAA of those PTPs that remain in a reduced state in a cellular context following the stimulus. We have shown that the purified PTP1B is permanently inactivated through alkylation by IAA (Fig. 2). To test whether the same mechanism effectively inactivates multiple cellular PTPs, we applied a similar approach in a cell culture model. Rat-1 fibroblast cells were lysed in the presence of

Principle

We wished to develop a technique that is capable of identifying those PTPs that are oxidized in response to an extracellular stimulus. The results shown in Fig. 2, Fig. 3 provided a solid foundation for the use of IAA to formulate such a technique. We propose that IAA, which effectively alkylates the free thiol of active Cys in the reduced PTPs, is incapable of reacting with the oxidized sulfenic acid form of the active site Cys residue in oxidized PTPs (Fig. 1). We tested this hypothesis by

Concluding remarks

The concept that ROS function as second messengers in signal transduction has been proposed for more than a decade. It was well documented that physiological ligands, including cytokines [22] and peptide growth factors acting through receptor tyrosine kinases [24], [25] or G-protein coupled receptors [23], [26], stimulate the production of intracellular ROS. This increase in the level of ROS in cells has been linked to the activation of various signaling pathways. It was shown that the

Acknowledgments

This work was supported by a grant from the National Science Council of Taiwan (NSC 92-2312-B-001-004) to T.C.M., and grants from the National Institute of Health of USA (CA53840 and GM55989) to N.K.T.

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