An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases

Abstract

Regulation of protein tyrosine phosphatases (PTPs) through reversible oxidation of the active site cysteine is emerging as a general, yet poorly characterized, mechanism for control of the activity of this important group of enzymes. This regulatory mechanism was initially described after in vitro treatment of PTPs with oxidizing agents. However, accumulating evidence has substantiated the notion that this mechanism is also operating in vivo, e.g., in association with the transient increase in H₂O₂ production which occurs after activation of receptor tyrosine kinases. A novel generic antibody-based method for monitoring of PTP oxidation is described. The sensitivity of this strategy has been validated by the demonstration of oxidation of endogenously expressed PTPs after stimulation of cells with growth factors. The method was also instrumental in providing the first evidence for intrinsic differences between PTP domains with regard to sensitivity to oxidation.

Introduction

Reversible tyrosine phosphorylation is a major regulatory mechanism for control of fundamental cellular processes such as proliferation, migration, and metabolism. The steady-state tyrosine phosphorylation is governed by the balanced action of tyrosine kinases and protein tyrosine phosphatases (PTPs).

PTPs constitute a structurally diverse family of enzymes, subjected to multiple mechanisms of control, with important regulatory functions in physiological and pathological situations (for recent reviews see [1], [2], [3]). The super-family of PTPs is broadly divided into dual-specificity PTPs, low molecular weight PTPs, and classical tyrosine-specific PTPs. The latter sub-group, which is the topic of this article, is composed of receptor-like and cytosolic PTPs. Classical PTPs contain a highly conserved catalytic signature motif, (V/I) H C S X G X G R (S/T) G, residing at the bottom of the active site cleft, as determined by analyses of crystallized PTPs. The invariant cysteine in this motif is crucial for catalysis. The catalytic reaction involves formation of a cysteinyl-phosphate intermediate, which is subsequently hydrolyzed.

The catalytic cysteine of PTPs occurs as a thiolate anion at physiological pH, as a consequence of charge interactions with the microenvironment [4]. This form is responsible for the catalytic activity, and is also highly sensitive to oxidation. Modification of the active site cysteine by reversible oxidation, with concomitant inhibition of PTP activity, was originally described after in vitro treatment of PTPs with H₂O₂ [5]. Further in vitro studies demonstrated that pervanadate treatment of PTPs was associated with an oxidation of the active site cysteine to an irreversibly oxidized sulfonic acid (S–O₃H), thus explaining the potent PTP-inhibitory activity of pervanadate [6].

These in vitro findings prompted studies on the possibility that oxidation of PTPs represented a physiological regulatory mechanism. Activation of tyrosine kinase receptors is associated with a transient production of H₂O₂, which is required for full receptor phosphorylation and activation of downstream signalling (reviewed in [7]) (Fig. 1). It was therefore investigated if receptor tyrosine kinase activation was associated with PTP inhibition. The first evidence that such a mechanism might operate took advantage of the fact that the reduced active forms of the active site cysteine is efficiently alkylated by incubation with, iodoacetic acid (IAA) [8]. EGF stimulation of cells was found to be associated with a transient decrease in the fraction of PTP-1B which reacted with IAA. Subsequently, evidence was provided for inactivation of PTP-1B and LMW-PTP after stimulations of cells with insulin or PDGF [9], [10]. Also, transient oxidation of SHP-2 was observed after PDGF stimulation [11], [12]. Together these observations, summarized in Fig. 1, suggest that H₂O₂-mediated oxidation of PTPs is a physiological mechanism for transient inactivation of PTPs in association with activation of receptor tyrosine kinases.

Reversible oxidation of PTPs also appears to be one of the mechanisms whereby UV-irradiation exerts its biological effects. Production of reactive oxygen species is a consequence of UV-irradiation of cells. Inhibition of PTP activity in cells after UV-irradiation was originally shown by the demonstration that cell membranes derived from UV-irradiated cell contained a reduction in PTP activity [13]. Indications that this inhibition occurred through reversible oxidation of PTPs were provided by the observation that UV-induced inhibition of PTPs was sensitive to reducing agents. Additional studies have subsequently shown inactivation of individual PTPs, such as DEP-1, RPTPα, and RPTσ, after UV-irradiation of cells [12], [14].

In vivo oxidation of PTPs is a reversible phenomenon. A series of chemical variants of reversibly oxidized active site cysteines have been described. Conversion of the active site cysteine to a sulfenic acid (–S–OH) has been demonstrated after in vitro treatment of PTPs. In addition, formation of disulfide bonds between the active site cysteine and other cysteines has been implied in oxidized species of LMW-PTP and cdc25 [9], [15]. Also, glutathionylated forms of the active site cysteine were identified by mass spectrometric analyses of PTP-1B which had been oxidized in cultured cells [16]. Finally, the analyses of crystallized reversibly oxidized PTP-1B revealed the presence of a novel sulfenyl-amide form generated through a covalent bond between the sulfur atom of the cysteine and the main chain nitrogen of the adjacent serine residue [17], [18]. It thus still remains to be characterized which of these forms dominates in vivo, and if different PTPs preferentially form one of these variants.

The discovery of reversible oxidation as a general mechanism for regulation of PTPs has stimulated efforts to develop sensitive and specific methods whereby changes in the oxidation state of PTPs can be monitored. The methods that have been used can broadly be divided into assays that monitor a reduction in the fraction of reduced PTPs or, alternatively, monitor the increase in the amounts of oxidized forms.

The two assays that detect reductions in the levels of active forms are labelling of reduced forms with iodoacetic acid, which reacts specifically with the reduced form of the active site cysteine, and measurements of in vitro PTP activity after extraction of cellular fractions or individual PTPs [8], [10], [14], [19]. One common flaw with both these assays is that they measure a decrease in the active reduced fraction, rather than an increase of oxidized forms. Since reduced forms appear to dominate in cells, these assays are intrinsically less sensitive. This aspect can be particularly problematic when only a biologically relevant subfraction of a given PTP is subjected to oxidation, as suggested in the case of the specific inactivation of PDGF receptor-associated SHP-2 after PDGF stimulation [11]. Additionally, the assays based on activity measurements require that the balance of reduced and oxidized forms is maintained throughout the assay procedure. It is a common experience that oxidation of PTPs occurs during in vitro handling, and in vitro PTP assays are traditionally performed in the presence of reducing agents like DTT. This can obviously not be done when the purpose is to monitor oxidation-induced changes that occurred prior to cell lysis. This problem has partially been overcome by performing cell lysis and assays in the presence of weaker reducing agents, like 5 mM N-acetylcysteine, which appear to maintain the original ratio of oxidized and reduced forms [12].

The two assays that have been used to monitor an increase in the oxidized inactive fraction of PTPs are a modified in-gel PTP assay and an antibody-based assay [11], [12]. In the in-gel PTP assay, reduced forms of PTPs are irreversibly alkylated by addition of an alkylating agent, e.g., iodoacetic acid. After SDS–PAGE of cellular fractions or immunoprecipitated PTPs in gels containing a PTP substrate, the activity of PTPs that occurred as reversibly oxidized forms can be recovered through in-gel refolding and treatment with reducing agents. This assay provides the opportunity to monitor changes in PTP oxidation without prior isolation through immunoprecipitation. As noted in the original description of this method, it relies on the successful in-gel refolding of PTPs [11]. This has clearly been demonstrated to work for cytosolic PTPs. However, it is still not convincingly shown that the method can be used for detection of oxidized receptor-like PTPs which, as larger molecules, are less efficiently refolded in SDS–PAGE gels.

The principle for the antibody-based assay format is described in Fig. 2. Like the in-gel assay this assay takes advantage of the sensitivity of reduced forms of PTPs to alkylation. Following alkylation of reduced forms, reversibly oxidized PTPs are converted to stable sulfonic acid forms by oxidation with pervanadate. These species are finally detected with antibodies raised against a peptide corresponding to the conserved PTP active site in which the cysteine is oxidized to sulfonic acid.

The development of the oxPTP antibodies, and the experimental procedures for their use are described in detail below.