Elsevier

Free Radical Biology and Medicine

Volume 33, Issue 8, 15 October 2002, Pages 1121-1132
Free Radical Biology and Medicine

Original contribution
Inhibition of PTPs by H2O2 regulates the activation of distinct MAPK pathways

https://doi.org/10.1016/S0891-5849(02)01000-6Get rights and content

Abstract

It has been shown that endogenous production of reactive oxygen species (ROS) during T cell activation regulates signaling events including MAPK activation. Protein tyrosine phosphatases (PTPs) have been regarded as targets of ROS which modify the catalytic cysteine residues of the enzymes. We have analyzed the interplay between the inhibition of PTPs and the activation of MAPK by H2O2. Stimulation of Jurkat T cells with H2O2 induces the phosphorylation of ERK, p38, and JNK members of MAPK family. H2O2 stimulation of T cells was found to inhibit the PTP activity of CD45, SHP-1, and HePTP. Transfection of cells with wtSHP-1 decreased H2O2-induced ERK and JNK phosphorylation without affecting p38 phosphorylation. Transfection with wtHePTP inhibited H2O2-induced ERK and p38 phosphorylation without inhibiting JNK phosphorylation. The Src-family kinase inhibitor, PP2, inhibited the H2O2-induced phosphorylation of ERK, p38, and JNK. The phospholipase C (PLC) inhibitor, U73122, or the protein kinase C (PKC) inhibitor, Ro-31-8425, blocked H2O2-induced ERK phosphorylation, whereas the same treatment did not inhibit p38 or JNK phosphorylation. Taken together, these results suggest that inhibition of PTPs by H2O2 contributes to the induction of distinct MAPK activation profiles via differential signaling pathways.

Introduction

Reactive oxygen species (ROS) are generated by the incomplete reduction of oxygen during various biological processes [1]. It has been known that ROS mediate diverse effects on the function of the cells [2]. Because ROS can be generated rapidly in response to extracellular stimuli and can be degraded efficiently, they have been regarded as potential second messengers [3]. Supporting this concept, various growth factor receptors, cytokine receptors, and GPCRs (G-protein coupled receptors) have been shown to produce ROS including H2O2 when cognate ligand binds the receptor [4].

It has been shown that T cells produce endogenous ROS in response to various physiological stimuli. ROS production in thymocytes stimulated with concanavalin A has been suggested to modulate JNK activation [5]. CD28 costimulation has been shown to produce ROS by a lipoxygenase, resulting in IL-2 gene transcription through NF-κB activation [6]. Recently, it has been demonstrated that endogenously produced ROS during T cell receptor (TCR) activation regulate ERK activation and Fas ligand expression [7]. In addition to the endogenous production of ROS, exogenously provided ROS have been shown to affect T cell function at inflammatory sites, where activated phagocytes release a high amount of ROS through the NADPH oxidase system [8]. Amine oxidase in the endothelial cells has also been proposed as a source of H2O2 during T cell migration [9]. Abnormal regulation of T cell function in complex diseases, such as rheumatoid arthritis [10], atherosclerosis [11], AIDS [12], and cancer [13], has been attributed to the oxidative environment in pathological situations. Although it is clear that endogenously or exogenously produced ROS affect T cell signaling, the target molecules of ROS and the mechanism of ROS regulation have not been precisely defined.

Compared to the other members of ROS, H2O2 is more stable and membrane permeable leading to the proposal that H2O2 can function as a second messenger. Both exogenous treatment and endogenous production of H2O2 has been suggested to contribute to cellular signaling by inhibiting protein tyrosine phosphatases (PTPs) [14]. PTPs contain an essential catalytic cysteine residue in their active sites with a lower pKa (∼5.5) than the pKa (∼8) of other cysteine residues in most proteins [15]. The low pKa makes the thiolate anion especially susceptible to the inhibitory action of H2O2. H2O2 has been shown to inhibit PTPs in vitro [16] as well as inside cells [17]. For example, it has been shown that H2O2 treatment substantially reduces total cellular PTP activity in the MO7e [18] and HER14 cells [19]. Specifically, H2O2 was found to inhibit PTP activity of CD45 in Jurkat T lymphocytes [20] and neutrophils [21]. PTP activity of SHP-1 was also found to be inhibited by H2O2 in SHP-1-transfected HELA cells [22]. Recent reports have shown that endogenously produced ROS inactivate PTP1B when insulin receptor [23] or EGF receptor [24] were triggered, suggesting that inhibition of PTPs by H2O2 may be a physiologically important event during cell signaling.

CD45, SHP-1, and HePTP are PTPs predominantly expressed in T cells, and it has been known that the activities of these PTPs are important in T cell activation. CD45 is the most abundant PTP in T cells, accounting for about 75% PTP activity in T cell membrane [25]. CD45 activity is critical for T cell activation and T cells deficient in CD45 expression failed to generate signals responding to TCR-engaging antibodies [26], [27], [28]. One requirement for CD45 in T cell activation has been understood to be the dephosphorylation of the inhibitory tyrosine residue of Src-family kinases such as Lck and Fyn [29]. However, it has been reported that CD45 may also dephosphorylate the activation loop tyrosine residue of Src-family tyrosine kinases [30], [31], suggesting that the role of CD45 in T cell signaling may depend on the types of stimuli and the accessibility of the substrates to the phosphatase. SHP-1 is a PTP that is predominantly expressed in hematopoietic cells [32]. Thymocytes from SHP-1-deficient mice (designated moth-eaten) showed the enhancement of constitutive as well as induced tyrosine phosphorylation of the TCR complex [33], suggesting a negative role of SHP-1 in TCR signaling. Supporting this concept, SHP-1 has been shown to dephosphorylate SLP76 [34] and Zap70 [35], which are important signaling molecules in T cell activation. HePTP is expressed exclusively in hematopoietic cells [36]. Overexpression of HePTP in T cells resulted in downregulation of ERK activation and IL-2 promoter activation, suggesting that HePTP has a negative role in T cell activation [37]. It has been shown that HePTP dephosphorylates the activating tyrosine residue of ERK [38], and PMA- and TCR-induced ERK activation is increased in spleen cells from HePTP knockout mice [39], indicating that ERK is an authentic substrate of HePTP.

One well-documented result of cell stimulation with H2O2 is MAPK activation [40]. The MAPK family members considered in this report include ERK, p38, and JNK. ERK activation has been implicated mainly in proliferation in response to growth factors, whereas p38 and JNK activation are more important to stress responses like inflammation [41]. It has been suggested that the combination of the magnitude and kinetics of activation of each member of MAPK family determines the appropriate response of the cell according to the specific stimulus [42]. In the case of T cells, the distinct activation profile of three members of the MAPK family has been shown to influence the specific stages of thymocyte development as well as the precise effector function of mature T cells [43].

In this report, the role of PTPs on H2O2-induced MAPK activation was analyzed by measurement of PTP activity immunoprecipitated from intact cells and by transfection of PTP vectors. Treatment of T cells with H2O2 inhibited PTP activity of CD45, SHP-1, and HePTP, suggesting that PTPs are targets of H2O2. Ectopic expression of wtSHP-1 inhibited H2O2-induced ERK and JNK phosphorylation without inhibiting p38 phosphorylation. On the other hand, ectopic expression of HePTP specifically affected H2O2-induced ERK and p38 phosphorylation without affecting JNK phosphorylation. The differential effect of PTP transfection suggests that each PTP controls the distinct signaling pathway leading to MAPK phosphorylation. The activity of Src-family tyrosine kinase was necessary for H2O2-induced ERK, p38, and JNK phosphorylation, whereas PLC and PKC activity was dispensable in the case of p38 and JNK phosphorylation induced by H2O2. Taken together, results in this study suggest that inhibition of PTPs by H2O2 contributes to the distinct activation profile of three members of the MAPK family.

Section snippets

Reagents and antibodies

H2O2 was purchased from Sigma Chemical Co. (St. Louis, MO, USA). PP2, U73122, and Ro-31-8425 were obtained from Calbiochem (La Jolla, CA, USA). Antibodies used in this study were obtained as follows: phospho-ERK (New England Biolabs, Beverly, MA, USA); phospho-p38 and phospho-JNK (Promega, Madison, WI, USA); ERK2, JNK, normal IgG, and CD45 for immunoprecipitation (Santa Cruz Biotechnology, Santa Cruz, CA, USA); CD45 for Western blotting and SHP-1 (Transduction Laboratories, Lexington, KY, USA);

H2O2 induces activation-related phosphorylation of ERK, p38, and JNK MAPKs

The goal of this study is to understand the mechanism of H2O2-induced signaling leading to MAPK phosphorylation and activation. The phosphorylation level of MAPKs was measured by Western blotting with antibodies that specifically recognize both phospho-Tyr and phospho-Thr residues that are regarded to be necessary and sufficient for the activation of MAPKs. As shown in Fig. 1, stimulation of Jurkat T cells with H2O2 induced specific phosphorylation of all three members of the MAPK family (ERK,

Discussion

The level of tyrosine phosphorylation inside the cell is determined by the balance between the activity of protein tyrosine kinases (PTKs) and of protein tyrosine phosphatases [52]. Although various potential schemes for the regulation of PTKs have been proposed, rigorously verified regulatory mechanisms for PTPs are poorly understood [53]. Inhibition of PTP activity by ROS is regarded as one physiological means of regulation of PTPs. It has been documented that ROS are produced endogenously

Acknowledgements

This study was supported by the National Institutes of Health, grant number AI42794, and by the Jean P. Schultz Endowed Oncology Research Fund. The authors wish to acknowledge the generous gift of SHP-1 and HePTP vectors from Dr. T. Mustelin (La Jolla Cancer Research Center, The Burnham Institute).

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