Selective regulation of hydrogen peroxide signaling by receptor tyrosine phosphatase-α

Abstract

Reactive oxygen species (ROS) are constantly produced in the human body and are involved in the pathogenesis of aging, cardiovascular diseases, and cancer. Emerging evidence indicates that oxidation and inhibition of protein tyrosine phosphatases (PTPs) are critical for ROS signal transduction. However, the role of individual PTPs in ROS signaling remains unclear. Here, we demonstrated that the receptor-like PTPα (RPTPα) was an effector of H₂O₂, the most stable form of ROS. H₂O₂ at nontoxic concentration rapidly induced the association of RPTPα with Src family kinases, platelet-derived growth factor receptor-β, and protein kinase D in various cultured cells, although it markedly suppressed RPTPα phosphorylation on Tyr-789. We further identified that RPTPα selectively regulated the signal transduction pathways induced by H₂O₂. Particularly, RPTPα was required for the activation of protein kinase D and for the modulation of p130Cas tyrosine phosphorylation in response to H₂O_2. In contrast, the H₂O₂-induced inactivation of Src family kinases and suppression of paxillin phosphorylation on Tyr-118 were both largely independent of RPTPα. Our findings indicate that H₂O₂ signaling pathways are selectively regulated by RPTPα in cells, which may provide new insights into the functional regulation of ROS signal transduction by PTPs.

Introduction

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), superoxide (O₂⁻), and hydroxyl radical (HO), are constantly produced in the human body under physiological conditions, such as metabolism and activation of cell surface receptors, to maintain cell survival [1], [2], [3], and in pathophysiological settings, such as inflammation, infection, tissue injury and hypercholesterolemia [4], [5], [6]. Both superoxide and hydroxyl radical are highly reactive, extremely short-lived, hardly permeable, and unlikely to mediate specific signals remote from the site of production. On the other hand, H₂O₂ is uncharged, relatively stable, and freely diffusible across the membrane to adjacent cells or tissues, serving as an autocrine and a paracrine mediator. Conventional thought has generally regarded ROS as being harmful to cells and tissues, leading to the pathogenesis of cardiovascular diseases, cancer, and neurodegenerative disorders [7], [8], [9]. However, controlled clinical trials have failed to show a consistent benefit of antioxidants on these diseases [10], [11], [12], [13]. A growing body of evidence suggests that ROS may be both protective and deleterious depending on concentration [6], [14]. We recently reported a novel mechanism for the vascular protective effects of ROS through a rapid inactivation of Src family kinases (SFKs) by H₂O₂ at low to moderate concentrations (50–250 μM) in primary human endothelial cells [15].

ROS react with and oxidize protein thiol groups. Most cysteine residues in proteins are protonated at physiological pH, with a pK_a of ∼8.5. However, cysteine residue surrounded by basic amino acids has a low pK_a of ∼5.0 and is not protonated under physiological conditions, thus it is particularly susceptible to oxidation [16]. Protein tyrosine phosphatases (PTPs) contain such a cysteine residue with an extremely low pK_a (pK_a < 6) in their signature motif, HCXXGXXR(S/T). This cysteine residue can be readily and reversibly oxidized by ROS, which inactivate the enzymes [17]. Numerous lines of evidence, including our results, have demonstrated that PTPs can be directly inhibited by H₂O₂ in vitro and reversibly inhibited by H₂O₂ in intact cells [15], [18], [19]. Of note, Groen et al. [20] recently reported that PTPs were differentially oxidized in cells and that there was not a strict inverse correlation between catalytic activity and oxidizability. Because PTPs control the protein tyrosine phosphorylation that is a fundamental mechanism for many signal transduction pathways, a growing body of evidence suggests that the oxidation and inhibition of PTPs by ROS may be an initial and critical step for ROS signaling in cells [2], [15], [21]. However, the role of individual PTPs involved in ROS signaling remains unclear.

The receptor-like PTPα (RPTPα) is a widely expressed transmembrane PTP with a short heavily glycosylated extracellular domain and two tandem cytoplasmic PTP domains [22]. The membrane-proximal domain contains most catalytic activity, whereas the membrane-distal domain is catalytically inactive but has a regulatory role [23]. It has been shown that RPTPα is a positive regulator of SFKs and is required for the integrin-mediated cell spreading and migration [24], [25], [26]. Deficiency of RPTPα impairs the catalytic activities of SFKs and the integrin signaling [24], [26]. In addition, RPTPα interferes with insulin receptor signaling [27] and regulates the Kv1.2 potassium channel upon the activation of m1 muscarinic acetylcholine receptor [28]. Emerging evidence indicates that RPTPα can be oxidized and inhibited by H₂O₂ [23]. However, the role of RPTPα in ROS signaling is not known.

In the present study, we have demonstrated that RPTPα is required for the activation of protein kinase D (PKD; initially called atypical PKCμ) [29] and for the modulation of tyrosine phosphorylation of focal adhesion protein p130 Crk-associated substrate (p130Cas) in response to H₂O_2. In contrast, the H₂O₂-induced inactivation of SFKs and suppression of paxillin phosphorylation on Tyr-118 are both largely independent of RPTPα. Our findings indicate that the signal transduction pathways induced by H₂O₂ are selectively regulated by RPTPα in cells, which may provide new insights into the functional regulation of ROS signaling by PTPs.