Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling

Abstract

Lysophosphatidic acid (LPA) is a growth factor for many cells including prostate and ovarian cancer-derived cell lines. LPA stimulates H2O2 production which is required for growth. However, there are significant gaps in our understanding of the spatial and temporal regulation of H2O2-dependent signaling and the way in which signals are transmitted following receptor activation. Herein, we describe the use of two reagents, DCP-Bio1 and DCP-Rho1, to evaluate the localization of active protein oxidation after LPA stimulation by detection of nascent protein sulfenic acids. We found that LPA stimulation causes internalization of LPA receptors into early endosomes that contain NADPH oxidase components and are sites of H2O2 generation. DCP-Rho1 allowed visualization of sulfenic acid formation, indicative of active protein oxidation, which was stimulated by LPA and decreased by an LPA receptor antagonist. Protein oxidation sites colocalized with LPAR1 and the endosomal marker EEA1. Concurrent with the generation of these redox signaling-active endosomes (redoxosomes) is the H2O2- and NADPH oxidase-dependent oxidation of Akt2 and PTP1B detected using DCP-Bio1. These new approaches therefore enable detection of active, H2O2-dependent protein oxidation linked to cell signaling processes. DCP-Rho1 may be a particularly useful protein oxidation imaging agent enabling spatial resolution due to the transient nature of the sulfenic acid intermediate it detects.

Peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling

“…An elegant study of epidermal growth factor (EGF) stimulation of ER-localized Nox4 showed that the native ER-localized PTP1B became oxidatively inactivated and that the ER targeting of PTB1B was required for its inhibition and for effective EGF-stimulated proliferation [81]. Also, recent imaging of H 2 O 2 generation [83, 84] and protein oxidation [85, 86] show that localized buildup of H 2 O 2 occurs proximally to growth factor receptors and oxidatively-modified targets. In endosome-mediated growth factor and cytokine signaling, the H 2 O 2 generation localized proximal to these so-called “redoxosomes” is required for effective downstream signaling [74, 85].…”

“…Also, recent imaging of H 2 O 2 generation [83, 84] and protein oxidation [85, 86] show that localized buildup of H 2 O 2 occurs proximally to growth factor receptors and oxidatively-modified targets. In endosome-mediated growth factor and cytokine signaling, the H 2 O 2 generation localized proximal to these so-called “redoxosomes” is required for effective downstream signaling [74, 85]. Thus, models for Prx involvement in regulating H 2 O 2 -mediated cell signaling processes must be compatible with the necessity for H 2 O 2 to act locally during signal transduction.…”

Endogenous, regulatory cysteine sulfenylation of ERK kinases in response to proliferative signals

“…S3) during responses to LPA, a lipid growth factor produced by prostate cancers and found in the ascites fluid of many ovarian cancers [53, 54]. Our group has previously shown that LPA induces the intracellular formation of H 2 O 2 in these cell lines and that the H 2 O 2 is necessary for the proliferative response of these cell lines to LPA [9, 10]. In serum-starved SK-OV-3 cells, there is no detectable pattern for changes in sulfenic acid formation in the total population of ERK1/2 in response to LPA; instead, it appears that total ERK1/2 is continuously oxidized across the time course (Fig.…”

“…An important milestone in support of ROS as regulators of cell signaling was the discovery in the mid-1990s by Sundaresan et al that H 2 O 2 generation was required to elicit appropriate proliferative responses to Platelet-Derived Growth Factor (PDGF) in primary rat vascular smooth muscle cells [5]. Roles for H 2 O 2 as a signaling mediator have now been demonstrated in many cell types and in vivo models [6–8], including our recent work establishing the important role of H 2 O 2 in the proliferative responses to lysophosphatidic acid (LPA), a bioactive lipid synthesized by ovarian and prostate cancer cells and cell lines [9, 10]. ROS have also been implicated in diverse signaling processes such as migration by endothelial cells [11], stem cell pluripotency and differentiation [12], and chondrocyte response to fibronectin fragments [13].…”

“…1B) [27, 28]. This probe has been used to identify multiple novel targets of oxidation, such as Akt2 [9, 14, 29], responding to extracellular signals [11, 28, 30, 31]. Based on preliminary data from experiments suggesting that oxidation of mitogen-activated protein kinases ERK1 and ERK2 (designated ERK1/2) occurs during proliferation (unpublished) and migration [11], we undertook a project to further investigate the redox regulation of these proteins during cell signaling.…”