Tyr42 phosphorylation of RhoA GTPase promotes tumorigenesis through nuclear factor (NF)-κB

https://doi.org/10.1016/j.freeradbiomed.2017.07.013Get rights and content

Hightlights

  • Hydrogen peroxide induces Tyr42 phosphorylation of RhoA via Src.

  • P-Tyr42 RhoA activates IKKβ on IKKγ, leading to NF-κB activation.

  • P-Tyr42 RhoA is essential for cell proliferation upon hydrogen peroxide.

  • P-Tyr42 RhoA is critical for tumorigenesis.

Abstract

Dysregulation of reactive oxygen species (ROS) levels is implicated in the pathogenesis of several diseases, including cancer. However, the molecular mechanisms for ROS in tumorigenesis have not been well established. In this study, hydrogen peroxide activated nuclear factor-κB (NF-κB) and RhoA GTPase. In particular, we found that hydrogen peroxide lead to phosphorylation of RhoA at Tyr42 via tyrosine kinase Src. Phospho-Tyr42 (p-Tyr42) residue of RhoA is a binding site for Vav2, a guanine nucleotide exchange factor (GEF), which then activates p-Tyr42 form of RhoA. P-Tyr42 RhoA then binds to IκB kinase γ (IKKγ), leading to IKKβ activation. Furthermore, RhoA WT and phospho-mimic RhoA, RhoA Y42E, both promoted tumorigenesis, whereas the dephospho-mimic RhoA, RhoA Y42F suppressed it. In addition, hydrogen peroxide induced NF-κB activation and cell proliferation, along with expression of c-Myc and cyclin D1 in the presence of RhoA WT and RhoA Y42E, but not RhoA Y42F. Indeed, levels of p-Tyr42 Rho, p-Src, and p-65 are significantly increased in human breast cancer tissues and show correlations between each of the two components. Conclusively, the posttranslational modification of as RhoA p-Tyr42 may be essential for promoting tumorigenesis in response to generation of ROS.

Introduction

Oxygen (O2) is often converted to reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide anion (O2•-), and hydroxyl radical (OH), under normal or pathological conditions. ROS are generated through several sources, such as the electron transport chain in mitochondria and enzymes, including NADPH oxidase, lipoxygenase, and cyclooxygenase [1]. In turn, ROS produced in cells can be removed through superoxide dismutase (SOD), catalase, glutathione peroxidase, and peroxiredoxins. Notably, cellular ROS at optimal concentrations can play roles as second messengers in signal transduction pathways to regulate biological responses. However, excessive ROS compared to antioxidant capacity cause serious cellular injuries, leading to the pathogenesis of several diseases, such as chronic inflammation, cardiovascular disease, neurological disorders, fibrotic diseases, and cancer [2]. Actually, elevated ROS in almost all cancers promote tumor development and progression [3].

NF-κB plays a central role in the regulation of a variety of biological processes, including immune responses, development, cell proliferation and cell survival. Deregulated NF-κB has been linked to a variety of diseases, particularly inflammatory diseases and cancer [4], [5]. The NF-κB family is composed of p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), RelB, and c-Rel, which are regulated by inhibitor of NF-κB (IκB) family members. In unstimulated cells, NF-κB of a dimeric form is sequestered in the cytoplasm by IκB proteins, which undergo rapid ubiquitin-mediated proteasomal degradation after being phosphorylated at Ser32/36 residues upon the stimulation of cells. Eventually, the cytoplasmic dimer of NF-κB is released from IκB and translocates into the nucleus, leading to the expression of specific genes. The IκB kinase (IKK) complex, which phosphorylates IκB upon cell stimulation, is composed of kinase subunits IKKα (IKK1) and IKKβ (IKK2) and regulatory subunit IKKγ, which is also referred to as NF-κB essential modulator (NEMO). In the canonical pathway, IKKβ phosphorylates IκBα, whereas in the non-canonical pathway, p52 processed from p100 with phosphorylation by IKKα generates a p52-RelB heterodimer, which translocates into nucleus [6]. Remarkably, NF-κB is constitutively activated in many types of cancer [7]. Furthermore, a typical growth factor, epithelial growth factor (EGF) also activates NF-κB [8] and EGF activates NDAPH oxidase, leading to ROS production [9].

Rho GTPases belong to the Ras superfamily of small GTPases. Rho GTPases, including RhoA, Cdc42, and Rac1/2, are activated by guanine nucleotide exchange factors (GEFs) through GTP binding to Rho and are inactivated rapidly by hydrolysing GTP to GDP through GTPase activating proteins (GAPs). An inactive form of Rho GTPases is localized in the cytosol with RhoGDI (guanine nucleotide dissociation inhibitor), and the Rho GTPase-RhoGDI complex must be disrupted for Rho GTPases to be activated by GEFs [10], [11]. In addition to the well-known functions of Rho GTPases in regulating cytoskeletal rearrangement, Rac1/2-GTP is translocated to the plasma membrane to assemble and activate the NADPH oxidase complex, leading to superoxide production [12]. Moreover, Rho GTPases were reported to be involved in the regulation of NF-κB [13]. In particular, several Rho GTPases have been overexpressed in human tumors, and the GTPases correlate with cancer progression in some cases [14]. In addition, RhoA mutants were observed in several cancers [15], [16], [17].

However, the underlying molecular mechanism by which ROS induce activation of NF-κB through RhoA regulation, particularly during tumor progression, has not been elucidated. In this study, we investigated how ROS, such as hydrogen peroxide, activate NF-κB with regard to the regulation of RhoA during tumorigenesis. Herein, we found that RhoA is phosphorylated at the Tyr42 by Src in response to not only hydrogen peroxide. P-Tyr42 RhoA essentially bound to IKKγ/NEMO to activate IKKβ, leading to NF-κB activation. Moreover, we found that p-Ty42 RhoA is a key molecule to induce cell proliferation, and p-Tyr42 Rho was increased in cancer cell lines in the presence of hydrogen peroxide and in human breast cancer tissues of patients.

Section snippets

Materials

H2O2 (hydrogen peroxide), Nonidet P-40 (NP-40), N-acetyl-l-cysteine (NAC), bovine serum albumin (BSA), poly-l-lysine solution (P8920), and anti-β-actin antibody were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). PP2 (4-amino-5-(4-chlorophenyl)−7-(t-butyl)pyrazolo[3,4-d]pyrimidine), PP3, MG132, and Y27632 were obtained from Calbiochem (La Jolla, CA). Dulbecco's modified Eagle's medium F-12 (DMEM F-12), foetal bovine serum (FBS), penicillin and streptomycin were purchased from Cambrex

Phosphorylation of RhoA at Tyr42 upon hydrogen peroxide

Macrophages produced much ROS during phagocytosis process. Therefore, in the beginning we initiated researches to reveal ROS effects on RhoA GTPase in RAW264.7 cells. ROS was reported to inhibit p-Tyr phosphatase, leading to Tyr kinase activity [24]. Thus, we assessed the phosphorylation of Tyr in RhoA in response to hydrogen peroxide. Hydrogen peroxide augmented Tyr phosphorylation of RhoA when p-Tyr was immunoprecipitated and then RhoA was immunoblotted (Fig. 1A). Next, RhoA was

RhoA is phosphorylated at Tyr42 by Src and activated by Vav2 in response to hydrogen peroxide

Little is known about how an increase in intracellular ROS is sensed and transmitted to the signaling machinery to regulate cell proliferation. Nonetheless, there are several reports investigating the mechanism by which ROS activate NF-κB. The phosphorylation of Tyr42 and C-terminal PEST (Pro-Glu-Ser-Thr) domain of IκB plays an important role in NF-κB activation by ROS [30], [31], [32], [33]. In some cases, hydrogen peroxide directly activates IKK [34]. In addition, p65 is phosphorylated by

Conclusion

Hydrogen peroxide activates nuclear factor-κB (NF-κB) and RhoA GTPase through the phosphorylation of RhoA at Tyr42 via Src. Vav2 activated by Src binds to p-Tyr42 of RhoA, leading to RhoA activation. P-Tyr42 RhoA then binds to IKKγ and stimulates IKKβ, resulting in NF-κB activation. Activated NF-κB induced factors related to cell proliferation such as c-Myc and cyclin D1. The posttranslational modification of RhoA such as Tyr42 phosphorylation is critical for cell proliferation and tumor growth

Conflict of interest

None.

Acknowledgements

This research was supported by the Basic Science Research Programme of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF-2015R1D1A1A01060393), and Hallym University (HRF-S-53). We thank Dr. J. Ashwell at NIH for providing the pGEX-IKKγ/NEMO (Addgene plasmid 11965) and pET-IKKγ/NEMO (Addgene plasmid 11966) constructs. We thank Jae-Nam Seo (Department of Pathology, Hallym University) and Jun-Sub Jung (Department of Pharmacology,

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