Tyr42 phosphorylation of RhoA GTPase promotes tumorigenesis through nuclear factor (NF)-κB
Graphical abstract
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,
References (72)
- et al.
IkappaB kinase gamma/nuclear factor-kappaB-essential modulator (IKKgamma/NEMO) facilitates RhoA GTPase activation, which, in turn, activates Rho-associated KINASE (ROCK) to phosphorylate IKKbeta in response to transforming growth factor (TGF)-beta1
J. Biol. Chem.
(2014) - et al.
Transcription factors NRF2 and NF-kappaB are coordinated effectors of the Rho family, GTP-binding protein RAC1 during inflammation
J. Biol. Chem.
(2014) - et al.
Variegated RHOA mutations in adult T-cell leukemia/lymphoma
Blood
(2016) - et al.
Transforming growth factor-beta1 regulates macrophage migration via RhoA
Blood
(2006) - et al.
Determination of GTP loading on Rho
Methods Enzymol.
(2000) - et al.
Loss of Raf-1 kinase inhibitory protein in pancreatic ductal adenocarcinoma
Pathology
(2010) Redox redux: revisiting PTPs and the control of cell signaling
Cell
(2005)- et al.
Vav2 is an activator of Cdc42, Rac1, and RhoA
J. Biol. Chem.
(2000) - et al.
RhoA GTPase oxidation stimulates cell proliferation via nuclear factor-kappaB activation
Free Radic. Biol. Med.
(2017) - et al.
Silica induces nuclear factor-kappa B activation through tyrosine phosphorylation of I kappa B-alpha in RAW264.7 macrophages
Toxicol. Appl. Pharmacol.
(2000)
Phosphorylation of the PEST domain of IkappaBbeta regulates the function of NF-kappaB/IkappaBbeta complexes
J. Biol. Chem.
Tyrosine phosphorylation of I kappa B alpha activates NF kappa B through a redox-regulated and c-Src-dependent mechanism following hypoxia/reoxygenation
J. Biol. Chem.
Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II
J. Biol. Chem.
SH2 domains, interaction modules and cellular wiring
Trends Cell Biol.
Src in cancer: deregulation and consequences for cell behaviour
Biochim Biophys. Acta
Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells
Free Radic. Biol. Med.
Epidermal growth factor-dependent regulation of Cdc42 is mediated by the Src tyrosine kinase
J. Biol. Chem.
Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1
J. Biol. Chem.
Serine 34 phosphorylation of rho guanine dissociation inhibitor (RhoGDIalpha) links signaling from conventional protein kinase C to RhoGTPase in cell adhesion
J. Biol. Chem.
Hydrogen peroxide prolongs nuclear localization of NF-kappaB in activated cells by suppressing negative regulatory mechanisms
J. Biol. Chem.
NEMO ensures signaling specificity of the pleiotropic IKKbeta by directing its kinase activity toward IkappaBalpha
Mol. Cell
Structure of a NEMO/IKK-associating domain reveals architecture of the interaction site
Structure
Actin dynamics control SRF activity by regulation of its coactivator MAL
Cell
Ras-stimulated extracellular signal-related kinase 1 and RhoA activities coordinate platelet-derived growth factor-induced G1 progression through the independent regulation of cyclin D1 and p27
J. Biol. Chem.
Cellular mechanisms and physiological consequences of redox-dependent signalling
Nat. Rev. Mol. Cell Biol.
Reactive oxygen species: from health to disease
Swiss Med. Wkly.
Reactive oxygen species in cancer
Free Radic. Res.
NF-kappaB and cancer: a paradigm of Yin-Yang
Am. J. Cancer Res.
The diverse and complex roles of NF-kappaB subunits in cancer
Nat. Rev. Cancer
New regulators of NF-kappaB in inflammation
Nat. Rev. Immunol.
The complexity of NF-kappaB signaling in inflammation and cancer
Mol. Cancer
Epidermal growth factor activation of NF-kappaB is mediated through IkappaBalpha degradation and intracellular free calcium
Oncogene
Epidermal growth factor stimulates nuclear factor-kappaB activation and heme oxygenase-1 expression via c-Src, NADPH oxidase, PI3K, and Akt in human colon cancer cells
PLoS One
Rho GTPases: biochemistry and biology
Annu. Rev. Cell Dev. Biol.
Regulation of the phagocyte NADPH oxidase by Rac GTPase
Antioxid. Redox Signal.
Rho GTPases modulate malignant transformation of tumor cells
Small GTPases
Cited by (19)
Cell-in-Cell–Mediated Entosis Reveals a Progressive Mechanism in Pancreatic Cancer
2023, GastroenterologyExtracellular pyruvate kinase M2 induces cell migration through p-Tyr42 RhoA-mediated superoxide generation and epithelial-mesenchymal transition
2023, Free Radical Biology and MedicineThe role of NF-κB in breast cancer initiation, growth, metastasis, and resistance to chemotherapy
2023, Biomedicine and PharmacotherapyA current overview of RhoA, RhoB, and RhoC functions in vascular biology and pathology
2022, Biochemical PharmacologyRhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to β-catenin and contributes transcriptional regulation of vimentin upon Wnt3A
2021, Redox BiologyCitation Excerpt :RhoA WT, but either RhoA Y42E or Y42F mutants was much less associated with β-catenin irrespective of GDP and GTP GTPγS (Fig. 5C). Similarly, GST-RhoA phosphorylated by Src and ATP [18] also readily bound to β-catenin in vitro, irrespective of GDP- or GTPγS-preloaded RhoA, suggesting that phosphorylation of Tyr42, but not GTP/GDP-binding state is critical for interaction between RhoA and β-catenin (Fig. 5D). Herein, we presented and compared 3D structure of RhoA-GDP [38] and -GTPγS [39] by using Proteopedia site (http://proteopedia.org/wiki/index.php/).
Distinct dual roles of p-Tyr42 RhoA GTPase in tau phosphorylation and ATP citrate lyase activation upon different Aβ concentrations
2020, Redox BiologyCitation Excerpt :Indeed, Src was reported to induce Tyr216 phosphorylation of GSK-3β in PC3, prostate cancer cells [28]. In addition, Src also can phosphorylate the Tyr42 residue of RhoA [13]. Apart from Tyr416 phosphorylation, Src can also be activated by oxidation events, such as an intramolecular disulfide bond between Cys245 and Cys487.