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

Author’s Accepted Manuscript Tyr42 phosphorylation of RhoA GTPase promotes tumorigenesis through nuclear factor (NF)-κB Jae-Gyu Kim, Kyoung-Chan Choi, Chang-Won Hong, Hwee-Seon Park, Eun-Kyoung Choi, YongSun Kim, Jae-Bong Park www.elsevier.com

PII: DOI: Reference:

S0891-5849(17)30685-8 http://dx.doi.org/10.1016/j.freeradbiomed.2017.07.013 FRB13390

To appear in: Free Radical Biology and Medicine Received date: 21 October 2016 Revised date: 19 June 2017 Accepted date: 11 July 2017 Cite this article as: Jae-Gyu Kim, Kyoung-Chan Choi, Chang-Won Hong, HweeSeon Park, Eun-Kyoung Choi, Yong-Sun Kim and Jae-Bong Park, Tyr42 phosphorylation of RhoA GTPase promotes tumorigenesis through nuclear factor ( N F ) - κB , Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2017.07.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tyr42 phosphorylation of RhoA GTPase promotes tumorigenesis through nuclear factor (NF)-κB Jae-Gyu Kim1, Kyoung-Chan Choi2, Chang-Won Hong3, Hwee-Seon Park1, Eun-Kyoung Choi4, YongSun Kim4,5, and Jae-Bong Park1,6* 1

Department of Biochemistry, Hallym University College of Medicine, Chuncheon, Kangwon-do, 24252,

Republic of Korea 2

Department of Pathology, Chuncheon Sacred Hospital Hallym University, Chuncheon 24252, Republic

of Korea 3

Department

of

Physiology,

Kyungpook

National

University

School

of

Medicine,

Daegu,

Gyeongsangbuk-do 41944, Republic of Korea 4

Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do 14066, Republic of Korea

5

Department of Microbiology, Hallym University College of Medicine, Chuncheon, Kangwon-do, 24252,

Republic of Korea 6

Institute of Cell Differentiation and Ageing, Hallym University College of Medicine, Chuncheon,

Kangwon-do, 24252, Republic of Korea *

Corresponding author at: Department of Biochemistry, Hallym University College of Medicine,

Chuncheon, Kangwon-Do, Republic of Korea, 200-702. Tel.: +82 33 248 2542; fax: +82 33 244 8425. [email protected]

Abstract Dysregulation of reactive oxygen species (ROS) levels is implicated in the pathogenesis of several diseases, including cancer. However, the molecular mechanisms of ROS in tumorigenesis have not been clearly elucidated. Hydrogen peroxide activated nuclear factor-κB (NF-κB) and RhoA GTPase. In particular, we found that hydrogen peroxide phosphorylated RhoA at Tyr42 via Src. P-Ty42 residue of RhoA was a binding site of Vav2, a guanine nucleotide exchange factor (GEF) that activates p-Tyr42 RhoA. P-Tyr42 RhoA then bound to IκB kinase γ (IKKγ), leading IKKβ activation. Furthermore, RhoA WT and Y42E promoted tumorigenesis, whereas RhoA Y42F suppressed it. In addition, hydrogen peroxide induced NF-κB activation, cell proliferation, and expression of c-Myc and cyclin D1 in the presnce of RhoA WT and RhoA Y42E (a phosphomimic), but not of RhoA Y42F (a dephospho-mimic). Indeed, p-Tyr42 Rho, p-Src, and p-65 were significantly increased in human breast cancer tissues and 1

showed correlations between each two components. Conclusively, the posttranslational modification of RhoA (p-Tyr42) may be essential for promoting tumorigenesis in response to possibly ROS.

Graphical abstract

The abbreviations used are as follows CA, constitutively active; DMEM, Dulbecco’s modified Eagle’s medium; DN, dominant negative; GAP, GTPase activating protein; GDF, GDI displacement factor; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; GSH, glutathione; Rhotekin-RBD, Rhotekin Rhobinding domain; ROCK, Rho-dependent coiled-coil kinase; ROS, reactive oxygen species; WT, wildtype. NF-κB, nuclear factor-κB; IκB, inhibitor of NF-κB; IKK, IκB kinase; NEMO, NF-κB essential modulator; PKA, protein kinase A; PKC, protein kinase C; SRF, serum response factor; TAM, tumor associated macrophage; WT, wild type Keywords RhoA, Tyr phosphorylation, Src, IKK, cancer, ROS 1. 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, 2

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 p52RelB 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, 3

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 tumours, and the GTPases correlate with cancer progression in some cases [14]. In addition, RhoA mutants were observed in several cancers [15-17]. 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.

2. Materials and Methods 2.1.

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, 4

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 (Verviers, Belgium). Anti-p65, anti-phospho-IκB at Ser32/36, anti-RhoA, anti-Src, antiIKKα/β, and anti-Vav2, p-Vav2 antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Antibodies against p-IKKα/β, p-p65 at Ser536, p-Src at Tyr416, and p-Ser were purchased from Cell Signaling Technology Inc. (MA, USA). Mouse anti-IKKα/β and anti-IKKγ monoclonal antibodies were obtained from BD Bioscience (Mountain View, CA). Methanol free-formaldehyde was obtained from Pierce (Rockford, IL). Anti-p-Tyr (4G10), and anti-IKKβ antibodies were purchased from Upstate. (Lake Placid, NY). Alexa Fluor-488 goat anti-mouse IgG, 4',6-diamidino-2-phenylindole (DAPI), and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA, USA). Anti-ProLong-Gold antifade mount solution, Alexa Fluor-568, and Alexa Fluor-594 were obtained from Molecular Probes (Eugene, OR). Polyvinylidene difluoride (PVDF) membranes were purchased from Millipore (Billerica, MA, USA). Attractene (301005) and Hyperfect (301802) transfection reagents were from Qiagen (Valencia, CA). Human active Src purified from sf21 cells was purchased from MERCK Millipore Daejeon Office (South Korea). Recombinant human Vav2 purified from HEK293 cells was purchased from OriGene Technologies, Inc. (Rockville, MD). Glutathione-Sepharose 4B or agarose and protein A/G-agarose beads were purchased from Amersham Biosciences (Piscataway, NJ). Tissue chips of human breast cancer tissues containing 63 patients’ samples [AccuMax array, A312(II)] were purchased from ISU ABXIS (Seoul, Korea).

2.2

Cell culture

RAW264.7 (murine macrophage cell line), HEK293 (human embryo kidney cell), 4T1 (mouse breast cancer cell line), and HT-29 (human colon cancer cell line) were maintained in DMEM F-12 in the case of RAW264.7 or DMEM in the case of HEK293, 4T1, HT-29, and CT26 (human colon cancer cell line) 5

supplemented with 5% heat-inactivated FBS and antibiotics (100 U/ml of penicillin, 100 μg/ml of streptomycin) in a 5% CO2, 95% air humidified atmosphere at 37°C. MCF7 and MD-MBA231 (human breast cancer cell lines) were cultured in DMEM containing 10% FBS.

2.3.

Western blot analysis.

After performing experiments, cells were washed once with PBS, harvested, lysed in RIPA buffer (20 mM Tris pH 7.5, 1 mM EDTA, 1% (v/v) NP-40, 125 mM NaCl), and centrifuged at 12,000×g for 15 min at 4 °C. To determine the protein concentration of the cell extracts Pierce BCA protein assay kit was used (Thermo Fisher Scientific, Rockford, IL, USA). Proteins of cell extracts (20 μg proteins) were separated using 12-14% SDS-PAGE, and SDS-PAGE gels were incubated in transfer buffer containing 20% methanol for 20 min and then transferred to a PVDF membrane. Non-specific binding was blocked by a 2 h incubation of the PVDF membrane at RT with Tris-buffered saline solution (Tris 10 mM, NaCl 150 mM, pH 7.4) containing 5% non-fat dried milk and 0.05% Tween-20 (general protocol from BioRad Inc.). Target proteins were probed with specific antibodies at 4 °C overnight, and the bound primary antibodies were detected by incubation with peroxidase-conjugated goat anti-mouse IgG secondary antibody. The PVDF membrane was soaked in chemiluminescent ECL reagents (Amersham, Arlington Heights, IL) and exposed to X-ray film (Agfa, VWR, USA).

2.4.

Preparation of cytosolic and nuclear fractions.

Cytosolic and nuclear fractions were separated following the protocol from Pierce (Rockford, IL). Breifly, RAW 264.7 cells were stimulated with H2O2 (0.1 mM) for various periods. Cells were harvested in ice-cold 1x PBS, pelleted by centrifuging at 13,000x g for 10 min and lysed for 30 min on ice in hypotonic buffer A (20 mM HEPES pH 7.4, 10 mM KCl, 1 mM MgCl2, 0.1% Triton X-100, and 20% glycerol). Lysates were centrifuged at 15,000x g for 15 min, and the resulting supernatants 6

constituted the cytosolic fractions. The pellets were washed three times with hypotonic buffer A, resuspended in hypertonic buffer B (20 mM HEPES pH 7.4, 1 mM EDTA, 400 mM NaCl, 0.1% Triton X-100, 20% glycerol), incubated for 30 min on ice and centrifuged at 15,000x g for 15 min. The supernatant was used as nuclear extracts. Fractions were analysed by immunoblotting with anti-p65 and anti-lamin A/C antibodies.

2.5.

NF-κB luciferase reporter activity

Cells (1×105) were plated onto 12-well plates and incubated for 24 h at 37°C in a CO2 incubator. The NF-κB-luciferase reporter construct (Stratagene, 2 μg) was transfected into cells using Lipofectamine 2000 (Invitrogen) for 3 h according to the manufacturer’s protocol. To calibrate the variation in transfection efficiency, cells were transfected with 0.5 μg of pCMV-Gal, an expression plasmid with the E. coli β-galactosidase gene. One day after the transfection, cells were lysed in 1x reporter lysis buffer (Promega), and cell debris was removed by centrifugation. The supernatant was used for measuring the relative luciferase activity according to the protocol from Promega using a luminometer (Lumat LB 9057, EG and GBertold).

2.6.

Immunoprecipitation.

Cells (1×107) were washed with 1× PBS, and cell lysates were prepared using a cell lysis buffer (20 mM Tris pH 7.4, 120 mM NaCl, 1% Nonidet P-40) containing 10 μg/ml each of leupeptin and aprotinin, and 1 mM sodium fluoride, 1 mM Na3VO4, and 5 mM MgCl2. Cell lysates were cleared by centrifugation at 15,000x g for 20 min at 4°C. The supernatant was then pre-cleared with protein A/G-agarose beads for 30 min. The supernatant was added with anti-IKKγ or anti-IKKβ (1:1000 dilution) antibody overnight at 4°C. Then, protein A/G-agarose beads (30 μl) were added to the lysate, and the mixture was incubated with shaking at 4°C for 2 h. The beads were collected using centrifugation and washed three times with 7

a lysis buffer. Proteins bound to the beads were eluted by adding 30 μl of 5x sample buffer and analysed using immunoblotting with specific primary antibodies [18].

2.7.

Mutagenesis and purification of recombinant proteins

HA-RhoA 34YF, Y42F, Y66F, Y74F, and Y156F mutants in pCDNA3.1 plasmid were constructed by using point mutagenesis kit (Intron Biotechnology, Gyeonggi, Korea) (Jeon et al., 2012). Recombinant proteins of GST-RhoA, GST-RhoA Y42E, GST-RhoA Y42F, GST-IκB, GST-IKKβ, and GST-IKKγ in pGEX4T.1 plasmid were expressed in E. coli BL21 and the proteins were purified by using GSH-beads [11].

2.8.

GTP-RhoA pull-down assay.

Cells were incubated in DMEM F-12 without serum for 12 h prior to cell stimulation. Cells were stimulated with hydrogen peroxide and washed with ice-cold 1x PBS and lysed in lysis buffer A (25 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM MgCl2, 5% glycerol and protease inhibitors including 1 mM PMSF and 1 μg/ml each of leupeptin, aprotinin, and pepstatin A). Lysates were clarified by centrifugation and equalized for total volume and protein concentration. After incubation with GSTRhotekin-Rho-binding domain (RBD) beads and three washes with ice-cold lysis buffer B (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5 mM MgCl2, 100 µM orthovanadate, protease inhibitors), the bound fraction (active RhoA-GTP) was separated using SDS-PAGE [19]. Total RhoA levels were similarly analysed using a reserved aliquot of whole cell lysate. Active RhoA and total RhoA were analysed by Western blotting with an anti-RhoA antibody (monoclonal antibody 26C4; Santa Cruz, CA). The relative amount of active RhoA was determined by taking the ratio of RhoA sedimented by GSTRBD beads (active RhoA) divided by the amount of total RhoA in the whole cell lysate. The results were quantified using Photoshop 7.01 software (Adobe Inc.). Statistical significance was determined using 8

PRISIM 4.0 software (GraphPad).

2.9.

In vitro kinase assay.

Purified Vav2 or RhoA (0.1 μg) was incubated with Src (0.1 μg) in a kinase assay buffer (20 mM HEPES pH 7.5, 2 mM β-glycerophosphate, 20 mM MgCl2, and 20 μM ATP) for 30 min at 25°C. In some cases, 10 μM H2O2 was used to pretreat the RhoA protein, and 1 U catalase was administered to remove excessive H2O2 after reaction. P-Tyr was detected with a Western blot [11].

2.10.

Determination of cell proliferation

Cells (2 x 104) were cultured in DMEM, 5% FBS, and 1% P/S and then incubated in FBS-free media for 12 h. H2O2 (100 μM) was used to treat cells, and samples were incubated for 24-48 h at 37°C in a CO2 incubator. Cells were fixed with 4% formaldehyde (200 μl) for 10 min at RT. Nuclei of cells were stained with 1 μg/ml DAPI (1:200) for 10 min at RT. Cell proliferation was quantified by determining the fluorescence of cell density using fluorescence microscopy. At least four locations were selected to calculate the average cell density; the fluorescence intensity was measured using ImageJ Pro software [20]. Cell proliferation was also monitored by the MTT assay [21].

2.11.

Preparation of anti-phospho-Tyr42-Rho antibody

The anti-p-Tyr42 Rho antibody was produced by Young-In Frontier (Seoul, Korea). Briefly, p-Tyr42 Rho peptide (epitope peptide T37VFEN(phospho-)Y42VADIE47) was synthesized using phospho-Tyr42 precusor. Fluorenylmethyloxycarbonyl (Fmoc)-Tyr(PO(Nme2)2)-OH was used as a precursor amino acid derivative to protecte phosphate of Tyr42 from reaction and the protective groups (N(Me2)2) were removed 9

after synthesis of peptide containing p-Tyr42. The peptide was purified byusing C18 column and confirmed

with Mass analysis. The peptide was conjugated to BSA, which was injected into rabbit to produce polyclonal anti-p-Tyr42 Rho antibodies. The serum containing antibody was purified through three steps: proteinA-bead, non-phospho peptide-bead to exclude dephospho-Rho antibody, and then p-Tyr42 peptide-beads were exploited to purify specific p-Tyr42 Rho GTPase antibody.

2.12.

Animals and murine tumor model.

BALB/c (female, 4-6 weeks old) mice were purchased from Samtako (Osan, Republic of Korea). Animal experiments were approved by the Institutional Animal Care and Use Committee of Hallym University (HIRB-2016-52). BALB/c mice were injected on the right leg with 4T1 cells (1 × 105). Tumor growth was measured every 3 days. Tumors were allowed to growth for 21 days.

2.13.

Evaluation and scoring of cancer stages Immunoreactivities of P-Tyr42 Rho, p-Src, and p-p65 were analysed using a semi-quantitative scoring method as previously described [22]. Cytoplasmic and/or membranous staining was considered to indicate positive expression. Briefly, the score was the sum of the percentage of positive tumor cells proportion (0: none; 1: 1–25%; 2: 26-50%; 3: 51-75%, 4: 76–100%) and staining intensity (0, none; 1, weak; 2, moderate; 3, strong). Scores were determined as a sum of intensity and positive tumor cell proportion. All slides were examined and scored by two 10

independent pathologists, who were blinded to the clinicopathological data and patient identity. Disagreements between the two pathologists were resolved by consensus.

2.14.

Statistical analysis Generally, the experiments were performed in three times, each experiment was carried out with duplicate or triplicate samples, and data are presented as the mean of three independent experiments ± SE. Statistical comparisons were made with Student’s t-test and ANOVA analysis was utilized for multiple comparisons using GraphPad Prism4.0 software (Graph Pad, San Diego, CA). The difference between two groups was statistically significant if p values were less than 0.05 (*p<0.05, and **p<0.01). Pearson’s correlation coefficient analysis was performed also using GraphPad Prism4.0 software [23].

3. Results 3.1. 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 immunoprecipitated and p-Tyr was identified by immunoblotting from cells stimulated by hydrogen peroxide. Howver, PP2, Src inhibitor abolished Tyr phosphorylation of immunoprecipitated RhoA (Fig.1B). Then, we tried to identify which Tyr residue of RhoA is phosphorylated in response to hydrogen peroxide. RhoA has five Tyr residues, including Tyr34, Tyr42, Tyr66, Tyr74, and Tyr156. 11

Therefore, WT and Y34F, Y42F, Y66F, Y74F, and Y156F RhoA DNA constructs containing an HA tag were transfected into HEK293 cells, and ectopically expressed HA-RhoA was immunoprecipitated with an anti-HA antibody after cells were stimulated with hydrogen peroxide, followed by p-Tyr immunoblotting. The phosphorylation of RhoA Y42F was abolished (Fig. 1C), suggesting that Tyr42 is a major phosphorylated residue upon hydrogen peroxide. Then, we produced an anti-p-Tyr42 Rho polyclonal antibody from rabbit. Because the peptide sequence (amino acid (aa) 37-47) of RhoA containing Tyr42 is exactly the same as those of RhoB in mammal (Fig. 1D), the p-Tyr42 Rho antibody may not distinguish p-Tyr42 RhoA and p-Tyr42 RhoB in mammalian cells. Concurrently, p-Tyr42 Rho GTPase detected by the p-Tyr42 Rho antibody was augmented by hydrogen peroxide in a timedependent manner (Fig. 1E). We revealed that by using the antibody PP2 dampened Tyr42 phosphorylation of Rho GTPase in response to hydrogen peroxide (Fig. 1F). Even low concentration (1 μM) of hydrogen peroxide for 1 h induced Tyr42 phosphorylation of Rho GTPase (Fig. 1G). In primary peritoneal macrophages from rat, hydrogen peroxide also induced p-Tyr42 Rho GTPase (Fig. 1H).

3.2. Src phosphorylates Tyr42 of RhoA To clarify whether hydrogen peroxide activates Src, p-Tyr416 Src was examined with Western blotting; hydrogen peroxide phosphorylated p-Tyr416 Src in a time-dependent manner (Fig. 2A). Along with Src activation, the co-immunoprecipitation of Src with RhoA was augmented (Fig. 2B). To confirm that Src is the actual kinase that phosphorylates RhoA, active Src protein was incubated with recombinant RhoA in the presence of ATP in vitro; the phosphorylation of RhoA at Tyr by Src was identified by an anti-p-Tyr antibody. Recombinant RhoA was readily phosphorylated at Tyr by Src in vitro. Notably, RhoA Tyr phosphorylation by Src enhanced after being oxidized by hydrogen peroxide (Fig. 2C). However, active recombinant Src could not phosphorylate RhoA Y42E and RhoA Y42F, but it phosphorylated WT RhoA in vitro (Fig. 2D), demonstrating again that Src is a kinase that phosphorylates Tyr42 of RhoA in 12

response to hydrogen peroxide. Furthermore, RhoA was readily phosphorylated at the Tyr residue by Src in the presence of GDP rather than GTPγS, whereas neither RhoA Y42E nor RhoA Y42F was phosphorylated by Src, regardless of GDP or GTPγS (Fig. 2E). The result suggests that inactive RhoAGDP rather than active RhoA-GTP is a favorable substrate of Src. Actually, the anti-p-Tyr42 Rho antibody recognized the phosphorylation of WT RhoA that was previously incubated with Src/ATP but could not recognize the phosphorylation of RhoA Y42E and RhoA Y42F (Fig. 2F). Next, we explored the effect of p-Tyr42 RhoA on the- RhoGDI complex. RhoA-GTPγS rarely interacted with RhoGDI, whereas RhoA WT-GDP and RhoA Y42F-GDP readily interacted with RhoGDI. However, phospho-mimic RhoA Y42E even in the presence of GDP rarely bound to RhoGDI in vitro (Fig. 2G), suggesting that Tyr42 phosphorylation dissociates RhoA from RhoGDI. Furthermore, hydrogen peroxide induced Tyr phosphorylation of RhoGDI (Fig. 2H), thereby leading to likely dissociation with RhoA [25].

3.3. Activation of p-Tyr42 RhoA by Vav2 in response to hydrogen peroxide To understand the relationship between p-Tyr42 RhoA and RhoA activity, we determined RhoA-GTP levels in RAW264.7 cells with hydrogen peroxide administration; hydrogen peroxide increased RhoAGTP levels in a time-dependent manner, but a Tyr kinase inhibitor, SU6656, prevented RhoA activation upon hydrogen peroxide (Fig. 3A). Next, we determined whether p-Tyr42 RhoA is active or inactive. RhoA-GTP, which was first pulled down with RBD-beads from cells stimulated by hydrogen peroxide, was immunoblotted with the p-Tyr42 Rho antibody; RhoA-GTP levels were positively correlated with pTyr42 Rho GTPase (Fig. 3B), suggesting that p-Tyr42 RhoA is an active form in vivo. Because Vav2 GEF is an activator of Cdc42, Rac1, and RhoA [26] and Vav2 preferentially activates RhoA, while Vav1 preferentially activates Rac1 [27], we examined the involvement of Vav2 in the activation of RhoA in response to hydrogen peroxide. Vav2 bound to RhoA WT and RhoA Y42E in response to hydrogen 13

peroxide, but it did not bind to RhoA Y42F (Fig. 3C). The result suggests that p-Tyr42 residue of RhoA is critically required for Vav2 binding to RhoA. In addition, co-immunoprecipitation of Vav2 with RhoA was enhanced in response to hydrogen peroxide whereas the Tyr kinase inhibitor PP2 abolished the co-immunoprecipitation of RhoA and Vav2 (Fig. 3D), suggesting that Tyr phosphorylation is required for the interaction of Vav2 and RhoA to activate RhoA. When we transfected si-Vav2 into RAW 264.7 cells, hydrogen peroxide failed to increase RhoA-GTP levels (Fig. 3E). The result suggests that Vav2 is indeed required for RhoA activation in response to ROS. Indeed, p-Tyr174 of Vav2 was detected in a time-dependent manner in response to hydrogen peroxide (Fig. 3F). Consistently, active Src protein induced the phosphorylation of Vav2 at Tyr174 in vitro (Fig. 3G). The results suggest that ROS activate Vav2 via Src. Tat-C3 did not inhibit Rho Tyr42 phosphorylation as determined by the anti-p-Tyr42 Rho antibody (Fig. 3H), suggesting that the inactive Rho GTPase is still a good substrate for Tyr42 phosphorylation. Next, we determined GTP levels of RhoA WT, Y42E, and Y42F upon hydrogen peroxide; RhoA WT and Y42E (a phospho-mimic) revealed upregulated GTP levels upon hydrogen peroxide, whereas Y42F (a dephospho-mimic) did not increase RhoA-GTP level (Fig. 3I). Even in the presence of Tat-C3, the pattern was similar, suggesting that Tat-C3 could not interfere with RhoA phosphorylation at Tyr42 and activation upon hydrogen peroxide [28]. However, RhoA could not bind to IKKγ/NEMO in the presence of Tat-C3 (Fig. 4N).

3.4. RhoA Tyr42 phosphorylation is essential for interaction with IKKγ We confirmed that hydrogen peroxide induced NF-κB activation, but Tat-C3 suppressed NF-κB activity in RAW264.7 cells and HEK293 cells (Fig. 4A). The result suggests that RhoA GTPase is essential for the regulation of NF-κB activation in response to hydrogen peroxide. Hereby, we investigated whether p-Tyr42 of RhoA is implicated in NF-κB activation; RhoA Y42F inhibited the phosphorylation of IκB and p65 upon hydrogen peroxide addition, whereas RhoA WT and RhoA Y42E induced the phosphorylation 14

of p65 and IκB (Fig. 4B), suggesting that p-Tyr42 RhoA is critically involved in NF-κB activation. To elucidate how RhoA regulates NF-κB, we assessed the interaction between RhoA and IKKγ because IKKγ was known to be a regulatory subunit of the IKK complex composed of IKKβ/IKKα, and oxidized RhoA was reported to bind to IKKγ [29]. IKKγ was immunoprecipitated, and RhoA, p-Tyr42 RhoA, IKKβ were then immunoblotted. Hydrogen peroxide increased co-immunoprecipitation of IKKγ and RhoA, pTyr42 RhoA, and IKKβ (Fig. 4C), suggesting that p-Tyr42 RhoA interacts with IKKβ/IKKγ complex. Notably, p-Tyr42 RhoA more fastly bound to IKKγ compared to total RhoA-GTP (Fig., 4C), suggesting that p-Tyr 42 RhoA has more affinity to IKKγ rather than total RhoA that is activated by hydrogen peroxide. Likewise, RhoA, p-Tyr42 RhoA, and IKKγ were co-immunoprecipitated with IKKβ by hydrogen peroxide in a time-dependent manner (Fig. 4D). Thus, we explored the involvement of p-Tyr42 residue of RhoA in the interaction with IKKγ; co-immunoprecipitation of RhoA and IKKγ was reduced by treatment with a phosphatase (calf intestinal alkaline phosphatase: CIP) (Fig. 4E) and a Src inhibitor, PP2 (Fig. 4F). Coimmunoprecipitation of dephospho-mimic RhoA Y42F with IKKγ/NEMO from cells stimulated with hydrogen peroxide was significantly reduced compared to WT RhoA (Fig. 4G). Using an in vitro system, recombinant RhoA WT and phospho-mimic RhoA Y42E oxidized by hydrogen peroxide interacted with GST-IKKγ, whereas dephospho-mimic RhoA Y42F did not bind to GST-IKKγ even in the presence of GTPγS (Fig. 4H). A small amount of RhoA WT bound to GST-IKKγ/NEMO, whereas neither phosphomimic RhoA Y42E nor dephospho-mimic RhoA Y42F bound to GST-IKKγ in the presence of GDP. However, much more RhoA Y42E and more RhoA Y42F bound to IKKγ in the presence of GTPγS rather than GDP (Fig. 4H). The results suggest that both Tyr42 phosphorylation of and GTP binding to RhoA are required for RhoA to readily bind to IKKγ/NEMO. Although IKKγ was reported to be essential for RhoA activation in TGF-β signaling [11], si-IKKγ did not prevent RhoA activation upon hydrogen peroxide treatment (Fig. 4I), suggesting that IKKγ is not critical for RhoA activation. To determine the domain of IKKγ/NEMO that interacts with p-Tyr42 RhoA, several GST-IKKγ/NEMO 15

domains were incubated with RhoA Y42E, a phospho-mimic form. RhoA Y42E-GTPγS in the presence of hydrogen peroxide bound to the domains aa 1-419, 101-200, and 101-419 and slightly bound to aa 44-111 of IKKγ/NEMO (Fig. 4J), suggesting that aa 101-200 of IKKγ/NEMO is a major binding domain for RhoA-GTP that is oxidized and phosphorylated at Tyr42. The phospho-mimic RhoA Y42E in the presence of GTPγS, IKKβ, IKKγ/NEMO, and hydrogen peroxide induced the most potent phosphorylation of IκB in vitro, whereas the dephospho-mimic RhoA Y42F rarely phosphorylated IκB (Fig. 4K, Fig. 4L). Herein we explored whether p-Tyr42 RhoA could directly activate IKKβ upon hydrogen peroxide. Notably, hydrogen peroxide did not induced phosphorylation of IKKβ, whereas LPS and TGF-β1 induced its phosphorylation (Fig. 4M), suggesting that IKKβ is activated by p-Tyr42 RhoA without IKKβ phosphorylation. Here, it is noteworthy that Tat-C3 impaired RhoA binding to IKKγ/NEMO and IKKβ (Fig. 4N), suggesting that ADP-ribosyl group could interfere with RhoA binding to IKKγ/NEMO, leading to impairment of NF-κB activation, although RhoA is phosphorylated at Tyr42 and activated in response to hydrogen peroxide.

3.5.Cell proliferation by hydrogen peroxide through p-Tyr42 RhoA Hereby, we investigated the physiological significance of RhoA phosphorylation at Tyr42, particularly in cancer. Hydrogen peroxide induced p-Tyr42 Rho (p-Rho Y42) as well as p-p65 and p-IκB in several cancer cell lines, including 4T1 and MCF7 (mouse and human breast cancer cell lines, respectively), HT-29 and CT-26 (human colon cancer cell lines), and RAW264.7 (mouse macrophage cell line) (Fig. 5A). Indeed, hydrogen peroxide stimulated proliferation of cells, whereas Tat-C3 (Rho inhibitor) and Bay 11-7082 (NF-κB inhibitor) suppressed proliferation of RAW264.7 and 4T1 cells (Fig. 5B). Mesurements of cell proliferation by the MTT assay revealed the similar result to that by DAPI staining (data not shown). Consistent with the previous results (Fig. 4B), RhoA WT, RhoA Y42E enhanced NF-κB activation, whereas RhoA Y42F significantly reduced NF-κB activation upon hydrogen peroxide in 4T1 16

cells (Fig. 5C). RhoA WT and phospho-mimic RhoA Y42E enhanced cell proliferation of mouse breast cancer cell line, 4T1 and human breast cancer cell line, MCF7 in response to hydrogen peroxide, whereas dephospho-mimic RhoA Y42F substantially inhibited cell proliferation (Fig. 5D, 5E). Here, transfection of RhoA WT, Y42E, and Y42F clones did not change the release of lactate dehydrogenase (LDH), suggesting that transfection of the DNA plasmid clones to cells are not harmful to cells (Fig. 5F). Consistent with the effect of RhoA on cell proliferation, RhoA WT and RhoA Y42E contributed to expression of C-Myc and cyclin D1, whereas RhoA Y42F diminished their expression (Fig. 5G).

3.6.Tumorigenesis by p-Tyr42 RhoA To understand the role of p-Tyr42 RhoA in tumorigenesis, we performed xenograft experiments by using 4T1 cells in BALB/c mouse. Using Western blotting RhoA was identified from 4T1 cells which was transfected with sh-RhoA and then RhoA WT, RhoA Y42E, and RhoA Y42F used for xenograft experiment (Fig. 6A). Mouse breast cancer cell line (4T1) knocked-down by sh-RhoA remarkably reduced tumor volume and weight, whereas cells reconstituted with WT RhoA and RhoA Y42E recovered tumorigenesis. Nonetheless, cells reconstituted with RhoA Y42F, a dephospho-mimic form, could not recover tumor formation (Fig. 6B, Fig. 6C, and Fig. 6D), which were also quantified and compared in bar graph (Fig. 6E, 6F). Here, we applied PMA on the site of BALB/c mouse injected with 4T1 cells to produce ROS in 4T1 cells. Actually, PMA produced much ROS in 4T1 cells (Fig. 6G), although PMA was generally known to be a promoter of carcinogenesis. Accordingly, PMA produced pTyr42 Rho GTPase in 4T1 cells (Fig. 6H). Ether RAW264.7 cells alone or 4T1 cells alone did not produce RO.However, co-culture of both cell lines remarkably produced ROS [29], whereas co-culture with either RAW264.7 cells or 4T1 cells exposed to UV could not produce ROS (Fig. 6I). Furthemore, co-culture of RAW264.7 and 4T1 cells ensured phosphorylation of Rho GTPase at Tyr42 without any other stimulation (Fig. 6J). 17

3.7. p-Tyr42 Rho in breast cancer tissue To explore the involvement of p-Y42 RhoA, activated Src, and activated NF-κB in human breast cancer tissues of patients, we performed immunohistochemistry staining using anti-p-Y42 Rho, -p-Src, -p-p65 antibodies in human breast cancer tissues. P-Y42 Rho, p-Src, and p-p65 were increased in human breast cancer tissues depending on stages (Fig. 7A). Therein, we explored correlation of p-Y42 Rho, pSrc, and p-p65 in human breast cancer tissues. Correlation between p-Src and p-Y42 RhoA, p-Src and p-p65, and p-Y42 Rho and p-p65 were significantly high (Fig. 7B).

4. Discussion 4.1. 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 (ProGlu-Ser-Thr) domain of IκB plays an important role in NF-κB activation by ROS [30-33]. In some cases, hydrogen peroxide directly activates IKK [34]. In addition, p65 is phosphorylated by hydrogen peroxide [35]. Moreover, a variety of other kinases including MAP kinase kinase kinase (MEKK) and protein kinase C (PKC) have been reported to be implicated in the activation of NF-κB following hydrogen peroxide stimulation [36]. In the alternative pathway, hydrogen peroxide phosphorylates p105, facilitating cleavage into an active form, p50 [37]. Nevertheless, in the present study we revealed a novel mechanism by which RhoA phosphorylated by Src at Tyr42 activates NF-κB in response to hydrogen peroxide. Although RhoA was reported to be 18

directly activated by the oxidation at Cys16/20 residues [38, 39], we revealed that a specific GEF, Vav2, which is also activated by Src, is required for RhoA activation in response to ROS (Fig. 3D and 3GE). Because RhoA-GDP, but not RhoA-GTP was readily phosphorylated by Src in vitro (Fig. 2E) and TatC3 did not disturb Tyr42 phosphorylation of Rho (Fig. 3H), we surmise that RhoA-GDP rather than RhoA-GTP is a favourable substrate of Src. Vav2 has a SH2 domain [40] and SH2 has a typical feature to bind a phosphorylated Tyr residue [41]. Because RhoA WT and RhoA Y42E bound, but RhoA Y42F did not bind to Vav2, we assume that p-Tyr42 is a binding site for SH2 domain of Vav2, thereby enhancing the interaction RhoA and Vav2 upon hydrogen peroxide (Fig. 3C, 3D). In general, Src is activated by dephosphorylatiion of Tyr527 (in chicken; Tyr530 in human) and by autophosphorylation of Tyr416 (in chicken; Tyr419 in human) or Tyr416 phosphorylation by other kinases [42]. Furthermore, Src is also directly activated by hyperoxidization of Cys245 and Cys487, leading to Tyr416 phosphorylation [43]. In an indirect manner, a specific unknown protein tyrosine phosphatase (PTP) is inactivated by ROS, resulting in Tyr phosphorylation of Src [44]. In addition, Csk, which phosphorylates Tyr527 of Src, is inactivated by ROS, leading to Src activation [45]. Although Src is an important oncogenic protein, the mechanism of tumorigenesis by Src was not completely elucidated [46]. Here, we provided evidence that RhoA and Vav2 are critical substrates of Src oncoprotein. Notably, although ROS were considered to directly oxidize target proteins, peroxiredoxins were reported to mediate the oxidation of target proteins such as ASK1 and STAT3 from hydrogen peroxide [47, 48]. Thus we could not exclude the possibility that peroxiredoxins may oxidize RhoA and Src inside cells upon hydrogen peroxide. Contrary to Tyr42 phosphorylation, the phosphorylation of RhoA at Ser188 by protein kinase A (PKA) increased its affinity for RhoGDI [49]. Notably, p-Tyr64 Cdc42 and p-Tyr64 Rac1 also bind readily to RhoGDI, leading to their inactivation [50, 51]. Moreover, the phosphorylation of Rac1 at Thr108 by ERK upon EGF signaling [52] and Ser71 by Akt [53] serves as a negative regulator of Rac1 activity. In 19

particular, RhoGDI phosphorylation at Tyr156 by Src and at Ser34 by PKC is also known to reduce its affinity for RhoA, leading to Rho GTPase activation [25, 54]. Indeed, we demonstrated that hydrogen peroxide induced RhoGDI phosphorylation at Tyr (Fig. 2H). Although the specific Tyr residue to be phosphorylated by ROS remains elusive, we speculate that Tyr156 is phosphorylated by Src [25].

4.2.P-Tyr42 RhoA binds to to IKKγ and activates IKKβ It is ambiguous that ROS stimulate NF-κB because hydrogen peroxide activates or inactivates NF-κB in different cases [55]. However, ROS activate NF-κB in many circumstances [56]. Furthermore, low concentration of hydrogen peroxide as low as 1 μM evidently activated NF-κB in RAW 264.7 (Fig. 1G). It suggests that in general appropriate concentration of ROS is required to activate NF-κB in various types of cells [3]. Although Rho GTPases were reported to regulate NF-κB [13], the molecular mechanism of RhoAmediated NF-κB activation in response to hydrogen peroxide has been largely unknown, particularly in tumorigenesis. Notably, RhoA oxidation was very recently reported to be implicated to NF-κB activation, cell proliferation, and tumorigenesis [29]. In the present study, it is evident that p-Tyr42 RhoA is also linked to NF-κB activation (Fig. 4). P-Tyr42 RhoA, which is supposed to be an active form upon hydrogen peroxide stimulation, readily bound to IKKγ (Fig. 4), but p-Tyr42 RhoA reduced its affinity to RhoGDI (Fig. 2G). Because the Tyr42 residue of RhoA is localized in the switch I region, its phosphorylation seems to confer selectivity on RhoA to interact with IKKγ instead of RhoGDI. The binding domain of IKKγ for RhoA Y42E upon hydrogen peroxide is likely the aa 101-200 domain (Fig. 4J). However, native RhoA-GDP, but not RhoA-GTP binds to aa 1-43 domain of IKKγ/NEMO [11]. The result suggests that Tyr42 phosphorylation of RhoA may change its structure, facilitating RhoAGTP interaction with IKKγ. It was known that IκB is obligated to bind to the C-terminal domain (aa 389419) of IKKγ [57] to be phosphorylated by IKKβ that binds to aa 44-111 domain of IKKγ [58]. Therefore, we surmise that p-Tyr42 RhoA is juxtaposed with IKKβ on IKKγ, and directly stimulates IKKβ activity to 20

phosphorylate IκB at Ser32/36. Actually, IKKβ was co-immunoprcipitated with p-Tyr42 RhoA and IKKγ (Fig. 4C and 4D). The oxidation of RhoA presumably at Cys16/20 residues [39] enhanced p-Tyr42 RhoA activity to stimulate IKKβ (Fig. 4K). Notably, IKKβ itself was not phosphorylated by hydrogen peroxide (Fig. 4M), but showed kinase activity to phosphorylate IκB in the presence of RhoA WT and RhoA Y42E (a phosphomimic), but not RhoA Y42F (Fig. 4K). The result suggests that p-Tyr42 oxidized RhoA could directly activate IKKβ without IKKβ phosphorylation (Fig. 4M), although specific kinases such as TGF-β activated kinase (TAK) are generally considered to activate IKKβ by Ser 177/181 phosphorylation [35]. We previously reported that the RhoA-GDP/RhoGDI complex is dissociated by RhoA-GDP binding to IKKγ; IKKγ plays a role as a GDI displacement factor (GDF). In this case, IKKγ is essentially required for RhoA activation in response to TGF-β1 [11]. However, IKKγ is not critical for RhoA activation by ROS. Instead, IKKγ may be considered to play a role as a scaffold protein to recruit IKKβ, p-Tyr42 RhoA-GTP, and IκB together (Fig. 7C).

4.3.Tumorigenesis through p-Tyr42 RhoA RhoA was reported to activate the transcription factor, serum response factor (SRF) by actin filament formation. The SRF co-activator, MAL (megakaryocytic acute leukemia), originally binding to G-actin is released from F-actin which is induced by RhoA activation upon cell stimulation. Consequently, MAL is translocated to the nucleus where it forms a complex with SRF to induce the expression of target genes [59]. Here, we propose a novel activation mechanism of another transcription factor, NF-κB through RhoA upon ROS stimulation: hydrogen peroxide → Src → p-Vav2/p-Tyr42 RhoA → p-Tyr42 RhoA-GTP → IKKβ/p-Tyr42 RhoA-GTP/ IKKγ complex → p-IκB/degradation → NF-κB activation → cyclin D1 and C-Myc expression → cell proliferation/tumorigenesis (Fig. 7C). Indeed, p-Src, p-Tyr42 Rho, and p-p65 exhibited the correlations in human breast cancer tissues (Fig. 7B).

21

Rho GTPases have been related to tumorigenesis in many human cancers through their overexpression [14]. With respect to the molecular mechanism, RhoA was reported to induce cyclin D1 expression [60], c-Myc expression [61], and p21Waf1/Cip1 and p27Kip1 repression [62-64]. Notably, p27Kip1 binds to RhoA, thereby inhibiting RhoA activation by interfering with the interaction between RhoA and its GEF [65]. Additionally, NF-κB was reported to induce cyclin D1 and c-Myc expression [66, 67]. NFκB is constitutively activated in many types of cancer, leading to a variety of pro-tumorigenic functions [7]. However, the manifest signaling pathways of these components were not fully addressed. From the literature and our results, we inferred and integrated the signaling pathway of p-Tyr42 RhoA/NFκB/cyclin D1 and c-Myc to enhance cell proliferation in response to ROS in cancer. In particular, we underline that Tyr42 phosphorylation of RhoA by Src is essential for the process. ROS are highly produced in cancer cells in response to growth factors and cytokines [3]. We assume that ROS produced from a number of causes including increased cellular receptor activity, oncogene activity, increased metabolic activity, mitochondrial dysfunction, peroxisome activity, ROSgenerating enzyme activity, and tumor-associated macrophages (TAMs) [3] might stimulate proliferation of tumor cells through p-Tyr42 RhoA and NF-κB activation. It is well known that macrophages produce high levels of ROS, called an oxidative burst, through NADPH oxidase [68]. Furthermore, ROS play an essential role in the differentiation of M2 macrophages and the occurrence of TAMs [69]. Intriguingly, we observed that co-culture of macrophages and cancer cells enhanced ROS production (Fig. 6I) and expressed p-Tyr42 Rho GTPase (Fig. 6H). ROS production may not be attributed by physical interaction between RAW264.7 and 4T1 cells, in that either cell exposed to UV did not generate ROS in co-culture system (Fig. 6I). The result suggests that soluble factor(s) secreted from either cell during communication between macrophage and cancer cell contributes to ROS production. Notably, macrophage and tumor cell contact induce RhoA activity in tumor cells, facilitating tumor cell migration through EGF secreted from macrophages [70, 71]. In addition, inflammation that is mainly regulated by NF-κB plays a critical role in cancer progression [72]. 22

This study is a novel report, to our knowledge, that posttranscriptional modification of RhoA such as Tyr42 phosphorylation in addition to overexpression and mutation of RhoA may be required for tumorigenesis. This study will be useful for the development of therapeutic drugs for cancers evoked by ROS, RhoA and NF-κB activation.

5. 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 in response to ROS.

Conficts of interest None.

Acknowledgement This research was supported by the Basic Science Research Programme of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, the Ministry of Science, ICT, & 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, Hallym University) for technical assistance for immunostaining of cancer tissues and xenograft experiment.

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Figure Legends Fig. 1. Hydrogen peroxide induces the phosphorylation of RhoA at Tyr42. (A) RAW264.7 cells were stimulated with 100 μM H2O2. P-Tyr was immunoprecipitated from the cell lysates, and RhoA was identified with a Western blot. (B) RAW264.7 cells were stimulated with 100 μM H2O2 without or with 10 μM PP2, and RhoA was immunoprecipitated, and then p-Tyr was immunoblotted. (C) pCDNA3.1 plasmids containing HA-RhoA WT, Y34F, Y42F, Y66F, Y74F, and Y156F (4 μg) were transfected into HEK293 cells for 16 h. The cells were stimulated with 100 μM H2O2 for 1 h. HA-RhoA proteins were immunoprecipitated, and p-Tyr, HA, and IKKγ were identified with Western blots. (D) Amino acid sequences of Rho family GTPases were compared. Anti-p-Tyr42 Rho was produced from rabbit by using the aa 37-47 peptide containing p-Tyr42 amino acid. (E) RAW264.7 cells pretreated with 100 μM H2O2. P-Tyr42 Rho was identified with Western blots using prepared p-Tyr42 Rho antibody. (F) PP2 (10 μM) was pretreated to RAW264.7 cells for 1 h and and then 100 μM H2O2 was tereated for 1 h. P-Tyr42 Rho GTPase was immunoblotted with prepared p-Tyr42 Rho antibody. (G) Hydrogen peroxide of various concentrations (1-100 μM) was treated to RAW264.7 cells for 1 h, and p-Tyr42 Rho GTPase was identified with western blot. (H) Peritoneal macrophages were prepared from rat, stimulated with H2O2 (100 μM), and immunoblotted with p-Tyr42 Rho antibody.

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Fig. 2. Src phosphorylates Tyr42 of RhoA. (A) RAW264.7 cells were stimulated with 100 μM H2O2, and p-Y416 Src was identified with a Western blot. (B) RAW264.7 cells were stimulated with 100 μM H2O2. Src was immunoprecipitated from the cell lysates, and co-immunoprecipitated RhoA was identified with a Western blot. (C) GST-RhoA (0.1 μg) was pretreated with 10 μM H2O2 for 1 h and then catalase (1 U) was added for 30 min to remove H2O2. An in vitro kinase assay was then performed; ATP (20 μM) and purified active Src protein (0.1 μg) were incubated with the RhoA, and p-Tyr was identified with a Western blot. (D) Purified recombinant GST-RhoA WT, RhoA Y42F, and RhoA Y42E (0.1 μg) were incubated with 10 μM H2O2 for 1 h, and catalase (1 U) was incubated with the samples for 1 h to remove H2O2. In vitro kinase assays were performed; active Src (0.1 μg/50 μl) and 20 μM ATP were added to the RhoA proteins, and p-Tyr, p-Src, were identified with Western blots. (E) GST, recombinant RhoA WT, RhoA Y42F, and RhoA Y42E proteins (0.5 μg/50 μl) were mixed with 1 mM GDP or 0.1 mM GTPγS for 30 min at 30°C. P-Tyr and p-Src were identified with Western blots. Input GST, RhoA WT, RhoA Y42F, and RhoA Y42E proteins are presented in the lower panel with Coomasie-blue staining. (F) Purified recombinant RhoA WT, RhoA Y42E, and RhoA Y42F (0.1 μg/50 μl) were incubated with active 30

Src protein (0.1 μg) in the presence of ATP (10 mM) and MgCl2 (60 mM). P-Tyr42 RhoA was identified with p-Tyr42 Rho antibody. (G) Purified recombinant RhoA WT, RhoA Y42E, and RhoA Y42F (0.1 μg each) were preloaded with 1 mM GDP or 0.1 mM GTPγS for 30 min at 30°C. GST-RhoGDI conjugated with beads was incubated with the RhoA proteins, and the RhoA proteins binding to GST-RhoGDI were identified by Western blotting. (H) RAW264.7 cells were stimulated with H2O2 (100 μM) and p-Tyr proteins were immunoprecipitated, and then RhoGDI was detected with Western blot. (I) Cells were transfected with HA-RhoA WT, Y42E, and Y42F, and was preincubated with Tat-C3 for 60 min. RhoAGTP was precipitated using a pull-down assay, and HA was detected with Western blotting.

Fig. 3. Tyr42 of RhoA is a binding site of Vav2. (A) RAW264.7 cells were pretreated with 10 μM SU6656 (Src inhibitor) for 1 h and then treated with 100 μM H2O2, and RhoA-GTP levels were determined with a pull-down assay using GST-Rhotekin-RBD. RhoA was visualized with a Western blot. (B) HEK293 cells were treated with H2O2, and RhoA-GTP was determined with a pull-down assay and Western blot. Simultaneously, p-Tyr42 Rho was identified with p-Tyr42 Rho antibody. (C) HA-RhoA WT, RhoA Y42E and RhoA Y42F DNAs (4 μg) were transfected into HEK293 cells, and 100 μM H2O2 was added to the cells. HA was immunoprecipitated, and co-immunoprecipitated Vav2 was immunoblotted. (D) RAW264.7 cells were stimulated with 100 μM H2O2. Vav2 was immunoprecipitated using a Vav2 antibody from the cell lysates (500 μg protein) pretreated with 10 μM PP2 (Src inhibitor) for 1 h. Coimmunoprecipitated RhoA was identified with a Western blot. (E) RAW264.7 cells were transfected with control-si and si-Vav2 (10 nM) for 72 h, and the cells were stimulated with 100 μM H2O2 for 1 h. RhoAGTP levels were determined from the cells. (F) RAW264.7 cells were treated with 100 μM H2O2, and then P-Tyr174 Vav2 was immunoblotted. (G) Recombinant Vav2 (0.1 μg) and recombinant active Src (0.1 μg) were incubated at 25°C for 30 min in presence of ATP (1 mM) and MgCl2 (5 mM) in 50 μl total volume. P-Vav2 was identified with a Western blot using anti-p-Vav2 (p-Tyr 174) antibody. (H) 31

RAW264.7 cells pretreated with 1 μg/ml Tat-C3 for 1 h, and then stimulated with 100 μM H2O2, and then P-Tyr42 Rho was identified with Western blot. (I) HA-RhoA WT, Y42E, and Y42F were transfected into HEK293 cells for 72 h, and Tat-C3 was treated for 1 h. Then the cells were stimulated with hydrogen peroxide. RhoA-GTP was assessed by a pull-down assay with GST-RBD beads, and then HA-RhoA and p-Tyr42 RhoA were detected with Western blotting bu using HA and p-Tyr42 antibodies. The result was shown as the mean ± deviation of two independent experiments and

Fig. 4. p-Tyr42 RhoA binds to IKKγ and then activates IKKβ, leading to IκB phosphorylation. (A) 32

RAW264.7 and HEK293 cells were treated with Tat-C3 (1 μg/ml) and then stimulated with H2O2 (100 μM). NF-κB-luciferase activity was measured using a luminometer. (B) RhoA WT, RhoA Y42E, RhoA Y42F, and mock vector DNA were transfected into HEK293 cells, and the cells were incubated for 12 h. The cells were stimulated with 100 μM H2O2, and p-IκB and p-p65 were identified with Western blots. (C, D) RAW264.7 cells were stimulated with 100 μM H2O2. IKKγ (C) and IKKβ (D) were immunoprecipitated, and co-immunoprecipitated RhoA, p-Ty42 Rho GTPase and IKKβ or IKKγ were identified with Western blots. (E) RAW264.7 cells were stimulated with 100 μM H2O2 for 1 h. Calf intestinal alkaline phosphatase (CIP) (1 U) without or with heating at 60°C (*) for 30 min was incubated the cell lysate for 30 min. IKKγ was immunoprecipitated, and co-immunoprecipitated RhoA was identified with a Western blot. (F) PP2 and PP3 (each 10 μM) were used to pretreat RAW264.7 cells for 1 h. IKKγ was immunoprecipitated from the cell lysates, and co-immunoprecipitated RhoA was identified with Western blotting. (G) HA-RhoA WT, RhoA Y42F, and mock vector DNA (4 μg) were transfected into HEK293 cells. HA was immunoprecipitated, and co-immunoprecipitated IKKγ was identified by Western blotting. (H) Purified RhoA WT, RhoA Y42E, and RhoA Y42F proteins (0.1 μg each) were preloaded with 1 mM GDP or 0.1 mM GTPγS for 30 min at 30°C and then 10 μM H2O2 was added for 1 h. Agarose bead-GST-IKKγ (0.5 μg protein) was mixed with RhoA proteins and incubated for 30 min at 25°C. RhoA bound to bead-GST-IKKγ was identified with Western blots. (I) si-IKKγ (10 nM) was transfected into RAW264.7 cells and incubated for 72 h. H2O2 (100 μM) was administered to the cells, and RhoA-GTP levels were determined with a pull-down assay using GST-Rhotekin-RBD beads. (J) Purified recombinant RhoA Y42E (0.1 μg) was preloaded with 0.1 mM GTPγS for 30 min at 30°C. The RhoA was incubated with H2O2 (10 μM) for 1 h and then incubated with GST-IKKγ domains (aa 1-419, 1-43, 44-111, 1-100, 101-200, 201-350, 351-419, and 101-419) conjugated to beads for 30 min at 25°C. RhoA Y42E bound to GST-IKKγ was identified by Western blotting. (K) Purified recombinant RhoA WT, RhoA Y42E, and RhoA Y42F (0.1 μg each) were pretreated with H2O2 (10 μM) for 1 h. The RhoA proteins were mixed and incubated with purified recombinant GST-IKKβ (0.1 μg), GST-IKKγ (0.1 μg), and IκB (1 μg) in the presence of 20 μM ATP for 30 min at 25°C. P-Ser32/36-IκB was identified with Western blots using anti-p-Ser32/36-IκB antibody. (L) Purified recombinant RhoA WT, RhoA Y42E, and RhoA Y42F (0.1 μg each) were preloaded with 1 mM GDP or 0.1 mM GTPγS for 30 min at 30°C. RhoA was mixed and incubated with purified GST-IKKβ (0.1 μg), GST-IKKγ (0.1 μg), and IκB (1 μg) in the presence of 20 μM ATP for 30 min at 25°C. P-Ser32/36-IκB was identified with Western blots using anti-p-Ser32/36-IκB antibody. (M) RAW264.7 cells were stimulated with LPS (1 μg/ml), TGF-β (5 ng/ml), and H2O2 (100 μM), and then p-IKKβ were immunoblotted. (N) RAW264.7 cells were pretreated with Tat-C3 for 60 min, and then treated with H2O2. IKKγ or IKKβ was immunoprecipitated and then co-immunoprecipitated RhoA was immunoblotted. 33

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Fig. 5. P-Tyr42 RhoA promotes proliferation of cancer cell lines. (A) Cancer cell lines (4T1, HT-29, MCF-7, CT-26, RAW264.7, and AGS) were treated with H2O2 (100 μM), and p-Tyr42 Rho, p-p65, and p-IκB were identified with Western blots. (B) RAW264.7, and 4T1 cells were pretreated with Tat-C3 (1 μg/ml) or Bay 117082 (NF-κB inhibitor, 10 μM) for 1 h, and the number of proliferating cells with or without 100 μM H2O2 was determined after DAPI staining. (C) 4T1 cells were transfected with sh-RhoA with mock vector, RhoA WT, RhoA Y42E, and Y42F, and then stimulated with H2O2 (100 μM). NF-κB reporter assay was performed to measure NF-κB activation. (D, E) Breast cancer cells (4T1 and MCF7) were transfected with mock vector, RhoA WT, RhoA Y42E, and RhoA Y42F (4 μg DNA) and then stimulated with 100 μM H2O2 for 2 days. Cell proliferation was determined by DAPI staining. (F) LDH released from cells was measured after transfecting mock vector, RhoA WT, Y42E, and Y42F clones to examine cell viability. (G) Mouse breast cancer cell, 4T1 was transfected with sh-RhoA with RhoA WT, Y42E, and Y42F, and c-Myc and cyclin D1 were determined upon H2O2 (100 μM) with Western blotting. The result was shown as a representative of two experiments with same pattern.

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Fig. 6. P-Tyr42 RhoA of 4T1 cells increases tumorigenesis in xenografts. (A) Cancer cell (4T1) was transfected with sh-RhoA and then with RhoA WT, RhoA Y42E, and RhoA Y42F. RhoA was detected by Western blot. The 4T1 cells were used for xenograft experiment. (B) A breast cancer cell line (4T1) was transfected with sh-RhoA (4 μg DNA) and reconstituted with RhoA WT, RhoA Y42E, and RhoA Y42F (4 μg DNA). Cells (4T1, 1x105) were injected into the right leg of BALB/c mice (n=4). PMA (1 μg/50 μl DMSO) was applied to the injected site every day. (C) Tumors in mice were visualized after three weeks. (D, E) Tumor volume size was measured for 3 weeks and after 3 weeks. (F) Tumor weight was measured. (G) ROS produced by PMA (1 μM) in 4T1 cells were detected with hydroethidine. (H) 4T1 cells were treated with PMA and then p-Y42 Rho was detected with Western blot. (I) RAW264.7 cells and 4T1 or ether cell exposed to 300J UV for 10 min were co-cultured, and ROS detected with hydroethidine. (J) p-Tyr42 Rho was immunoblotted from co-cultured RAW264.7 and 4T1 cells.

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Fig. 7. P-Tyr42 RhoA, p-Src, and p-p65 in human breast cancer tissues. (A) Human berast cancer tissues were stained with p-Ty42 Rho, p-Src at Tyr416, p-p65 at Ser 527 antibodies. Scores of each stage were analysed and quantified. (B) Correlations of p-Src, p-Tyr42 RhoA, and p-p65 were analysed and plotted. Samples of blue number were normal tissues around tumor, and samples of red number were tumor tissues. (C) Proposed schematic illustration of tumorigenesis mechanism through p-Tyr42 RhoA and NF-κB activation in response to ROS was shown.

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.

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