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P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK Kim Cuong Cap a, b, Jae-Gyu Kim a, c, Amir Hamza a, Jae-Bong Park a, c, d, * a
Department of Biochemistry, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 24252, Republic of Korea Institute of Research and Development, Duy Tan University, Danang, 550000, Viet Nam c Institute of Cell Differentiation and Aging, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 24252, Republic of Korea d Clinical and Translational Science Institute, eLmed Inc, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 24252, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2019 Accepted 5 January 2020 Available online xxx
Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase pTyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression. © 2020 Elsevier Inc. All rights reserved.
Keywords: p-Tyr42 RhoA Superoxide PMA p47phox ROCK
1. Introduction NADPH oxidase (NOX) produces superoxide from oxygen, using NADPH as the electron donor [1]. There are several types of NOX, including NOX1, NOX2, NOX3, NOX4, NOX5, Duox1, and Duox2 [2]. For NADPH oxidase complex of phagocytes, it is composed of catalytic subunits as membrane-bound components of a heterodimer of NOX2/gp91phox and p22phox and regulatory subunits as cytosolic components of p47phox, p67phox, and p40phox [3]. Stimulation of phagocytes typically induces phosphorylation of p47phox [4] and there are a number of reported kinases that phosphorylate NADPH oxidase, in particular its p47phox subunit. These kinases
* Corresponding author. Department of Biochemistry, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 24252, Republic of Korea. E-mail address: [email protected] (J.-B. Park).
include protein kinase A, mitogen-activated protein kinase like p38 and ERK [5,6]. Protein kinase C (PKC) has also been reported to phosphorylate p47phox, in response to phorbol myristate acetate (PMA), an activator of PKC, and causing the p47phox translocation to the membrane cytoskeleton [7]. Upon phosphorylation, p47phox becomes associated with membrane-associated p22phox and gp91phox along with p40phox and p67phox [8] (see Scheme 1). Ras-related Rho GTPases regulate a range of cellular functions through a variety of signaling pathways in cells in response to extracellular stimuli. Rho GTPases including RhoA, Cdc42, and Rac1 primarily regulate dynamic rearrangement of cytoskeletal structures including actin filaments [10]. Rac1/2 GTPases are essential for activating NADPH oxidase by binding to p67phox as active GTPases [9] with Rac1 being critically required for superoxide production. RhoA has also been reported to be involved in superoxide formation, the mechanism of which has not been clearly elaborated [11].
https://doi.org/10.1016/j.bbrc.2020.01.001 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Abbreviations Apo DPI IOZ IPTG NAC PMA ROCK SOZ sh Y42E Y42F
apocynin diphenyleneiodonium IgG-opsonized zymosan isopropyl b-D-1-thiogalctopyranoside N-acetyl-L-cysteine phorbol amyristic acetate RhoA dependent coiled coil kinase serum-opsonized zymosan short hairpin Tyr42Glu mutant Tyr42Phe mutant
Recently, we reported that hydrogen peroxide induces tyrosine 42 phosphorylation (p-Tyr42 RhoA) and cysteine 16/20 oxidation of RhoA, all being critically involved in NF-kB activation and increased tumorigenesis [12,13]. As such, in this study, we investigated whether p-Tyr42 RhoA was involved in regulation of superoxide production in cancer cells. Following stimulation of A549 lung cancer cells by PMA, an activator of PKC, superoxide was produced in the cells along with increased levels of p-Tyr42 Rho and pp47phox. In addition, RhoA Y42F, a dephospho-mimic of RhoA, interfered with superoxide generation upon PMA stimulation. We then demonstrate that p-Tyr42 RhoA levels increase following PMA treatment due to the production of superoxide and Src activation. These in turn amplify superoxide production through activation of ROCK and p47phox, thus generating a positive feedback loop for superoxide generation and tumor progression. 2. Materials and methods 2.1. Materials Tat-C3 fusion protein was purified from Escherichia coli BL-21 [14]. Y27632 was obtained from Millipore-Sigma (Burlington, USA). N-acetyl-L-cysteine (NAC), phosphatase inhibitor cocktail, diphenyleneiodonium (DPI), isopropyl b-D-1-thiogalctopyranoside (IPTG), PP2, DMSO, recombinant activated ROCK II fragment protein, ATP, GDP, GTPgS, H2O2 and apocynin were purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM), and penicillin-streptomycin antibiotics were from GibcoBRL (New York, USA). The protease/ phosphatase inhibitor cocktail was purchased from ApexBio (Boston, USA). NSC 23766 compound was from Calbiochem (La Jolla, USA). PMA (TPA, 12-O-tetradecanoylphorbol-13-acetate) was purchased from MedChemExpress (New York, USA). Si-RhoA and control si-RNA were from Santa Cruz Biotechnology. Sh-RhoA and scramble (Scr) RNA were obtained from Bioneer (Daejeon, Korea). IgG-opsonized zymosan (IOZ) and serum-opsonized zymosan (SOZ) were prepared as in a previous report [15]. Anti -IgG, ROCK I, ROCK II, -RhoA, -actin, -HA-probe were purchased from Santa Cruz Biotechnology (Texas, USA). Anti -p-Y416 Src was from Cell Signaling Technology (Danvers, USA). P-p47phox was from Invitrogen (Waltham, USA).
Scheme 1. A novel mechanism of p-Tyr42 RhoA contributes to amplifying superoxide production and tumor progression.
2.3. Cell culture Cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and were maintained in DMEM F-12 for RAW264.7 murine macrophage cell line, in DMEM for A549 human colon cancer cells and also AGS human gastric cancer cell line. The media were all supplemented with 5% heat-inactivated FBS and antibiotics (100 U/ ml of penicillin, 100 mg/ml of streptomycin). 2.4. Mutagenesis and purification of recombinant proteins HA-RhoA WT, HA-RhoA Y42E, and HA-RhoA Y42F mutants in pCDNA3.1 plasmid constructs were made using a point mutagenesis kit (Intron Biotechnology, Gyeonggi, Korea). Recombinant proteins GST-RhoA WT, GST-RhoA Y42E, and GST-RhoA Y42F were first expressed using pGEX4T.1 vectors and then purified using glutathione-conjugated agarose beads with details provided previously [13]. For p47phox, its coding region was first subcloned into pGEX4T1 plasmid using EcoRI and SalI restriction enzyme sites. 2.5. In vitro kinase assay The assay buffer contained recombinant p47phox protein (3 mg), recombinant activated ROCK II fragment protein (10 ng) plus 30 mM ATP in 50 ml of kinase buffer (20 mM HEPES pH 7.5, 20 mM MgCl2, 20 mM b-mercaptoethanol, 1 mM EDTA). The reaction was allowed to proceed for 2 h at 25 C. Levels of p-p47phox were then identified by western blotting using anti-p-Ser345 p47phox antibody. 2.6. Sh/si-RNA, DNA transfection Cells seeded at 30%e40% confluency were transfected with sh/ si-RNAs using Lipofectamine 3000 transfection reagent and according to the manufacturer’s instructions. Briefly, 2.5 ml of transfection reagent was added to 125 ml of serum-free medium containing 50 nM of sh/siRNA or 4 mg of plasmid DNA followed by incubation for 20 min at room temperature. The complex was then added to the cells and was allowed to incubate for 48e72 h before the next treatment.
2.2. Preparation of anti-phospho-Tyr42-Rho antibody
2.7. ROS assay
Anti-p-Tyr42 Rho antibody was raised by immunization using a p-Tyr42 Rho peptide (epitope peptide T37VFEN (phospho-) Y42VADIE47) that was synthesized from the phospho-Tyr42 precursor (Young-In Frontier, Seoul, Korea) [12].
Superoxide was directly measured in the live cells using the dihydroethidium assay kit (D11347, Invitrogen). Briefly, cells at 2 105 were stimulated with the selected treatment in serum-free medium, washed and then fixed in 4% formaldehyde for 15 min at
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Fig. 1. PMA induces p-Tyr42 RhoA through superoxide. (AeC) RAW264.7, A549 and AGS cells were treated with DMSO vehicle or PMA (1 mM) for 1 h, followed by measuring superoxide levels in the live cells using the dihydroethidium assay. (D, E) A549 and AGS cells were treated with H2O2 (100 mM), and RhoA and p-Tyr42 Rho were detected by western blotting. (FeH) RAW264.7, A549 and AGS cells were treated with PMA (1 mM) for 1 h and relative RhoA and p-Y42 RhoA levels were determined by western blotting. (I, J) RAW264.7 cells were challenged with SOZ (I), IOZ (J) particles (5 105) for the indicated time and RhoA and p-Tyr42 Rho level changes were detected with western blotting. (K) A549 cells were pre-treated with Apocinin (1 mM), NAC (10 mM), or DPI (10 mM) for 1 h, then stimulated with PMA (1 mM) for 1 h and RhoA and p-Y42 RhoA level changes were detected with western blotting.
room temperature (RT). For generating fluorescence, the cells were treated with 50 mM hydroethidine in DMSO (500 ml) for 15 min at RT, and then twice washed with 1 PBS. Fluorescence images were recorded with a fluorescence microscope with a filter of 540e552 nm for excitation and with a filter of above 590 nm for emission. 2.8. Cell proliferation assay Cells were first seeded either in 12-well dishes (1 105 cells/ well) or in 6-well dishes (4 105 cells/well). At the point of the assay, nuclei of cells were stained with 1 mg/ml DAPI (1:200) for 15 min at RT and their intensities were measured as previously [12]. 2.9. Immunoprecipitation After treatment, cells at 1 107 were washed with 1 PBS and lysed in lysis buffer (20 mM Tris pH 7.4, 120 mM NaCl, 1 mM MgCl2
1% Nonidet P-40) supplemented with 1% of protease/phosphatase inhibitor cocktail (ApexBio). The cell lysates were cleared by centrifugation at 13,000g for 20 min at 4 C. The supernatants were then pre-cleared with protein A/G-agarose beads for 1 h and incubated with anti-IgG or anti-p-p47phox (1:1000 dilution) antibodies for 3 h at 4 C. Protein A/G agarose beads (30 ml) were then added to the lysates and incubated with shaking at 4 C for 3 h, followed by washing. 2.10. Wound healing assay A549 cells (1 105 cells/60 15 mm dishes) were seeded and incubated for 24 h and then treated according to the treatment sets (si-RNA transfection, DNA plasmid transfection or PMA stimulation). After becoming confluent, the cell monolayer was scratched using a plastic pipette tip. Migration of the cells at the edge of the scratch was analyzed by capturing and analyzing images using the available microscope (Axiovert 200, Zeiss).
Fig. 2. p-Tyr42 RhoA GTPase is involved in superoxide production. (A) A549 cells were pretreated with DMSO (1 mM), NSC23766 (50 mM), Tat-C3 (1 mg/ml) for 1 h and superoxide levels were measured using the dihydroethidium assay. (B) A549 cells were transfected with scrambled RNA (Scr, 50 nM) or sh-RhoA (50 nM) and with the add-back by pCDNA3.1 plasmids containing HA-RhoA WT, Y42E, Y42F for 48 h, then stimulated with or without 1 mM PMA for 1 h. The superoxide levels were determined using the dihydroethidium assay.
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Fig. 3. Src, RhoA, and ROCK affect phosphorylation of p47phox. (A) A549 cells were pretreated with PP2 (10 mM), stimulated with PMA (1 mM) for 1 h and p-Ser345 p47phox and p-Tyr416 Src levels were then determined by western blotting. (B) A549 cells were pretreated with apocinin (1 mM), NAC (10 mM), or DPI (10 mM) for 1 h, then stimulated with PMA (1 mM) for 1 h and p-Ser345 p47phox level changes were then detected by western blotting. (C) A549 cells were pretreated with Tat-C3 (1 mg/ml) or Y27633 (10 mM) for 1 h. They were subsequently stimulated with 1 mM PMA for 1 h and p-Tyr416 Src and p-Ser345 p47phox level changes were identified by western blots. (D) A549 cells were transfected with or without si-RhoA, followed by add-back with HA-RhoA WT, Y42E, and Y42F in pCDNA3.1 for 48 h; they were then stimulated with 1 mM PMA for 1 h and p-Ser345 p47phox changes were determined as before. (E) A549 cells were treated with Y27633 (10 mM) for 1 h and superoxide production was measured using the dihydroethidine assay. (F) Amino acid sequences neighboring the phosphorylation site Ser345 of p47PHOX (red color) were compared to previous known amino acid sequences around the phosphorylation sites Thr696 and Thr853 of myosin phosphatase target subunit 1 (MYPT1) (red color). Same sequence of amino acids (blue color) and similar ones (faint blue color) are noted (Asp, D and Glu, E). (G) Recombinant purified p47phox (3 mg), recombinant purified active ROCK II fragment protein (10 ng), and 30 mM ATP in 50 ml of kinase buffer were incubated for 2 h at 25 C. P-p47phox levels were then determined by running the post-reaction mix in a western blot. (H) A549 cells were stimulated with 1 mM PMA for 1 h and p-Ser345 p47phox was immunoprecipitated or co-immunoprecipitated via ROCK. ROCK I and ROCK II were identified as previously. (I) RAW cell lysates were incubated with 3 mg purified GST-RhoA WT, GST-RhoA Y42E or GST-RhoA Y42F in presence of 1 mM GDP or 0.1 mM GTPgS for 2 h at 4 C. ROCK II band was confirmed by western blotting. Input of GST-RhoA WT, Y42E and Y42F proteins was visualized with Coomassie blue staining. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2.11. Western blotting Cells rinsed in 1 PBS were harvested and lysed in RIPA buffer (50 mM Tris HCl pH 7.5, 1 mM MgCl2, 1% Nonidet P40, 150 mM NaCl) supplemented with 1% phosphatase/protease inhibitor cocktail (ApexBio). Cell lysates were centrifuged at 13,000g for 20 min at 4 C. Protein samples were subjected to SDS-PAGE at 20e30 mg/lane and transferred to PVDF membranes. These blots were then probed with the selected antibodies. 2.12. Statistical analysis Protein band imaging and statistical significance calculations were done with Photoshop CC2018 (Adobe, San Jose, USA) and PRISM 8.0 software (GraphPad, San Diego, USA), respectively. Generally, the data are shown as mean ± SE of at least 3 independent experiments with the protein bands shown as being representative of at least three independent experiments. Statistical analysis of significance was based on 1- or 2-way ANOVA, in which *p < 0.05 was considered significant, **p < 0.01 more significant, and ***p < 0.001 being highly significant. 3. Results PMA increases p-Tyr42 RhoA levels through superoxide production. PMA induced ROS in RAW264.7 (mouse macrophage
cell line), A549 (human lung cancer cell line) and AGS (human gastric cancer cell line) cells (Fig. 1A, 1B and 1C, respectively). We observed that hydrogen peroxide also induces Tyr42 phosphorylation of Rho in A549 and AGS cells (Fig. 1D and 1E, respectively). PMA has been reported to activate PKC, which in turn phosphorylates p47phox, leading to NADPH oxidase and superoxide production [16e18]. Thereby, we examined the effect of PMA on p-Tyr42 Rho levels and PMA indeed induced Tyr42 phosphorylation of Rho in RAW264.7, A549 and AGS cells (Fig. 1F, 1G and 1H, respectively). The previous report of superoxide formation during phagocytosis of opsonized zymosans [11] prompted us to explore the Ty42 phosphorylation changes in Rho in RAW274.7 cells exposed to serum-opsonized and IgG-opsonized zymosan particles (SOZ and IOZ, respectively). It is revealed that SOZ and IOZ particles induced Ty42 phosphorylation of Rho in RAW274.7 cells (Fig. 1I and 1J, respectively). To verify whether p-Tyr42 Rho induced by PMA was due to ROS production, we examined the effects of ROS scavenger and NADPH oxidase inhibitors on the event. As expected, NAC (ROS scavenger) and DPI and apocynin (NADPH oxidase inhibitors) reduced p-Tyr42 RhoA levels in response to PMA (Fig. 1K). These results suggest that ROS induced from a variety of stimulants is generally able to contribute to phosphorylation of RhoA at Tyr42 residue in several types of cells. p-Tyr42 RhoA GTPase regulates superoxide production. Involvement of small GTPases in superoxide production in response to PMA was examined. Tat-C3 (Rho inhibitor) and NSC 23766 (Rac
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Fig. 4. p-Tyr42 Rho is critical for migration and proliferation of A549 cells. (A) A549 cells were transfected with or without si-RhoA and then with the add-back of HA-RhoA WT, Y42E, Y42F in pCDNA3.1 plasmids for 48 h. The cells were then stimulated with 1 mM PMA for 1 h. The cells were scratched using a plastic pipette tip and the migration of the cells was captured at 0, 24 and 48 h post scratch using a microscope. (B) AGS cells were stimulated with H2O2 (100 mM) or PMA (1 mM) for 24 or 48 h. A549 cell proliferation was quantified by the blue color staining of DAPI using a fluorescence microscope. (C) A549 cells were stimulated with PMA (1 mM) for 48 h. Images were acquired and then analyzed. Effect of si-RhoA for reducing Rho A levels and levels of transfected RhoA WT, Y42E and Y42F were determined by western blotting. (D) Proposing a novel mechanism by which pTyr42 RhoA contributes to amplifying superoxide production and tumor progression through a signaling loop following the activation of cells with PMA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
inhibitor) both abolished superoxide in response to PMA (Fig. 2A), suggesting that Rho and Rac GTPases are involved in regulation of superoxide formation. We then explored whether p-Tyr42 of RhoA was playing a role in regulation of superoxide formation. Sh-RhoA remarkably inhibited superoxide production, but reconstitution with RhoA WT and RhoA Y42E (phospho-mimic of RhoA, Tyr changed to Glu) dramatically restored superoxide formation. However, RhoA Y42F (dephospho-mimic of RhoA, Tyr changed to Phe) still had a reduced level of superoxide formation in A549 cells in response to PMA (Fig. 2B), suggesting that p-Tyr42 of RhoA is critical for regulation of superoxide production. Src, RhoA, and ROCK are involved in phosphorylation of p47phox. We next characterized the components of signaling pathway for superoxide production being mediated through pTyr42 RhoA. We examined whether PMA activates Src: PMA induced p-Tyr416 Src, which is an active form of Src; PP2, an inhibitor of Src, inhibited p-Tyr416 Src along with p-Ser345 p47phox levels (Fig. 3A). The superoxide production role on p-p47phox levels was also investigated: reducing superoxide levels by NAC, DPI or apocynin treatment of the cells also markedly inhibited pp47phox levels (Fig. 3B). We next confirmed the involvement of RhoA and ROCK in signaling pathway for superoxide production with Tat-C3 and Y27632 inhibiting p-Tyr416 Src and p-Ser345
p47phox (Fig. 3C). Remarkably, sh-RhoA and sh-RhoA plus RhoA Y42F also inhibited the phosphorylation of p47phox whereas reconstituted RhoA WT and RhoA Y42E induced the phosphorylation of p47phox, suggesting that p-Tyr42 RhoA was critical for Ser345 phosphorylation of p47phox (Fig. 3D). ROCK phosphorylates p47phox. We confirmed that ROCK inhibitor Y27632 also significantly inhibited superoxide production upon PMA treatment, suggesting that ROCK was involved in superoxide generation (Fig. 3E). We then compared the peptide sequences of p47phox, particularly those surrounding Ser345 for phosphorylation by ROCK [19] with known substrates of ROCK in myosin phosphatase target subunit 1 (MYPT1), including Thr696 and Thr853 [20]. We found that amino acids RRR (335e337) and Gly (347), Lys (350) and Glu (352) residues were homologous to the sequences of MYPT1 (Fig. 3F, sFig.1). Enzymatically active ROCK fragment was also able to phosphorylate recombinant p47phox protein in vitro, suggesting that ROCK activates p47phox by direct phosphorylation (Fig. 3G). P47phox also co-immunoprecipitated with ROCK II upon PMA treatment of the cells (Fig. 3H). Furthermore, ROCK bound to recombinant GST-RhoA WT and RhoA Y42E phosphomimic form, but not to RhoA Y42F dephosphomimic form, suggesting that ROCK is a true effector protein for p-Tyr42 RhoA (Fig. 3I).
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Superoxide levels and p-Tyr42 RhoA content affect cellular proliferation and migration. We then explored the role of superoxide produced by PMA and p-Tyr42 RhoA levels on cell migration. si-RhoA and reconstituted RhoA Y42F attenuated cell migration whereas reconstituted RhoA WT and RhoA Y42E restored cell migration upon PMA cell treatment in the wound healing assay (Fig. 4A). Also, both PMA and hydrogen peroxide induced proliferation of A549 cells (Fig. 4B); however, si-RhoA and reconstituted RhoA Y42F dephosphomimic abolished proliferation of A549 cells; on the contrary, cells reconstituted with RhoA WT and RhoA Y42E phosphomimic had restored levels of cell proliferation (Fig. 4C).
Acknowledgment
4. Discussion
References
p-Tyr42 RhoA as well as several other Ras-related GTPases are involved in regulation of superoxide generation. It has been reported that RhoA, Cdc42 and Rac1 GTPases are involved in phagocytosis of particles [21]. In addition, it has been well established that Rac1 and Rac2 are essential for NADPH oxidase activation through binding to p67phox of NADPH oxidase. Another small GTPase, Rap1, is also involved in regulation of superoxide generation, but in an indirect manner. Rap1 and RhoA seem to have complementary or additive functions in regulating superoxide production by activating Rac1 and subsequently increasing Rac1’s ability to translocate and bind to NADPH oxidase complex, particularly p67phox [22] RhoA protein is involved in superoxide generation during the phagocytosis of IOZ particles [11]. However, the molecular mechanism by which RhoA induces superoxide production remains to be elucidated. As an inducer of superoxide production, PMA was utilized to stimulate A549 cells [16]. A549 cells have also revealed a signaling pathway for PKC/NADPH oxidase/ROS production [23]. As we have previously found that superoxide can induce Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) [12], we attempted to examine whether p-Tyr42 RhoA, in turn, regulates superoxide formation. Consequently, we established a sequence of events starting at superoxide production and amplification through a positive feedback cycle: superoxide production / Src / p-Tyr42 RhoA / ROCK / p-p47phox / NADPH oxidase activation / superoxide production (Fig. 4D). Physiological role of superoxide. In this study, we examined the role of superoxide on proliferation and migration of cancer cells. Effects on PMA-induced cell proliferation and migration were tested upon modulation of Tyr42 phosphorylation state of RhoA as RhoA Y42F reduced cell proliferation and migration. A molecular mechanism by which superoxide regulates these cellular functions in cancer cells has been reported with superoxide activating NF-kB, which in turn induces expression of cyclin D1 and c-Myc, leading to cell proliferation [12,13]. ROS are highly produced in a variety of cancer cells, stimulated by several growth factors and cytokines [24]. ROS produced by a number of causes might also stimulate proliferation, migration and invasion of tumor cells, leading to tumor progression [24]. We found that a variety of stimulants including lipopolysaccharide (LPS), epithelial growth factor (EGF), and Wnt3A as well as PMA can increase superoxide levels (data not shown). It has already been well established that PMA can lead to tumor promotion [25]. Taken together, we propose that p-Tyr42 RhoA GTPase critically amplifies superoxide production through p47phox becoming phosphorylated by ROCK and promoting tumorigenesis.
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Declaration of competing interest We declare that there is no conflict interests.
This research was supported by the Basic Science Research Programme of the National Research Foundation of Korea (NRF) (2018R1A4A1020922; 2018R1D1A1B07049273) and Hallym University (HRF-201901007). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.001.
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
K.C. Cap et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx G. Hayem, O.N. Jensen, M.A. Gougerot-Pocidalo, J. El-Benna, A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites, J. Clin. Investig. 116 (7) (2006) 2033e2043. [20] M. Khasnis, A. Nakatomi, K. Gumpper, M. Eto, Reconstituted human myosin light chain phosphatase reveals distinct roles of two inhibitory phosphorylation sites of the regulatory subunit, MYPT1, Biochemistry 53 (16) (2014) 2701e2709. [21] J.B. Park, Phagocytosis induces superoxide formation and apoptosis in macrophages, Exp. Mol. Med. 35 (5) (2003) 325e335. [22] Y. Li, J.G. Kim, H.J. Kim, M.Y. Moon, J.Y. Lee, J. Kim, S.C. Kim, D.K. Song, Y.S. Kim, J.B. Park, Small GTPases Rap1 and RhoA regulate superoxide formation by
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Rac1 GTPases activation during the phagocytosis of IgG-opsonized zymosans in macrophages, Free Radic. Biol. Med. 52 (9) (2012) 1796e1805. [23] S.E. Cheng, I.T. Lee, C.C. Lin, W.L. Wu, L.D. Hsiao, C.M. Yang, ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptor-dependent STAT3 activation, PLoS One 8 (1) (2013), e54125. [24] G.Y. Liou, P. Storz, Reactive oxygen species in cancer, Free Radic. Res. 44 (5) (2010) 479e496. [25] T.M. Kolb, M.A. Davis, The tumor promoter 12-O-tetradecanoylphorbol 13acetate (TPA) provokes a prolonged morphologic response and ERK activation in Tsc2-null renal tumor cells, Toxicol. Sci. 81 (1) (2004) 233e242.
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK Kim Cuong Cap a, b, Jae-Gyu Kim a, c, Amir Hamza a, Jae-Bong Park a, c, d, * a
Department of Biochemistry, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 24252, Republic of Korea Institute of Research and Development, Duy Tan University, Danang, 550000, Viet Nam c Institute of Cell Differentiation and Aging, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 24252, Republic of Korea d Clinical and Translational Science Institute, eLmed Inc, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 24252, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2019 Accepted 5 January 2020 Available online xxx
Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase pTyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression. © 2020 Elsevier Inc. All rights reserved.
Keywords: p-Tyr42 RhoA Superoxide PMA p47phox ROCK
1. Introduction NADPH oxidase (NOX) produces superoxide from oxygen, using NADPH as the electron donor [1]. There are several types of NOX, including NOX1, NOX2, NOX3, NOX4, NOX5, Duox1, and Duox2 [2]. For NADPH oxidase complex of phagocytes, it is composed of catalytic subunits as membrane-bound components of a heterodimer of NOX2/gp91phox and p22phox and regulatory subunits as cytosolic components of p47phox, p67phox, and p40phox [3]. Stimulation of phagocytes typically induces phosphorylation of p47phox [4] and there are a number of reported kinases that phosphorylate NADPH oxidase, in particular its p47phox subunit. These kinases
* Corresponding author. Department of Biochemistry, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 24252, Republic of Korea. E-mail address: [email protected] (J.-B. Park).
include protein kinase A, mitogen-activated protein kinase like p38 and ERK [5,6]. Protein kinase C (PKC) has also been reported to phosphorylate p47phox, in response to phorbol myristate acetate (PMA), an activator of PKC, and causing the p47phox translocation to the membrane cytoskeleton [7]. Upon phosphorylation, p47phox becomes associated with membrane-associated p22phox and gp91phox along with p40phox and p67phox [8] (see Scheme 1). Ras-related Rho GTPases regulate a range of cellular functions through a variety of signaling pathways in cells in response to extracellular stimuli. Rho GTPases including RhoA, Cdc42, and Rac1 primarily regulate dynamic rearrangement of cytoskeletal structures including actin filaments [10]. Rac1/2 GTPases are essential for activating NADPH oxidase by binding to p67phox as active GTPases [9] with Rac1 being critically required for superoxide production. RhoA has also been reported to be involved in superoxide formation, the mechanism of which has not been clearly elaborated [11].
https://doi.org/10.1016/j.bbrc.2020.01.001 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Abbreviations Apo DPI IOZ IPTG NAC PMA ROCK SOZ sh Y42E Y42F
apocynin diphenyleneiodonium IgG-opsonized zymosan isopropyl b-D-1-thiogalctopyranoside N-acetyl-L-cysteine phorbol amyristic acetate RhoA dependent coiled coil kinase serum-opsonized zymosan short hairpin Tyr42Glu mutant Tyr42Phe mutant
Recently, we reported that hydrogen peroxide induces tyrosine 42 phosphorylation (p-Tyr42 RhoA) and cysteine 16/20 oxidation of RhoA, all being critically involved in NF-kB activation and increased tumorigenesis [12,13]. As such, in this study, we investigated whether p-Tyr42 RhoA was involved in regulation of superoxide production in cancer cells. Following stimulation of A549 lung cancer cells by PMA, an activator of PKC, superoxide was produced in the cells along with increased levels of p-Tyr42 Rho and pp47phox. In addition, RhoA Y42F, a dephospho-mimic of RhoA, interfered with superoxide generation upon PMA stimulation. We then demonstrate that p-Tyr42 RhoA levels increase following PMA treatment due to the production of superoxide and Src activation. These in turn amplify superoxide production through activation of ROCK and p47phox, thus generating a positive feedback loop for superoxide generation and tumor progression. 2. Materials and methods 2.1. Materials Tat-C3 fusion protein was purified from Escherichia coli BL-21 [14]. Y27632 was obtained from Millipore-Sigma (Burlington, USA). N-acetyl-L-cysteine (NAC), phosphatase inhibitor cocktail, diphenyleneiodonium (DPI), isopropyl b-D-1-thiogalctopyranoside (IPTG), PP2, DMSO, recombinant activated ROCK II fragment protein, ATP, GDP, GTPgS, H2O2 and apocynin were purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM), and penicillin-streptomycin antibiotics were from GibcoBRL (New York, USA). The protease/ phosphatase inhibitor cocktail was purchased from ApexBio (Boston, USA). NSC 23766 compound was from Calbiochem (La Jolla, USA). PMA (TPA, 12-O-tetradecanoylphorbol-13-acetate) was purchased from MedChemExpress (New York, USA). Si-RhoA and control si-RNA were from Santa Cruz Biotechnology. Sh-RhoA and scramble (Scr) RNA were obtained from Bioneer (Daejeon, Korea). IgG-opsonized zymosan (IOZ) and serum-opsonized zymosan (SOZ) were prepared as in a previous report [15]. Anti -IgG, ROCK I, ROCK II, -RhoA, -actin, -HA-probe were purchased from Santa Cruz Biotechnology (Texas, USA). Anti -p-Y416 Src was from Cell Signaling Technology (Danvers, USA). P-p47phox was from Invitrogen (Waltham, USA).
Scheme 1. A novel mechanism of p-Tyr42 RhoA contributes to amplifying superoxide production and tumor progression.
2.3. Cell culture Cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and were maintained in DMEM F-12 for RAW264.7 murine macrophage cell line, in DMEM for A549 human colon cancer cells and also AGS human gastric cancer cell line. The media were all supplemented with 5% heat-inactivated FBS and antibiotics (100 U/ ml of penicillin, 100 mg/ml of streptomycin). 2.4. Mutagenesis and purification of recombinant proteins HA-RhoA WT, HA-RhoA Y42E, and HA-RhoA Y42F mutants in pCDNA3.1 plasmid constructs were made using a point mutagenesis kit (Intron Biotechnology, Gyeonggi, Korea). Recombinant proteins GST-RhoA WT, GST-RhoA Y42E, and GST-RhoA Y42F were first expressed using pGEX4T.1 vectors and then purified using glutathione-conjugated agarose beads with details provided previously [13]. For p47phox, its coding region was first subcloned into pGEX4T1 plasmid using EcoRI and SalI restriction enzyme sites. 2.5. In vitro kinase assay The assay buffer contained recombinant p47phox protein (3 mg), recombinant activated ROCK II fragment protein (10 ng) plus 30 mM ATP in 50 ml of kinase buffer (20 mM HEPES pH 7.5, 20 mM MgCl2, 20 mM b-mercaptoethanol, 1 mM EDTA). The reaction was allowed to proceed for 2 h at 25 C. Levels of p-p47phox were then identified by western blotting using anti-p-Ser345 p47phox antibody. 2.6. Sh/si-RNA, DNA transfection Cells seeded at 30%e40% confluency were transfected with sh/ si-RNAs using Lipofectamine 3000 transfection reagent and according to the manufacturer’s instructions. Briefly, 2.5 ml of transfection reagent was added to 125 ml of serum-free medium containing 50 nM of sh/siRNA or 4 mg of plasmid DNA followed by incubation for 20 min at room temperature. The complex was then added to the cells and was allowed to incubate for 48e72 h before the next treatment.
2.2. Preparation of anti-phospho-Tyr42-Rho antibody
2.7. ROS assay
Anti-p-Tyr42 Rho antibody was raised by immunization using a p-Tyr42 Rho peptide (epitope peptide T37VFEN (phospho-) Y42VADIE47) that was synthesized from the phospho-Tyr42 precursor (Young-In Frontier, Seoul, Korea) [12].
Superoxide was directly measured in the live cells using the dihydroethidium assay kit (D11347, Invitrogen). Briefly, cells at 2 105 were stimulated with the selected treatment in serum-free medium, washed and then fixed in 4% formaldehyde for 15 min at
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Fig. 1. PMA induces p-Tyr42 RhoA through superoxide. (AeC) RAW264.7, A549 and AGS cells were treated with DMSO vehicle or PMA (1 mM) for 1 h, followed by measuring superoxide levels in the live cells using the dihydroethidium assay. (D, E) A549 and AGS cells were treated with H2O2 (100 mM), and RhoA and p-Tyr42 Rho were detected by western blotting. (FeH) RAW264.7, A549 and AGS cells were treated with PMA (1 mM) for 1 h and relative RhoA and p-Y42 RhoA levels were determined by western blotting. (I, J) RAW264.7 cells were challenged with SOZ (I), IOZ (J) particles (5 105) for the indicated time and RhoA and p-Tyr42 Rho level changes were detected with western blotting. (K) A549 cells were pre-treated with Apocinin (1 mM), NAC (10 mM), or DPI (10 mM) for 1 h, then stimulated with PMA (1 mM) for 1 h and RhoA and p-Y42 RhoA level changes were detected with western blotting.
room temperature (RT). For generating fluorescence, the cells were treated with 50 mM hydroethidine in DMSO (500 ml) for 15 min at RT, and then twice washed with 1 PBS. Fluorescence images were recorded with a fluorescence microscope with a filter of 540e552 nm for excitation and with a filter of above 590 nm for emission. 2.8. Cell proliferation assay Cells were first seeded either in 12-well dishes (1 105 cells/ well) or in 6-well dishes (4 105 cells/well). At the point of the assay, nuclei of cells were stained with 1 mg/ml DAPI (1:200) for 15 min at RT and their intensities were measured as previously [12]. 2.9. Immunoprecipitation After treatment, cells at 1 107 were washed with 1 PBS and lysed in lysis buffer (20 mM Tris pH 7.4, 120 mM NaCl, 1 mM MgCl2
1% Nonidet P-40) supplemented with 1% of protease/phosphatase inhibitor cocktail (ApexBio). The cell lysates were cleared by centrifugation at 13,000g for 20 min at 4 C. The supernatants were then pre-cleared with protein A/G-agarose beads for 1 h and incubated with anti-IgG or anti-p-p47phox (1:1000 dilution) antibodies for 3 h at 4 C. Protein A/G agarose beads (30 ml) were then added to the lysates and incubated with shaking at 4 C for 3 h, followed by washing. 2.10. Wound healing assay A549 cells (1 105 cells/60 15 mm dishes) were seeded and incubated for 24 h and then treated according to the treatment sets (si-RNA transfection, DNA plasmid transfection or PMA stimulation). After becoming confluent, the cell monolayer was scratched using a plastic pipette tip. Migration of the cells at the edge of the scratch was analyzed by capturing and analyzing images using the available microscope (Axiovert 200, Zeiss).
Fig. 2. p-Tyr42 RhoA GTPase is involved in superoxide production. (A) A549 cells were pretreated with DMSO (1 mM), NSC23766 (50 mM), Tat-C3 (1 mg/ml) for 1 h and superoxide levels were measured using the dihydroethidium assay. (B) A549 cells were transfected with scrambled RNA (Scr, 50 nM) or sh-RhoA (50 nM) and with the add-back by pCDNA3.1 plasmids containing HA-RhoA WT, Y42E, Y42F for 48 h, then stimulated with or without 1 mM PMA for 1 h. The superoxide levels were determined using the dihydroethidium assay.
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Fig. 3. Src, RhoA, and ROCK affect phosphorylation of p47phox. (A) A549 cells were pretreated with PP2 (10 mM), stimulated with PMA (1 mM) for 1 h and p-Ser345 p47phox and p-Tyr416 Src levels were then determined by western blotting. (B) A549 cells were pretreated with apocinin (1 mM), NAC (10 mM), or DPI (10 mM) for 1 h, then stimulated with PMA (1 mM) for 1 h and p-Ser345 p47phox level changes were then detected by western blotting. (C) A549 cells were pretreated with Tat-C3 (1 mg/ml) or Y27633 (10 mM) for 1 h. They were subsequently stimulated with 1 mM PMA for 1 h and p-Tyr416 Src and p-Ser345 p47phox level changes were identified by western blots. (D) A549 cells were transfected with or without si-RhoA, followed by add-back with HA-RhoA WT, Y42E, and Y42F in pCDNA3.1 for 48 h; they were then stimulated with 1 mM PMA for 1 h and p-Ser345 p47phox changes were determined as before. (E) A549 cells were treated with Y27633 (10 mM) for 1 h and superoxide production was measured using the dihydroethidine assay. (F) Amino acid sequences neighboring the phosphorylation site Ser345 of p47PHOX (red color) were compared to previous known amino acid sequences around the phosphorylation sites Thr696 and Thr853 of myosin phosphatase target subunit 1 (MYPT1) (red color). Same sequence of amino acids (blue color) and similar ones (faint blue color) are noted (Asp, D and Glu, E). (G) Recombinant purified p47phox (3 mg), recombinant purified active ROCK II fragment protein (10 ng), and 30 mM ATP in 50 ml of kinase buffer were incubated for 2 h at 25 C. P-p47phox levels were then determined by running the post-reaction mix in a western blot. (H) A549 cells were stimulated with 1 mM PMA for 1 h and p-Ser345 p47phox was immunoprecipitated or co-immunoprecipitated via ROCK. ROCK I and ROCK II were identified as previously. (I) RAW cell lysates were incubated with 3 mg purified GST-RhoA WT, GST-RhoA Y42E or GST-RhoA Y42F in presence of 1 mM GDP or 0.1 mM GTPgS for 2 h at 4 C. ROCK II band was confirmed by western blotting. Input of GST-RhoA WT, Y42E and Y42F proteins was visualized with Coomassie blue staining. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2.11. Western blotting Cells rinsed in 1 PBS were harvested and lysed in RIPA buffer (50 mM Tris HCl pH 7.5, 1 mM MgCl2, 1% Nonidet P40, 150 mM NaCl) supplemented with 1% phosphatase/protease inhibitor cocktail (ApexBio). Cell lysates were centrifuged at 13,000g for 20 min at 4 C. Protein samples were subjected to SDS-PAGE at 20e30 mg/lane and transferred to PVDF membranes. These blots were then probed with the selected antibodies. 2.12. Statistical analysis Protein band imaging and statistical significance calculations were done with Photoshop CC2018 (Adobe, San Jose, USA) and PRISM 8.0 software (GraphPad, San Diego, USA), respectively. Generally, the data are shown as mean ± SE of at least 3 independent experiments with the protein bands shown as being representative of at least three independent experiments. Statistical analysis of significance was based on 1- or 2-way ANOVA, in which *p < 0.05 was considered significant, **p < 0.01 more significant, and ***p < 0.001 being highly significant. 3. Results PMA increases p-Tyr42 RhoA levels through superoxide production. PMA induced ROS in RAW264.7 (mouse macrophage
cell line), A549 (human lung cancer cell line) and AGS (human gastric cancer cell line) cells (Fig. 1A, 1B and 1C, respectively). We observed that hydrogen peroxide also induces Tyr42 phosphorylation of Rho in A549 and AGS cells (Fig. 1D and 1E, respectively). PMA has been reported to activate PKC, which in turn phosphorylates p47phox, leading to NADPH oxidase and superoxide production [16e18]. Thereby, we examined the effect of PMA on p-Tyr42 Rho levels and PMA indeed induced Tyr42 phosphorylation of Rho in RAW264.7, A549 and AGS cells (Fig. 1F, 1G and 1H, respectively). The previous report of superoxide formation during phagocytosis of opsonized zymosans [11] prompted us to explore the Ty42 phosphorylation changes in Rho in RAW274.7 cells exposed to serum-opsonized and IgG-opsonized zymosan particles (SOZ and IOZ, respectively). It is revealed that SOZ and IOZ particles induced Ty42 phosphorylation of Rho in RAW274.7 cells (Fig. 1I and 1J, respectively). To verify whether p-Tyr42 Rho induced by PMA was due to ROS production, we examined the effects of ROS scavenger and NADPH oxidase inhibitors on the event. As expected, NAC (ROS scavenger) and DPI and apocynin (NADPH oxidase inhibitors) reduced p-Tyr42 RhoA levels in response to PMA (Fig. 1K). These results suggest that ROS induced from a variety of stimulants is generally able to contribute to phosphorylation of RhoA at Tyr42 residue in several types of cells. p-Tyr42 RhoA GTPase regulates superoxide production. Involvement of small GTPases in superoxide production in response to PMA was examined. Tat-C3 (Rho inhibitor) and NSC 23766 (Rac
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Fig. 4. p-Tyr42 Rho is critical for migration and proliferation of A549 cells. (A) A549 cells were transfected with or without si-RhoA and then with the add-back of HA-RhoA WT, Y42E, Y42F in pCDNA3.1 plasmids for 48 h. The cells were then stimulated with 1 mM PMA for 1 h. The cells were scratched using a plastic pipette tip and the migration of the cells was captured at 0, 24 and 48 h post scratch using a microscope. (B) AGS cells were stimulated with H2O2 (100 mM) or PMA (1 mM) for 24 or 48 h. A549 cell proliferation was quantified by the blue color staining of DAPI using a fluorescence microscope. (C) A549 cells were stimulated with PMA (1 mM) for 48 h. Images were acquired and then analyzed. Effect of si-RhoA for reducing Rho A levels and levels of transfected RhoA WT, Y42E and Y42F were determined by western blotting. (D) Proposing a novel mechanism by which pTyr42 RhoA contributes to amplifying superoxide production and tumor progression through a signaling loop following the activation of cells with PMA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
inhibitor) both abolished superoxide in response to PMA (Fig. 2A), suggesting that Rho and Rac GTPases are involved in regulation of superoxide formation. We then explored whether p-Tyr42 of RhoA was playing a role in regulation of superoxide formation. Sh-RhoA remarkably inhibited superoxide production, but reconstitution with RhoA WT and RhoA Y42E (phospho-mimic of RhoA, Tyr changed to Glu) dramatically restored superoxide formation. However, RhoA Y42F (dephospho-mimic of RhoA, Tyr changed to Phe) still had a reduced level of superoxide formation in A549 cells in response to PMA (Fig. 2B), suggesting that p-Tyr42 of RhoA is critical for regulation of superoxide production. Src, RhoA, and ROCK are involved in phosphorylation of p47phox. We next characterized the components of signaling pathway for superoxide production being mediated through pTyr42 RhoA. We examined whether PMA activates Src: PMA induced p-Tyr416 Src, which is an active form of Src; PP2, an inhibitor of Src, inhibited p-Tyr416 Src along with p-Ser345 p47phox levels (Fig. 3A). The superoxide production role on p-p47phox levels was also investigated: reducing superoxide levels by NAC, DPI or apocynin treatment of the cells also markedly inhibited pp47phox levels (Fig. 3B). We next confirmed the involvement of RhoA and ROCK in signaling pathway for superoxide production with Tat-C3 and Y27632 inhibiting p-Tyr416 Src and p-Ser345
p47phox (Fig. 3C). Remarkably, sh-RhoA and sh-RhoA plus RhoA Y42F also inhibited the phosphorylation of p47phox whereas reconstituted RhoA WT and RhoA Y42E induced the phosphorylation of p47phox, suggesting that p-Tyr42 RhoA was critical for Ser345 phosphorylation of p47phox (Fig. 3D). ROCK phosphorylates p47phox. We confirmed that ROCK inhibitor Y27632 also significantly inhibited superoxide production upon PMA treatment, suggesting that ROCK was involved in superoxide generation (Fig. 3E). We then compared the peptide sequences of p47phox, particularly those surrounding Ser345 for phosphorylation by ROCK [19] with known substrates of ROCK in myosin phosphatase target subunit 1 (MYPT1), including Thr696 and Thr853 [20]. We found that amino acids RRR (335e337) and Gly (347), Lys (350) and Glu (352) residues were homologous to the sequences of MYPT1 (Fig. 3F, sFig.1). Enzymatically active ROCK fragment was also able to phosphorylate recombinant p47phox protein in vitro, suggesting that ROCK activates p47phox by direct phosphorylation (Fig. 3G). P47phox also co-immunoprecipitated with ROCK II upon PMA treatment of the cells (Fig. 3H). Furthermore, ROCK bound to recombinant GST-RhoA WT and RhoA Y42E phosphomimic form, but not to RhoA Y42F dephosphomimic form, suggesting that ROCK is a true effector protein for p-Tyr42 RhoA (Fig. 3I).
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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K.C. Cap et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Superoxide levels and p-Tyr42 RhoA content affect cellular proliferation and migration. We then explored the role of superoxide produced by PMA and p-Tyr42 RhoA levels on cell migration. si-RhoA and reconstituted RhoA Y42F attenuated cell migration whereas reconstituted RhoA WT and RhoA Y42E restored cell migration upon PMA cell treatment in the wound healing assay (Fig. 4A). Also, both PMA and hydrogen peroxide induced proliferation of A549 cells (Fig. 4B); however, si-RhoA and reconstituted RhoA Y42F dephosphomimic abolished proliferation of A549 cells; on the contrary, cells reconstituted with RhoA WT and RhoA Y42E phosphomimic had restored levels of cell proliferation (Fig. 4C).
Acknowledgment
4. Discussion
References
p-Tyr42 RhoA as well as several other Ras-related GTPases are involved in regulation of superoxide generation. It has been reported that RhoA, Cdc42 and Rac1 GTPases are involved in phagocytosis of particles [21]. In addition, it has been well established that Rac1 and Rac2 are essential for NADPH oxidase activation through binding to p67phox of NADPH oxidase. Another small GTPase, Rap1, is also involved in regulation of superoxide generation, but in an indirect manner. Rap1 and RhoA seem to have complementary or additive functions in regulating superoxide production by activating Rac1 and subsequently increasing Rac1’s ability to translocate and bind to NADPH oxidase complex, particularly p67phox [22] RhoA protein is involved in superoxide generation during the phagocytosis of IOZ particles [11]. However, the molecular mechanism by which RhoA induces superoxide production remains to be elucidated. As an inducer of superoxide production, PMA was utilized to stimulate A549 cells [16]. A549 cells have also revealed a signaling pathway for PKC/NADPH oxidase/ROS production [23]. As we have previously found that superoxide can induce Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) [12], we attempted to examine whether p-Tyr42 RhoA, in turn, regulates superoxide formation. Consequently, we established a sequence of events starting at superoxide production and amplification through a positive feedback cycle: superoxide production / Src / p-Tyr42 RhoA / ROCK / p-p47phox / NADPH oxidase activation / superoxide production (Fig. 4D). Physiological role of superoxide. In this study, we examined the role of superoxide on proliferation and migration of cancer cells. Effects on PMA-induced cell proliferation and migration were tested upon modulation of Tyr42 phosphorylation state of RhoA as RhoA Y42F reduced cell proliferation and migration. A molecular mechanism by which superoxide regulates these cellular functions in cancer cells has been reported with superoxide activating NF-kB, which in turn induces expression of cyclin D1 and c-Myc, leading to cell proliferation [12,13]. ROS are highly produced in a variety of cancer cells, stimulated by several growth factors and cytokines [24]. ROS produced by a number of causes might also stimulate proliferation, migration and invasion of tumor cells, leading to tumor progression [24]. We found that a variety of stimulants including lipopolysaccharide (LPS), epithelial growth factor (EGF), and Wnt3A as well as PMA can increase superoxide levels (data not shown). It has already been well established that PMA can lead to tumor promotion [25]. Taken together, we propose that p-Tyr42 RhoA GTPase critically amplifies superoxide production through p47phox becoming phosphorylated by ROCK and promoting tumorigenesis.
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Declaration of competing interest We declare that there is no conflict interests.
This research was supported by the Basic Science Research Programme of the National Research Foundation of Korea (NRF) (2018R1A4A1020922; 2018R1D1A1B07049273) and Hallym University (HRF-201901007). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.001.
Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001
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Please cite this article as: K.C. Cap et al., P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.001