Reversible Glutathionylation Regulates Actin Polymerization in A431 Cells

Abstract

In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead to the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation of a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed that the glutathionylation site is Cys-374. Deglutathionylation of the G-actin leads to about a 6-fold increase in the rate of polymerization.In vivo studies revealed a 12% increase in F-actin content 15 min after EGF treatment, and F-actin was found in the cell periphery suggesting that in response to growth factor, actin polymerizationin vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by glutaredoxin (thioltranferase), because Cd(II), an inhibitor of glutaredoxin, inhibits intracellular actin deglutathionylation at 2 μm, comparable with its IC50 in vitro. Moreover, mass spectral analysis showed efficient transfer of GSH from immobilized S-glutathionylated actin to glutaredoxin. Overall, this study revealed a novel physiological relevance of actin polymerization regulated by reversible glutathionylation of the penultimate cysteine mediated by growth factor stimulation. In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead to the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation of a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed that the glutathionylation site is Cys-374. Deglutathionylation of the G-actin leads to about a 6-fold increase in the rate of polymerization.In vivo studies revealed a 12% increase in F-actin content 15 min after EGF treatment, and F-actin was found in the cell periphery suggesting that in response to growth factor, actin polymerizationin vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by glutaredoxin (thioltranferase), because Cd(II), an inhibitor of glutaredoxin, inhibits intracellular actin deglutathionylation at 2 μm, comparable with its IC50 in vitro. Moreover, mass spectral analysis showed efficient transfer of GSH from immobilized S-glutathionylated actin to glutaredoxin. Overall, this study revealed a novel physiological relevance of actin polymerization regulated by reversible glutathionylation of the penultimate cysteine mediated by growth factor stimulation. reactive oxygen species epidermal growth factor protein-tyrosine phosphatase phospholipase C-γ 2′,7′-dichlorodihydrofluorescein diacetate 5,5′-dithiobis-(2-nitrobenzoic acid) 2-vinylpyridine phosphate-buffered saline iodoacetamide 4-morpholineethanesulfonic acid 3-(cyclohexylamino)propanesulfonic acid 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid bovine serum albumin phorbol 12-myristate 13-acetate liquid chromatography-mass spectrometry electrospray ionization The mechanisms involving extracellular stimuli-induced ROS1 generation in cells and subsequent signal propagating events are under active investigation (1Sundaresan M., Yu, Z.-X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2305) Google Scholar, 2Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar, 3Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.T. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1424) Google Scholar, 4Lee S.R. Kwon K.S. Kim S.R. Rhee S.R. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 5Barrett W.C. DeGnore J.P. Keng Y.-F. Zhang Z.-Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). In response to growth factor, ROS, such as O2− or H2O2, effectively inhibit protein-tyrosine phosphatases (PTPs) by converting the active site cysteines to sulfenic acids. The sulfenic derivatives are readily S-glutathionylated by GSH, which is abundant in mammalian cells (6Cooper A.J.L. Rosenberg R.N. Prusiner S.B. DiMauro S. Barchi R.L. Kunk L.M. The Molecular and Genetic Basis of Neurological Diseases. Butterworth-Heinemann, Boston1997: 1195-1230Google Scholar), thereby avoiding further oxidation to irreversible sulfinic or sulfonic derivatives (5Barrett W.C. DeGnore J.P. Keng Y.-F. Zhang Z.-Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). PTP inhibition leads to elevation of tyrosine-phosphorylated proteins, thereby promoting signal transduction cascades. Reactivation of glutathionylated PTP is most likely catalyzed by glutaredoxin, characterized as the primary enzyme for protein-SSG deglutathionylation in cells (7Mieyal J.J. Gravina S.A. Mieyal P.A. Srinivasan U. Starke D.W. Packer L. Biothiols in Health and Disease. Marcel-Dekker, Inc., New York1995: 305-372Google Scholar, 8Chrestensen C.A. Starke D.W. Mieyal J.J. J. Biol. Chem. 2000; 275: 26556-26565Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). As the intracellular redox equilibrium shifts toward oxidation and protein phosphorylation in response to extracellular stimuli, cytoskeletal changes also occur. Actin, an abundant and ubiquitous cellular protein, plays a major role in mediating the infrastructure and dynamics of the cytoplasmic matrix (9Kabsch W. Vandekerckhove J. Annu. Rev. Biophys. Biomol. Struct. 1992; 21: 49-76Crossref PubMed Scopus (216) Google Scholar, 10Sheterline P. Clayton J. Sparrow J.C. Actins, A Complete Data Resource.in: 4th Edition. Academic Press, New York1996: 1-23Google Scholar). Actin polymerization is a dynamic process implicated in growth factor-mediated cytoskeletal changes (11Rijken P.J. Hage W.J. van Bergen en Henegouwen P.M.P. Verkleij A.J. Boonstra J. J. Cell Sci. 1991; 100: 491-499PubMed Google Scholar). Here we report both in vitro and in vivo data that indicate EGF-induced deglutathionylation of the penultimate cysteine residue of actin facilitates its polymerization and cytoskeletal changes. The human epidermal A431 cell line was from ATCC; bovine muscle actin, EGF, DTNB, NADPH, GSSG reductase, carboxymethyl-BSA, CM-Sepharose, Q-Sepharose, phenyl-Sepharose, GSSG, and GSH were from Sigma; rabbit muscle actin was a generous gift from Dr. Xiong Liu; monoclonal anti-GSH antibody was obtained from Virogen; pYFP-actin vector was from CLONTECH; DCF-Ac and Phalloidin-Oregon Green 488 were from Molecular Probes; precast SDS gels and polyvinylidene difluoride membranes were from Invitrogen; Fugene6 was from Roche Molecular Biochemicals; H2O2 was from Fisher; CdCl2 and 2-vinyl pyridine were from Aldrich. [14C]Iodoacetamide was from American Radiolabeled Chemicals. Recombinant human glutaredoxin was expressed in Escherichia coli (12Chrestensen C.A. Eckman C.B. Starke D.W. Mieyal J.J. FEBS Lett. 1995; 374: 25-28Crossref PubMed Scopus (41) Google Scholar) and purified as described (13Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (131) Google Scholar). Typical specific activity of pure enzyme is 100 units/mg in the standard assay with cysteinyl-glutathione disulfide and GSH as substrates (12Chrestensen C.A. Eckman C.B. Starke D.W. Mieyal J.J. FEBS Lett. 1995; 374: 25-28Crossref PubMed Scopus (41) Google Scholar). A431 cells were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum to 90% confluency. After washing and incubating with PBS (30–60 min at 37 °C), the cells were incubated with H2O2 or EGF (at the indicated concentrations) at room temperature for 30 or 10 min, respectively. Cells were lysed with 50 μl of 2× SDS sample buffer without reducing agents and subjected to SDS-PAGE (12%) and Western blot analysis using anti-GSH antibody. A431 cells were grown as described to 70% confluency. Then they were starved in Hanks’ balanced salt solution (containing 2 mmMg2+ and phenol red) at 37 °C for 30 min followed by addition of DCF-Ac (3 μm). After 45 min, excess DCF-Ac was washed out, and the cells were incubated in Hanks’ solution with or without EGF (0.17 μm). After 15 min at room temperature, fluorescent images were obtained as described (2Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar). A431 cells were grown to 90% confluency. After starvation in PBS for 2 h, cells were exposed to EGF (5 μm) or H2O2 (10 mm) or PBS (control) for 30 min at 37 °C, followed by lysis in 50 μl of buffer A (125 mm sodium phosphate, pH 7.5, 3 mm EDTA) containing 1% Triton X-100. Measurement of the [GSSG]/[Total GSH] ratio was adapted from Griffith (14Griffith O.W. Anal. Biochem. 1980; 106: 207-212Crossref PubMed Scopus (4000) Google Scholar). To determine cysteine pKa values, pH-dependent radiolabel incorporation into rabbit muscle actin from [14C] iodoacetamide was carried out analogous to previous studies (15Srinirasan U. Mieyal P.A. Mieyal J.J. Biochemistry. 1997; 36: 3199-3206Crossref PubMed Scopus (108) Google Scholar). Buffers (10 mm) (MES (pH 5–7), HEPPSO (pH 7–9), CAPS (pH 9–10)) were adjusted to 0.5 m ionic strength and contained carboxymethyl-BSA (as coprecipitant) in the absence (control) or presence of 6 μm actin and 18 μm[14C]IAM. The reaction proceeded for 10 min, and then the proteins were precipitated, washed, and the radioactivity was determined. Bovine muscle actin (10 μm) in buffer A (2 mm Tris-HCl, pH 7.5, 0.2 mm ATP, 0.2 mm CaCl2) was incubated in the absence or presence of 200 μm GSSG at room temperature for 5 min. Polymerization was initiated by the addition of 100 mm KCl and 2 mm MgCl2 and monitored by light scattering (550 nm). A431 cells were grown to 80% confluency. After starvation for 8 h in serum-free Dulbecco’s modified Eagle’s medium, the cells were equilibrated in PBS at 37 °C for 1 h and then incubated for 20 min more in the absence (control) or presence of EGF (2 μm). Medium was removed and the cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with phalloidin-Oregon Green 488. After extensive washing with PBS, 1 ml of 2% SDS was added to each well to dissolve cell contents (37 °C, 10 h), and absorbance at 280 nm and fluorescence (496 nm excitation and 520 nm emission) were measured. F-actin content was expressed as the ratio of fluorescence intensity toA 280 nm. A431 cells grown on a glass plate (70% confluency) were transfected with a yellow fluorescent protein fused to a β-actin (pYFP-actin) vector using Fugene6. Upon expression of the fused protein (3∼4 days later), cells were subjected to starvation in PBS for 12 h prior to EGF treatment (2 μm). Fluorescent images of a single cell were recorded before and after 3, 5, and 10 min incubations with EGF (see the legend for Fig. 4). A431 cells were grown to 90% confluency. After cells were incubated with PBS alone or with CdCl2 (2 μm) for 1 h at 37 °C, 1 μm EGF (or no EGF for controls) was added and incubated for 20 min more. Medium was removed, and the extent of actin glutathionylation was monitored using anti-GSH as described above. Rabbit muscle actin (∼ 100 μg) was immobilized onto ∼200-μl dry volume of DNaseI-coupled resin and glutathionylated with 1 mm GSSG in PBS for 1 h at room temperature. After thorough washing with PBS, the resin was suspended in 100 μl of PBS containing 0.2 nmol (2.3 μg) of glutaredoxin and incubated at 37 °C for 1 h before centrifugation. The supernatant was subjected to LC-MS analysis with a Finnigan LCQ (Thermoquest, San Jose, CA) mass spectrometer. Monoclonal anti-GSH antibody was used to investigate growth factor-mediated protein glutathionylation in vivo. Fig.1 A shows that H2O2 treatment induced glutathionylation of a 42-kDa protein in a concentration-dependent manner in human epidermal carcinoma A431 cells. With 10 mmH2O2 (lane 9), the extent of glutathionylation of the 42-kDa protein was enhanced about 10-fold relative to that observed without H2O2. Equal amounts of loaded protein were verified by Coomassie Blue staining of an equivalent SDS gel (data not shown). In contrast, treatment of the same cell line with increasing concentrations of EGF surprisingly resulted in a progressive decrease in the extent of glutathionylation of the same protein band (Fig. 1 B). At 0.5 μmEGF (lane 5), the glutathionylated 42-kDa protein was diminished to approximately one-third of the basal level. When EGF was raised to 2 μm or higher, glutathionylation was no longer observable (lanes 7 and 8). The unexpected decrease in glutathionylation of the 42-kDa protein prompted us to examine ROS generation in A431 cells in response to EGF. Using the membrane-permeable fluorescent ROS indicator DCF-Ac, we found that 0.17 μm EGF induced a rapid ROS production as indicated by enhancement of fluorescence intensity (data not shown). This is in agreement with Bae et al. (2Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar). Because the [GSSG]/[Total GSH] redox ratio affects the state of glutathionylated proteins, we also examined the effect of EGF-induced ROS production on the redox state of GSH (Fig.2). For control cells, the redox ratio was 3.4 ± 0.1%. When cells were treated with 1 μmEGF or 10 mm H2O2 for 30 min, the [GSSG]/[Total GSH] ratio increased to 6.2 ± 1% or 17 ± 2%, respectively. These results indicate that EGF did shift the cellular redox equilibrium toward more oxidative, so the observed deglutathionylation of the 42-kDa protein most likely is due to an active compensatory process that is required for cellular response. This unexpected dichotomy was also observed with HeLa cells. To identify the 42-kDa protein, we used HeLa cells to obtain a larger quantity of the protein. The protein was purified to homogeneity using consecutive FPLC chromatographic separations, which included CM-Sepharose, Q-Sepharose, and phenyl-Sepharose, followed by two-dimensional electrophoresis. The purified protein was subjected to trypsin digestion and peptide sequence analysis (Michigan State University Core Facility) and was thereby identified as the cytoplasmic actin. Glutathionylation of actin, among other proteins, has been observed in other cells under varying conditions of oxidative stress,e.g. gastric mucosal cells treated with H2O2 or diamide (16Rokutan K. Johnson Jr., R.B. Kawai K. Am. J. Physiol. 1994; 266: G247-G254PubMed Google Scholar) and PMA-stimulated neutrophils (17Chai Y.-C. Ashraf S.S. Rokutan K. Johnston Jr., R.B. Thomas J.A. Arch. Biochem. Biophys. 1994; 310: 273-281Crossref PubMed Scopus (196) Google Scholar). In these cases, increasedS-glutathionylation of actin was observed. However, the site(s) of adduction of the GS moiety was not identified, and its physiological implications were not investigated. Purified actin contains six cysteine residues. To investigate the relationship between actin structure/conformation and the physiological consequences of its S-glutathionylation, the stoichiometry and site(s) of glutathionylation were determined in vitro using rabbit muscle actin as the protein source because of its high purity and sequence homology. When the actin was incubated with 1 mm GSSG for 30 min, the modified actin showed a molecular mass increase of 305 Da relative to untreated actin, based on LC-MS analysis, indicating incorporation of one GSH per actin. The modified actin was then subjected to CNBr cleavage followed by mass spectrometric analysis of the peptides. The 305-Da GSH moiety was found to be associated with a peptide (WITKQEYDEAGPSIVHRKCF) encompassing the C-terminal penultimate cysteine. This result identified the penultimate cysteine (Cys-374) as the most likely glutathionylation site in vivo, consistent with findings that this cysteine appears to be the most was at physiological pH by P. Clayton J. Sparrow J.C. Actins, A Complete Data Resource.in: 4th Edition. Academic Press, New York1996: 1-23Google Scholar, M. J.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. Biochemistry. 21: PubMed Scopus Google Scholar). With as the of G-actin was found to be modified Biochem. J. 266: PubMed Scopus Google Scholar). This that the relative of the cysteines in actin not be determined by pKa values, by we the pH of the reaction of actin with The data revealed a single to a pKa of and one moiety was per actin iodoacetamide was at a excess relative to actin. However, actin was treated with a excess of at pH six were to the six cysteine per actin the relationship between the of on actin and its state of polymerization (16Rokutan K. Johnson Jr., R.B. Kawai K. Am. J. Physiol. 1994; 266: G247-G254PubMed Google Scholar, Y.-C. Ashraf S.S. Rokutan K. Johnston Jr., R.B. Thomas J.A. Arch. Biochem. Biophys. 1994; 310: 273-281Crossref PubMed Scopus (196) Google Scholar, P.A. Arch. Biochem. Biophys. 1991; PubMed Scopus Google Scholar, Google no of the effect of oxidative be from we the relative of polymerization of the glutathionylated and of bovine muscle G-actin induced by 2 mm and 100 mm by light the rate for actin polymerization was at that of the glutathionylated actin (Fig. These data are consistent with the that a in the actin in the of and the of this is polymerization Y. T. K. K. J. Biochem. 1994; PubMed Scopus Google Scholar). the GSH moiety at the penultimate cysteine is to the rate of actin in vitro of the of actin and of the to of actin J. Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The physiological effect of actin deglutathionylation on F-actin was both in and at the single cell level. When A431 cells, with or without 1 μm EGF, were incubated for 20 min and then and stained with the actin Green the results revealed that the F-actin increased by 12 ± with EGF findings a for the report of et al. (11Rijken P.J. Hage W.J. van Bergen en Henegouwen P.M.P. Verkleij A.J. Boonstra J. J. Cell Sci. 1991; 100: 491-499PubMed Google Scholar) that showed EGF treatment of cells actin For single cell A431 cells were transfected with actin fused with yellow fluorescent protein, and actin polymerization was monitored by The data that actin was in the prior to EGF treatment after a treatment with 1 μm EGF, the light intensity increased with the of the light from the cell periphery B). This fluorescent enhancement was observed 3 min after EGF was transiently observed and to at 5 min. these results indicate that EGF an increase in F-actin and its toward the periphery of the The that EGF treatment deglutathionylation of actin in both A431 and HeLa cells that this deglutathionylation reaction is well regulated and with cell is most likely the enzyme for this based on its and relative for protein-SSG deglutathionylation (7Mieyal J.J. Gravina S.A. Mieyal P.A. Srinivasan U. Starke D.W. Packer L. Biothiols in Health and Disease. Marcel-Dekker, Inc., New York1995: 305-372Google Scholar, 8Chrestensen C.A. Starke D.W. Mieyal J.J. J. Biol. Chem. 2000; 275: 26556-26565Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). data for this are from both in vitro and in vivo Rabbit muscle actin was immobilized on resin and treated with 1 mm GSSG in PBS to glutathionylated actin at the penultimate cysteine. The resin was washed and in PBS This immobilized glutathionylated actin was then incubated with pure human glutaredoxin. The from both control and glutathionylated were subjected to A shows that the control glutaredoxin the molecular mass of the glutaredoxin exposed to the immobilized glutathionylated actin an increase in mass of 305 Da (Fig. 5 consistent with transfer of a moiety from the immobilized actin to the glutaredoxin. To the in vivo role of glutaredoxin, we the of a glutaredoxin inhibitor that has been to inhibit intracellular deglutathionylation C.A. Starke D.W. Mieyal J.J. J. Biol. Chem. 2000; 275: 26556-26565Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). A431 cells were with 2 μm the IC50 observed in for 1 h prior to treatment with EGF. Western blot analysis revealed that effect on actin glutathionylation prior to EGF treatment (lanes 1 and However, EGF-induced actin deglutathionylation (lanes 2 and 4). these results indicate that glutaredoxin is for the deglutathionylation of actin in response to growth factor in A431 cells. Because to a for cellular and and changes of cells are by a rapid of actin (11Rijken P.J. Hage W.J. van Bergen en Henegouwen P.M.P. Verkleij A.J. Boonstra J. J. Cell Sci. 1991; 100: 491-499PubMed Google W. PubMed Scopus Google Scholar). To these cellular actin and rate of are regulated by a of proteins as well as by that also the proteins P. Clayton J. Sparrow J.C. Actins, A Complete Data Resource.in: 4th Edition. Academic Press, New York1996: 1-23Google Scholar). Actin phosphorylation S. Verkleij A.J. Boonstra J. van Bergen en Henegouwen P.M.P. FEBS Lett. 1995; PubMed Scopus Google Scholar) and K. J. Cell PubMed Scopus Google Scholar) been and these events are to the state of of the in the signal transduction not been investigated. was that the EGF is associated with actin in A431 cells J.C. P. A.J. Boonstra J. Cell 1992; PubMed Scopus Google Scholar). findings this growth factor-mediated to deglutathionylation of actin at its penultimate cysteine catalyzed by glutaredoxin, which leads to enhancement of the rate of actin polymerization and of the actin to the cell These results also the of reversible glutathionylation of protein as a mechanism. is that the same EGF results in changes in the intracellular state of glutathionylation of (5Barrett W.C. DeGnore J.P. Keng Y.-F. Zhang Z.-Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) and actin In light of the likely role of cytoskeletal in extracellular S.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), of the state of glutathionylation of actin and changes in its state of polymerization the and dynamic of specific signal transduction Dr. for with the of the and Dr. Xiong and Dr. for the generous of rabbit muscle actin and HeLa cell respectively.