edox regulation of akt phosphorylation in PTEN mouse embryonic fibroblasts

REDOX REGULATION OF AKT PHOSPHORYLATION IN PTEN-/- MOUSE EMBRYONIC FIBROBLASTS LUO LE B.SC (HONS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENT I would like to express my greatest gratitude to my supervisor, Associate Professor Marie-Véronique Clément, for giving me the opportunity to be trained as a young researcher in the “MVC-Lab”. I am grateful to her constant support and advice over the years. With her encouragement and patient guidance, working in the lab becomes a journey of adventure. She is always supportive and optimistic when the experiments are not working. I am also impressed that she can always extract some important information out of something that does not appear so fancy to me. I will always keep in mind her notion of “re”-search in my upcoming research life. I would like to express my sincere appreciation to my TAC members, Dr Tang Bor Luen and Dr Yeong Foong May, for their valuable suggestions throughout the project. My warmest thanks go to my lab colleagues and friends for making the lab a wonderful place to stay in. I want to thank our lab officer Ms Mui Khin for doing all the logistic works, which gives us the best support for running our experiments. I am thankful to my seniors Sharon, Michelle and Huey fern for guiding me and teaching me all the necessary skills. I shall not forget my brothers and sisters in the lab, San Min, Charis, Shi Jie, Ryan, and Kyaw for being by my side like real family members. Finally, my heartfelt gratitude to my parents for persistent support which allows me to fulfil my dream. I also need to thank my sister for backing me up to take care of daddy and mummy in these years. Although we are separated by distances, the family is forever together. i TABLE OF CONTENTS ACKNOWLEDGEMENT i  TABLE OF CONTENTS ii  SUMMARY vii  LIST OF TABLES .ix  LIST OF FIGURES x  LIST OF ABBREVIATIONS xiii CHAPTER INTRODUCTION .1  1.1 Reactive oxygen species in cell signalling .1  1.1.1 Overview of free radicals 1  1.1.2 Reactive oxygen species .1  1.1.3 Redox homeostasis 4  1.1.4 Redox signalling .5  1.1.4.A TNFα-induced ROS production 6  1.1.4.B PDGF-induced ROS production 7  1.1.4.C EGF-induced ROS production 8  1.1.4.D Angiotensin II-induced ROS production 9  1.2 Nox family 10  1.2.1 Nox isoforms .11  1.2.2 Nox-mediated ROS production .15  1.2.3 Nox in cell signalling 17  1.2.3.A MAPK pathway 17  1.2.3.B Akt pathway .17  1.3 Akt 18  1.3.1 Structure 19  1.3.2 Activation process of Akt .19  1.3.2.A Step 1: Membrane translocation .20  1.3.2.B Step 2: Phosphorylation 21  1.3.3 A second level of regulation on Akt phosphorylation and activity: protein phosphatases 25  1.3.3.A PP2A .25  ii 1.3.3.B PHLPP .28  1.3.4 Other regulators: Akt interacting proteins 29  1.4 Redox regulation of Akt .30  1.4.1 PI3K related redox regulation of Akt 30  1.4.2 Akt as the direct target for redox regulation .34  1.5 Rationale of the project .36 CHAPTER MATERIALS AND METHODS .38  2.1 Materials .38  2.1.1 Chemicals and reagents .38  2.1.2 Antibodies .39  2.1.3 Plasmids 40  2.1.4 Cell lines and cell culture 40  2.2 Methods 41  2.2.1 Plasmid amplification .41  2.2.2 Mammalian cell transfection .42  2.2.3 RNA Interference (RNAi) Assay 43  2.2.4 RNA extraction and PCR 43  2.2.5 Sodium Dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis 44  2.2.6 Immunoprecipitation assay .46  2.2.7 In vitro Akt kinase assay .47  2.2.8 Membrane fractionation 48  2.2.9 Mitochondrial fractionation 48  2.2.10 Nuclear fractionation 49  2.2.11 Determination of Akt oxidation by AMS assay 49  2.2.12 Cell viability estimation by crystal violet assay .50  2.2.13 Cell cycle 51  2.2.14 Intracellular superoxide measurement by Lucigenin chemiluminescence assay .51  2.2.15 PP2A activity assay .52  2.2.16 Intracellular reduced glutathione (GSH) measurement 53  2.2.17 Intracellular pH (pHi) Measurement and NHE activity Assay .54  2.2.18 Measurement of PIP3 by Immunofluorescence and Confocal Microscopy .55  2.2.19 Statistical analysis .56 iii CHAPTER RESULTS 57  3.1 A decrease in intracellular O2.- level induces dephosphorylation of the survival kinase Akt in MEFPTEN-/- cells 57  3.1.1 Characterization of MEFPTEN-/- cells .57  3.1.2 Hyperphosphorylation of Akt is detected in MEFPTEN-/- cells .58  3.1.3 Intracellular O2.- level is higher in MEFPTEN-/- cells compared to MEFWT cells 61  3.1.4 Akt is dephosphorylated upon the decrease in intracellular O2.- level by DPI62  3.1.4.A DPI treatment decreases the level of intracellular O2.- .62  3.1.4.B Akt phosphorylation level is reduced upon the decrease in intracellular O2.- levl by DPI 65  3.1.4.C DPI does not change the overall phosphorylation status 71  3.1.4.D Restoration of intracellular O2.- level by DDC in DPI-treated cells is associated with the recovery of Akt phosphorylation level …………………… 72  3.1.4.E Prolonged reduction in intracellular O2.- level disrupts cell proliferation 77  3.1.5 Akt is dephosphorylated upon reduction of intracellular O2.- level by siNox4 83  3.2 Decrease in the intracellular level of O2.- induces the dephosphorylation of cytosolic Akt 93  3.2.1 The kinase-mediated process is not related to the reduction in Akt phosphorylation 93  3.2.2 The dephosphorylation mainly occurs on cytosolic Akt than membrane Akt 101  3.2.3 The dephosphorylation of Akt is similarly observed in mitochondria and nucleus as in cytosol 107  3.3 The cytosolic dephosphorylation of Akt is dependent on PP2A 109  3.3.1 Thr308 is the more sensitive site for dephosphorylation 109  3.3.2 Ser129 is not involved in the current system 111  3.3.3 Akt dephosphorylation is dependent on PP2A 113  3.3.4 The PP2A-B55α subunit is involved in Akt dephosphorylation .119  3.3.5 PP2A-C and PP2A-B55α subunits are mainly present in cytosol 124  3.3.6 Changes in PP2A-Akt interaction might be related to the enhanced Akt dephosphorylation 125  3.4 Regulation of Akt phosphorylation by the redox sensitive oxidation status of Akt 128  3.4.1 Detection of Akt oxidation by AMS assay 128  3.4.2 Changes of Akt oxidation status in response to changes in O2.- level .132  3.4.3 Akt phosphorylation level is associated with the changes in Akt oxidation status in MEFPTEN-/- cells 136  iv 3.4.4 Akt phosphorylation level is associated with the changes in Akt oxidation status in MEFWT and LNCaP cells .137  3.4.5 Serum deprivation induces Akt oxidation in both MEFPTEN-/- and MEFWT cells 141  3.4.6 Changes in the cellular redox environment after DPI- or siNox4-mediated changes in intracellular O2.- level .144  3.5 Regulation of Akt phosphorylation by NHE1 151  3.5.1 NHE1 positively regulates Akt phosphorylation 151  3.5.2 Akt-NHE1 interaction is detected .156  3.5.3 Akt dephosphorylation induced by LY294002 is delayed by the overexpression of NHE1 159  3.5.4 Redox regulation of the Akt-NHE1 signalling 161 CHAPTER DISCUSSION 167  4.1 The hyperphosphorylation of Akt in MEFPTEN-/- cells is downregulated upon a decrease in the intracellular level of O2.- 168  4.1.1 Reduction of intracellular O2.- level is achieved by DPI or siNox4 168  4.1.2 Akt is dephosphorylated upon the decrease in O2.- level by DPI and siNox. 172  4.1.3 Akt is dephosphorylated on both Thr308 and Ser473: interdependency of the two phosphorylation sites? .175  4.2 Cytosolic regulation of Akt: an important aspect in maintaining Akt phosphorylation 180  4.2.1 PIP3, the key messenger at the membrane, is not affected by the decrease in the level of intracellular O2.- in MEFPTEN-/- cells 180  4.2.2 Membrane recruitment of Akt or PDK1 is not affected .181  4.2.3 Cytosolic Akt is more sensitive to dephosphorylation than membrane Akt: localization matters 183  4.3 Akt oxidation status is regulated by O2.- .185  4.3.1 Akt oxidation is reversely correlated with intracellular O2.- level in MEFPTEN-/cells 185  4.3.2 The possible reducing/oxidation powers in O2.- -mediated alteration in Akt oxidation: present and future works .186  4.4 Regulation of Akt phosphorylation can be achieved via Akt oxidation 188  4.5 O2.- and Akt in cell proliferation and survival 192  4.5.1 Pro-proliferation activities of O2.- .192  v 4.5.2 O2.- mediated regulation of Akt .193  4.5.3 The possible implication of O2.- dependent Akt regulation in tumour cells 196  4.6 Scaffolding functionality of NHE1 in relation to Akt 197  4.7 Conclusions .200 APPENDIX A…………………………………………………………203 APPENDIX B…………………………………………………………204 REFERENCES……………………………………………………….205  PUBLICATION AND PRESENTATION…………………………. 230 vi SUMMARY Over the years, studies have demonstrated the emerging roles of the superoxide anion (O2.-) as an essential signalling molecule. The involvement of O2.- in cell proliferation and cell growth and has been demonstrated in different systems. Moreover, there are accumulating evidence pointing to the anti-apoptotic role of O2.-. Our group has shown that an increase in intracellular O2.- endows tumour cells with a survival advantage against a variety of apoptotic triggers. In line with the pro-survival role of O2.-, our group recently demonstrated the role of O2.- in regulating the survival kinase Akt via an oxidative inhibition of PTEN by S-nitrosylation. During the course of this study, it was noticed that in mouse embryonic fibroblasts that not express the tumour suppressor PTEN (MEFPTEN-/- cells), a decrease in the intracellular level of O2.- abrogated the hyperphosphorylation of Akt that was observed in these cells. Therefore, we hypothesize that O2.- may regulate the PI3K-Akt pathway not only through the inhibition of PTEN but also through a novel pathway that may be critical for the maintenance of the hyperphosphorylated Akt observed in MEFPTEN-/- cells. In the current project, the PTEN-independent pathways involved in the regulation of Akt phosphorylation by O2.- in MEFPTEN-/- cells is investigated. Using diphenyleneiodonium chloride (DPI), the inhibitor for the O2.--producing NADPH oxidases and silencing of the Nox4 isoform by small interference RNA, we show that the reduction of intracellular level of O2.- in MEFPTEN-/- cells results in a decrease in the phosphorylation level of the otherwise hyperphosphorylated Akt kinase. In investigating how O2.- regulates Akt phosphorylation level in MEFPTEN-/- cells, we provide evidence that the dephosphorylation of Akt is not dependent on any vii alterations in the level of PIP3, an important secondary messenger regulating Akt phosphorylation. Instead, the Akt molecules present in the cytosol are the primary target of this O2.- -mediated regulation, which is achieved via PP2A-dependent dephosphorylation. Furthermore, we also show that Akt oxidation status is inversely correlated with the level of intracellular O2.-. The proposed regulation of Akt phosphorylation by O2.- is possibly dependent on the shift between reduced-Akt and oxidized-Akt, which is associated with the susceptibility of Akt to the PP2A phosphatase. In addition to the cytosolic regulation of Akt phosphorylation by O2.-, we have also reported NHE1 as a regulator of Akt phosphorylation at the membrane. We showed for the first time that NHE1 interacts directly with Akt. This interaction allows NHE1 to serve as an additional anchor point for Akt recruitment to the membrane. Moreover, complex formation between NHE1 and Akt is disrupted by a reduction in intracellular O2.- level, which further illustrates the importance of O2.- in regulating Akt phosphorylation. Taken together, our data show that O2.- can regulate the level of Akt phosphorylation in various ways in MEFPTEN-/- cells. This suggests that in addition to be due to the elevated level of PIP3, hyperphosphorylation of Akt in PTEN-defective cells could be dependent on intracellular O2.- level as well. viii LIST OF TABLES Table 1: Examples of reactive oxygen species including radicals and non-radicals .2  Table 2: Tissue distribution and intracellular localization of Nox proteins .12  Table 3: Summary of PP2A isoforms for each subunit .26  Table 4: A summary of studies showing the effect of DPI on Akt phosphorylation.173  Table 5: A summary of siNox4 studies on Akt phosphorylation .174  Table 6: Selected studies addressing the interdependency of Thr308 and Ser473 phosphorylation 177  Table 7: Sequence search on Akt isoforms 191  ix Biol Med 44, 921-937. Kissner, R., Nauser, T., Kurz, C., and Koppenol, W. H. (2003). Peroxynitrous acid-where is the hydroxyl radical? IUBMB Life 55, 567-572. Kitagawa, K., Takasawa, K., Kuwabara, K., Sasaki, T., Tanaka, S., Mabuchi, T., Sugiura, S., Omura-Matsuoka, E., Matsumoto, M., and Hori, M. (2002). Differential Akt phosphorylation at Ser473 and Thr308 in cultured neurons after exposure to glutamate in rats. Neurosci Lett 333, 187-190. Klaunig, J. E., Kamendulis, L. M., and Hocevar, B. A. (2010). Oxidative stress and oxidative damage in carcinogenesis. Toxicol Pathol 38, 96-109. Knaus, U. G., Heyworth, P. G., Evans, T., Curnutte, J. T., and Bokoch, G. M. (1991). Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science 254, 1512-1515. Knaus, U. G., Heyworth, P. G., Kinsella, B. T., Curnutte, J. T., and Bokoch, G. M. (1992). Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase. J Biol Chem 267, 23575-23582. Kohn, A. D., Takeuchi, F., and Roth, R. A. (1996). Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J Biol Chem 271, 21920-21926. Kranzhöfer, R., Schmidt, J., Pfeiffer, C. A., Hagl, S., Libby, P., and Kübler, W. (1999). Angiotensin induces inflammatory activation of human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 19, 1623-1629. Kumar, B., Koul, S., Khandrika, L., Meacham, R. B., and Koul, H. K. (2008). Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res 68, 1777-1785. Kumari, S., Liu, X., Nguyen, T., Zhang, X., and D'Mello, S. R. (2001). Distinct phosphorylation patterns underlie Akt activation by different survival factors in neurons. Brain Res Mol Brain Res 96, 157-162. Kuo, Y. C., Huang, K. Y., Yang, C. H., Yang, Y. S., Lee, W. Y., and Chiang, C. W. (2008). Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt. J Biol Chem 283, 1882-1892. Kuribayashi, F., Nunoi, H., Wakamatsu, K., Tsunawaki, S., Sato, K., Ito, T., and Sumimoto, H. (2002). The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase. EMBO J 21, 6312-6320. Kuroda, J., Nakagawa, K., Yamasaki, T., Nakamura, K. I., Takeya, R., Kuribayashi, F., Imajoh-Ohmi, S., Igarashi, K., Shibata, Y., and Sueishi, K., et al. (2005). The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells. Genes Cells 10, 1139-1151. 215 Kwon, J., Lee, S. R., Yang, K. S., Ahn, Y., Kim, Y. J., Stadtman, E. R., and Rhee, S. G. (2004). Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors. Proc Natl Acad Sci U S A 101, 1641916424. Laine, J., Künstle, G., Obata, T., Sha, M., and Noguchi, M. (2000). The protooncogene TCL1 is an Akt kinase coactivator. Mol Cell 6, 395-407. Lambeth, J. D. (2004). NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4, 181-189. Lassègue, B., Sorescu, D., Szöcs, K., Yin, Q., Akers, M., Zhang, Y., Grant, S. L., Lambeth, J. D., and Griendling, K. K. (2001). Novel gp91(phox) homologues in vascular smooth muscle cells : nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ Res 88, 888-894. Lee, S. R., Kwon, K. S., Kim, S. R., and Rhee, S. G. (1998). Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J Biol Chem 273, 15366-15372. Lee, S. R., Yang, K. S., Kwon, J., Lee, C., Jeong, W., and Rhee, S. G. (2002). Reversible inactivation of the tumor suppressor PTEN by H2O2. J Biol Chem 277, 20336-20342. Lener, B., Kozieł, R., Pircher, H., Hütter, E., Greussing, R., Herndler-Brandstetter, D., Hermann, M., Unterluggauer, H., and Jansen-Dürr, P. (2009). The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells. Biochem J 423, 363-374. Leslie, N. R. (2006). The redox regulation of PI 3-kinase-dependent signaling. Antioxid Redox Signal 8, 1765-1774. Leslie, N. R., Bennett, D., Lindsay, Y. E., Stewart, H., Gray, A., and Downes, C. P. (2003). Redox regulation of PI 3-kinase signalling via inactivation of PTEN. EMBO J 22, 5501-5510. Leto, T. L., Adams, A. G., and de Mendez, I. (1994). Assembly of the phagocyte NADPH oxidase: binding of Src homology domains to proline-rich targets. Proc Natl Acad Sci U S A 91, 10650-10654. Li, J., Stouffs, M., Serrander, L., Banfi, B., Bettiol, E., Charnay, Y., Steger, K., Krause, K. H., and Jaconi, M. E. (2006). The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation. Mol Biol Cell 17, 3978-3988. Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S. I., Puc, J., Miliaresis, C., Rodgers, L., and McCombie, R., et al. (1997). PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943-1947. 216 Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J. A., and Robinson, J. P. (2003). DPI induces mitochondrial superoxide-mediated apoptosis. Free Radic Biol Med 34, 465-477. Li, X., Liu, J., and Gao, T. (2009). beta-TrCP-mediated ubiquitination and degradation of PHLPP1 are negatively regulated by Akt. Mol Cell Biol 29, 6192-6205. Li, Y., and Trush, M. A. (1998). Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production. Biochem Biophys Res Commun 253, 295-299. Li, Y., Zhu, H., Kuppusamy, P., Roubaud, V., Zweier, J. L., and Trush, M. A. (1998). Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. J Biol Chem 273, 2015-2023. Liao, Y., and Hung, M. C. (2010). Physiological regulation of Akt activity and stability. American journal of translational research 2, 19-42. Lijnen, P., Petrov, V., van Pelt, J., and Fagard, R. (2008). Inhibition of superoxide dismutase induces collagen production in cardiac fibroblasts. American journal of hypertension 21, 1129-1136. Lillig, C. H., Potamitou, A., Schwenn, J. D., Vlamis-Gardikas, A., and Holmgren, A. (2003). Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins. J Biol Chem 278, 22325-22330. Lim, S., and Clément, M. V. (2007). Phosphorylation of the survival kinase Akt by superoxide is dependent on an ascorbate-reversible oxidation of PTEN. Free Radic Biol Med 42, 1178-1192. Lin, K. I., Pasinelli, P., Brown, R. H., Hardwick, J. M., and Ratan, R. R. (1999). Decreased intracellular superoxide levels activate Sindbis virus-induced apoptosis. J Biol Chem 274, 13650-13655. Liu, L. Z., Hu, X. W., Xia, C., He, J., Zhou, Q., Shi, X., Fang, J., and Jiang, B. H. (2006). Reactive oxygen species regulate epidermal growth factor-induced vascular endothelial growth factor and hypoxia-inducible factor-1alpha expression through activation of AKT and P70S6K1 in human ovarian cancer cells. Free Radic Biol Med 41, 1521-1533. Lo, Y. Y., and Cruz, T. F. (1995). Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem 270, 11727-11730. Lopes, L. R., Dagher, M. C., Gutierrez, A., Young, B., Bouin, A. P., Fuchs, A., and Babior, B. M. (2004). Phosphorylated p40PHOX as a negative regulator of NADPH oxidase. Biochemistry 43, 3723-3730. 217 Lu, Z., and Xu, S. (2006). ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life 58, 621-631. Luetjens, C. M., Bui, N. T., Sengpiel, B., Münstermann, G., Poppe, M., Krohn, A. J., Bauerbach, E., Krieglstein, J., and Prehn, J. H. (2000). Delayed mitochondrial dysfunction in excitotoxic neuron death: cytochrome c release and a secondary increase in superoxide production. J Neurosci 20, 5715-5723. Luikenhuis, S., Perrone, G., Dawes, I. W., and Grant, C. M. (1998). The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species. Mol Biol Cell 9, 1081-1091. Lynch, D. K., Ellis, C. A., Edwards, P. A., and Hiles, I. D. (1999). Integrin-linked kinase regulates phosphorylation of serine 473 of protein kinase B by an indirect mechanism. Oncogene 18, 8024-8032. Maehama, T., and Dixon, J. E. (1998). The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5trisphosphate. J Biol Chem 273, 13375-13378. Maehara, Y., Miyano, K., and Sumimoto, H. (2009). Role for the first SH3 domain of p67phox in activation of superoxide-producing NADPH oxidases. Biochem Biophys Res Commun 379, 589-593. Mahadev, K., Motoshima, H., Wu, X., Ruddy, J. M., Arnold, R. S., Cheng, G., Lambeth, J. D., and Goldstein, B. J. (2004). The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. Mol Cell Biol 24, 1844-1854. Mahadev, K., Wu, X., Zilbering, A., Zhu, L., Lawrence, J. T., and Goldstein, B. J. (2001). Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes. J Biol Chem 276, 48662-48669. Mahadev, K., Zilbering, A., Zhu, L., and Goldstein, B. J. (2001). Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade. J Biol Chem 276, 21938-21942. Maira, S. M., Galetic, I., Brazil, D. P., Kaech, S., Ingley, E., Thelen, M., and Hemmings, B. A. (2001). Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane. Science 294, 374-380. Malo, M. E., and Fliegel, L. (2006). Physiological role and regulation of the Na+/H+ exchanger. Can J Physiol Pharmacol 84, 1081-1095. Mamane, Y., Petroulakis, E., LeBacquer, O., and Sonenberg, N. (2006). mTOR, translation initiation and cancer. Oncogene 25, 6416-6422. Masereel, B., Pochet, L., and Laeckmann, D. (2003). An overview of inhibitors of Na(+)/H(+) exchanger. Eur J Med Chem 38, 547-554. 218 McCord, J. M., and Fridovich, I. (1969). Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244, 6049-6055. Meier, B. (2001). Superoxide generation of phagocytes and nonphagocytic cells. Protoplasma 217, 117-124. Meier, B., Radeke, H. H., Selle, S., Younes, M., Sies, H., Resch, K., and Habermehl, G. G. (1989). Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha. Biochem J 263, 539-545. Meier, R., Thelen, M., and Hemmings, B. A. (1998). Inactivation and dephosphorylation of protein kinase Balpha (PKBalpha) promoted by hyperosmotic stress. EMBO J 17, 7294-7303. Meima, M. E., Webb, B. A., Witkowska, H. E., and Barber, D. L. (2009). The sodium-hydrogen exchanger NHE1 is an Akt substrate necessary for actin filament reorganization by growth factors. J Biol Chem 284, 26666-26675. Meng, T. C., Buckley, D. A., Galic, S., Tiganis, T., and Tonks, N. K. (2004). Regulation of insulin signaling through reversible oxidation of the protein-tyrosine phosphatases TC45 and PTP1B. J Biol Chem 279, 37716-37725. Meng, T. C., Fukada, T., and Tonks, N. K. (2002). Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell 9, 387-399. Menon, S. G., Sarsour, E. H., Spitz, D. R., Higashikubo, R., Sturm, M., Zhang, H., and Goswami, P. C. (2003). Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle. Cancer Res 63, 2109-2117. Mesquita, F. S., Dyer, S. N., Heinrich, D. A., Bulun, S. E., Marsh, E. E., and Nowak, R. A. (2010). Reactive oxygen species mediate mitogenic growth factor signaling pathways in human leiomyoma smooth muscle cells. Biol Reprod 82, 341-351. Miller, F. J., Filali, M., Huss, G. J., Stanic, B., Chamseddine, A., Barna, T. J., and Lamb, F. S. (2007). Cytokine activation of nuclear factor kappa B in vascular smooth muscle cells requires signaling endosomes containing Nox1 and ClC-3. Circ Res 101, 663-671. Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. (1999). Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci 24, 186-191. Mirshafiey, A., and Mohsenzadegan, M. (2008). The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. Iran J Allergy Asthma Immunol 7, 195-202. Misra, H. P. (1979). Reaction of copper-zinc superoxide dismutase with diethyldithiocarbamate. J Biol Chem 254, 11623-11628. Mittal, M., Roth, M., König, P., Hofmann, S., Dony, E., Goyal, P., Selbitz, A. C., 219 Schermuly, R. T., Ghofrani, H. A., and Kwapiszewska, G., et al. (2007). Hypoxiadependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature. Circ Res 101, 258-267. Miyamoto, S., Rubio, M., and Sussman, M. A. (2009). Nuclear and mitochondrial signalling Akts in cardiomyocytes. Cardiovasc Res 82, 272-285. Mochizuki, T., Furuta, S., Mitsushita, J., Shang, W. H., Ito, M., Yokoo, Y., Yamaura, M., Ishizone, S., Nakayama, J., and Konagai, A., et al. (2006). Inhibition of NADPH oxidase activates apoptosis via the AKT/apoptosis signal-regulating kinase pathway in pancreatic cancer PANC-1 cells. Oncogene 25, 3699-3707. Mori, K., Shibanuma, M., and Nose, K. (2004). Invasive potential induced under long-term oxidative stress in mammary epithelial cells. Cancer Res 64, 7464-7472. Mumbengegwi, D. R., Li, Q., Li, C., Bear, C. E., and Engelhardt, J. F. (2008). Evidence for a superoxide permeability pathway in endosomal membranes. Mol Cell Biol 28, 3700-3712. Muniyappa, H., Song, S., Mathews, C. K., and Das, K. C. (2009). Reactive oxygen species-independent oxidation of thioredoxin in hypoxia: inactivation of ribonucleotide reductase and redox-mediated checkpoint control. J Biol Chem 284, 17069-17081. Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K., and Kondo, T. (2003). Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt. J Biol Chem 278, 50226-50233. Murillo, M. M., Carmona-Cuenca, I., Del Castillo, G., Ortiz, C., Roncero, C., Sánchez, A., Fernández, M., and Fabregat, I. (2007). Activation of NADPH oxidase by transforming growth factor-beta in hepatocytes mediates up-regulation of epidermal growth factor receptor ligands through a nuclear factor-kappaB-dependent mechanism. Biochem J 405, 251-259. Murphy, H. S., Shayman, J. A., Till, G. O., Mahrougui, M., Owens, C. B., Ryan, U. S., and Ward, P. A. (1992). Superoxide responses of endothelial cells to C5a and TNFalpha: divergent signal transduction pathways. Am J Physiol 263, L51-L59. Myers, J. M., Antholine, W. E., and Myers, C. R. (2008). Hexavalent chromium causes the oxidation of thioredoxin in human bronchial epithelial cells. Toxicology 246, 222-233. Myhre, O., Andersen, J. M., Aarnes, H., and Fonnum, F. (2003). Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol 65, 1575-1582. O'Donnell, B. V., Tew, D. G., Jones, O. T., and England, P. J. (1993). Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem J 290 ( Pt 1), 41-49. 220 O'toole, A., Moule, S. K., Lockyer, P. J., and Halestrap, A. P. (2001). Tumour necrosis factor-alpha activation of protein kinase B in WEHI-164 cells is accompanied by increased phosphorylation of Ser473, but not Thr308. Biochem J 359, 119-127. Oberley, L. W. (2005). Mechanism of the tumor suppressive effect of MnSOD overexpression. Biomed Pharmacother 59, 143-148. Oberley, T. D., Schultz, J. L., Li, N., and Oberley, L. W. (1995). Antioxidant enzyme levels as a function of growth state in cell culture. Free Radic Biol Med 19, 53-65. Orii, K. E., Lee, Y., Kondo, N., and McKinnon, P. J. (2006). Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development. Proc Natl Acad Sci U S A 103, 10017-10022. Ouyang, Y. B., Zhang, X. H., He, Q. P., Wang, G. X., Siesjö, B. K., and Hu, B. R. (2000). Differential phosphorylation at Ser473 and Thr308 of Akt-1 in rat brain following hypoglycemic coma. Brain Res 876, 191-195. Paramio J.M., Segrelles C., Ruiz S., Jorcano J.L. (2001). Inhibition of protein kinase B (PKB) and PKCzeta mediates keratin K10-induced cell cycle arrest. Mol Cell Biol. 21, 7449-59. Park, S. E., Song, J. D., Kim, K. M., Park, Y. M., Kim, N. D., Yoo, Y. H., and Park, Y. C. (2007). Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells. FEBS Lett 581, 180-186. Parkos, C. A., Dinauer, M. C., Walker, L. E., Allen, R. A., Jesaitis, A. J., and Orkin, S. H. (1988). Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci U S A 85, 3319-3323. Pearce, L. R., Huang, X., Boudeau, J., Pawłowski, R., Wullschleger, S., Deak, M., Ibrahim, A. F. M., Gourlay, R., Magnuson, M. A., and Alessi, D. R. (2007). Identification of Protor as a novel Rictor-binding component of mTOR complex-2. Biochem J 405, 513-522. Pedruzzi, E., Guichard, C., Ollivier, V., Driss, F., Fay, M., Prunet, C., Marie, J. C., Pouzet, C., Samadi, M., and Elbim, C., et al. (2004). NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol 24, 10703-10717. Pei, H., Li, L., Fridley, B. L., Jenkins, G. D., Kalari, K. R., Lingle, W., Petersen, G., Lou, Z., and Wang, L. (2009). FKBP51 affects cancer cell response to chemotherapy by negatively regulating Akt. Cancer Cell 16, 259-266. Pekarsky, Y., Koval, A., Hallas, C., Bichi, R., Tresini, M., Malstrom, S., Russo, G., Tsichlis, P., and Croce, C. M. (2000). Tcl1 enhances Akt kinase activity and mediates its nuclear translocation. Proc Natl Acad Sci U S A 97, 3028-3033. Pelicano, H., Carney, D., and Huang, P. (2004). ROS stress in cancer cells and 221 therapeutic implications. Drug Resist Updat 7, 97-110. Pelicano, H., Xu, R. H., Du, M., Feng, L., Sasaki, R., Carew, J. S., Hu, Y., Ramdas, L., Hu, L., and Keating, M. J., et al. (2006). Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism. J Cell Biol 175, 913-923. Persad, S., Attwell, S., Gray, V., Mawji, N., Deng, J. T., Leung, D., Yan, J., Sanghera, J., Walsh, M. P., and Dedhar, S. (2001). Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem 276, 27462-27469. Pervaiz, S., and Clement, M. V. (2007). Superoxide anion: oncogenic reactive oxygen species? Int J Biochem Cell Biol 39, 1297-1304. Pervaiz, S., and Clément, M. V. (2002). A permissive apoptotic environment: function of a decrease in intracellular superoxide anion and cytosolic acidification. Biochem Biophys Res Commun 290, 1145-1150. Pervaiz, S., Cao, J., Chao, O. S., Chin, Y. Y., and Clément, M. V. (2001). Activation of the RacGTPase inhibits apoptosis in human tumor cells. Oncogene 20, 6263-6268. Pervaiz, S., Ramalingam, J. K., Hirpara, J. L., and Clément, M. V. (1999). Superoxide anion inhibits drug-induced tumor cell death. FEBS Lett 459, 343-348. Peshavariya, H., Dusting, G. J., Jiang, F., Halmos, L. R., Sobey, C. G., Drummond, G. R., and Selemidis, S. (2009). NADPH oxidase isoform selective regulation of endothelial cell proliferation and survival. Naunyn Schmiedebergs Arch Pharmacol 380, 193-204. Pullar, J. M., and Hampton, M. B. (2002). Diphenyleneiodonium triggers the efflux of glutathione from cultured cells. J Biol Chem 277, 19402-19407. Putney, L. K., Denker, S. P., and Barber, D. L. (2002). The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions. Annu Rev Pharmacol Toxicol 42, 527-552. Qiao, M., Iglehart, J. D., and Pardee, A. B. (2007). Metastatic potential of 21T human breast cancer cells depends on Akt/protein kinase B activation. Cancer Res 67, 52935299. Qin, S., and Chock, P. B. (2003). Implication of phosphatidylinositol 3-kinase membrane recruitment in hydrogen peroxide-induced activation of PI3K and Akt. Biochemistry 42, 2995-3003. Quinn, M. T., Evans, T., Loetterle, L. R., Jesaitis, A. J., and Bokoch, G. M. (1993). Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components. J Biol Chem 268, 20983-20987. Quinn, M. T., and Gauss, K. A. (2004). Structure and regulation of the neutrophil 222 respiratory burst oxidase: comparison with nonphagocyte oxidases. J Leukoc Biol 76, 760-781. Resjö, S., Göransson, O., Härndahl, L., Zolnierowicz, S., Manganiello, V., and Degerman, E. (2002). Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes. Cell Signal 14, 231-238. Roberts, C. K., and Sindhu, K. K. (2009). Oxidative stress and metabolic syndrome. Life Sci 84, 705-712. Roux, P. P., and Blenis, J. (2004). ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68, 320-344. Sakon, S., Xue, X., Takekawa, M., Sasazuki, T., Okazaki, T., Kojima, Y., Piao, J. H., Yagita, H., Okumura, K., and Doi, T., et al. (2003). NF-kappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death. EMBO J 22, 3898-3909. Sanders, S. A., Eisenthal, R., and Harrison, R. (1997). NADH oxidase activity of human xanthine oxidoreductase--generation of superoxide anion. Eur J Biochem 245, 541-548. Sarbassov, D. D., Guertin, D. A., Ali, S. M., and Sabatini, D. M. (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098-1101. Sarbassov, D. D., Ali, S. M., and Sabatini, D. M. (2005). Growing roles for the mTOR pathway. Curr Opin Cell Biol 17, 596-603. Sarfstein, R., Gorzalczany, Y., Mizrahi, A., Berdichevsky, Y., Molshanski-Mor, S., Weinbaum, C., Hirshberg, M., Dagher, M. C., and Pick, E. (2004). Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras. J Biol Chem 279, 16007-16016. Sathyamoorthy, M., de Mendez, I., Adams, A. G., and Leto, T. L. (1997). p40(phox) down-regulates NADPH oxidase activity through interactions with its SH3 domain. J Biol Chem 272, 9141-9146. Sato, S., Fujita, N., and Tsuruo, T. (2000). Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci U S A 97, 10832-10837. Saunders, J. A., Rogers, L. C., Klomsiri, C., Poole, L. B., and Daniel, L. W. (2010). Reactive oxygen species mediate lysophosphatidic acid induced signaling in ovarian cancer cells. Free Radic Biol Med Sayre, L. M., Smith, M. A., and Perry, G. (2001). Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr Med Chem 8, 721-738. 223 Scaife, R. M. (2005). Selective and irreversible cell cycle inhibition by diphenyleneiodonium. Mol Cancer Ther 4, 876-884. Scheid, M. P., Marignani, P. A., and Woodgett, J. R. (2002). Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B. Mol Cell Biol 22, 6247-6260. Schreiner, C. E., Kumerz, M., Gesslbauer, J., Schachner, D., Joa, H., Erker, T., Atanasov, A. G., Heiss, E. H., and Dirsch, V. M. (2010). Resveratrol blocks Akt activation in angiotensin II or EGF-stimulated vascular smooth muscle cells redoxindependently. Cardiovasc Res Schröder, K., Helmcke, I., Palfi, K., Krause, K. H., Busse, R., and Brandes, R. P. (2007). Nox1 mediates basic fibroblast growth factor-induced migration of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 27, 1736-1743. Schröder, K., Wandzioch, K., Helmcke, I., and Brandes, R. P. (2009). Nox4 acts as a switch between differentiation and proliferation in preadipocytes. Arterioscler Thromb Vasc Biol 29, 239-245. Schönthal, A. H. (1998). Role of PP2A in intracellular signal transduction pathways. Front Biosci 3, D1262-D1273. Seo, J. H., Ahn, Y., Lee, S. R., Yeol Yeo, C., and Chung Hur, K. (2005). The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI3 kinase) in the PI-3 kinase/Akt pathway. Mol Biol Cell 16, 348-357. Serrander, L., Cartier, L., Bedard, K., Banfi, B., Lardy, B., Plastre, O., Sienkiewicz, A., Fórró, L., Schlegel, W., and Krause, K. H. (2007). NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation. Biochem J 406, 105-114. Shay, K. P., and Hagen, T. M. (2009). Age-associated impairment of Akt phosphorylation in primary rat hepatocytes is remediated by alpha-lipoic acid through PI3 kinase, PTEN, and PP2A. Biogerontology 10, 443-456. Shi, J., Vlamis-Gardikas, A., Aslund, F., Holmgren, A., and Rosen, B. P. (1999). Reactivity of glutaredoxins 1, 2, and from Escherichia coli shows that glutaredoxin is the primary hydrogen donor to ArsC-catalyzed arsenate reduction. J Biol Chem 274, 36039-36042. Shiose, A., Kuroda, J., Tsuruya, K., Hirai, M., Hirakata, H., Naito, S., Hattori, M., Sakaki, Y., and Sumimoto, H. (2001). A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem 276, 1417-1423. Sies, H. (1997). Oxidative stress: oxidants and antioxidants. Exp Physiol 82, 291-295. Simpson, L., and Parsons, R. (2001). PTEN: life as a tumor suppressor. Exp Cell Res 264, 29-41. 224 Slepkov, E. R., Rainey, J. K., Sykes, B. D., and Fliegel, L. (2007). Structural and functional analysis of the Na+/H+ exchanger. Biochem J 401, 623-633. Snabaitis, A. K., Cuello, F., and Avkiran, M. (2008). Protein kinase B/Akt phosphorylates and inhibits the cardiac Na+/H+ exchanger NHE1. Circ Res 103, 881890. Sparks, C. A., and Guertin, D. A. (2010). Targeting mTOR: prospects for mTOR complex inhibitors in cancer therapy. Oncogene 29, 3733-3744. Staal, S. P. (1987). Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci U S A 84, 5034-5037. Staal, S. P., Hartley, J. W., and Rowe, W. P. (1977). Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma. Proc Natl Acad Sci U S A 74, 3065-3067. Stambolic, V., Suzuki, A., de la Pompa, J. L., Brothers, G. M., Mirtsos, C., Sasaki, T., Ruland, J., Penninger, J. M., Siderovski, D. P., and Mak, T. W. (1998). Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29-39. Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K., Lin, H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T., and Davis, T., et al. (1997). Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 15, 356-362. Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G. F., Holmes, A. B., Gaffney, P. R., Reese, C. B., McCormick, F., and Tempst, P., et al. (1998). Protein kinase B kinases that mediate phosphatidylinositol 3,4,5trisphosphate-dependent activation of protein kinase B. Science 279, 710-714. Storz, P. (2005). Reactive oxygen species in tumor progression. Front Biosci 10, 1881-1896. Stuehr, D. J., Fasehun, O. A., Kwon, N. S., Gross, S. S., Gonzalez, J. A., Levi, R., and Nathan, C. F. (1991). Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J 5, 98-103. Sturrock, A., Huecksteadt, T. P., Norman, K., Sanders, K., Murphy, T. M., Chitano, P., Wilson, K., Hoidal, J. R., and Kennedy, T. P. (2007). Nox4 mediates TGF-beta1induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 292, L1543L1555. Suh, Y. A., Arnold, R. S., Lassegue, B., Shi, J., Xu, X., Sorescu, D., Chung, A. B., Griendling, K. K., and Lambeth, J. D. (1999). Cell transformation by the superoxidegenerating oxidase Mox1. Nature 401, 79-82. 225 Sundaresan, M., Yu, Z. X., Ferrans, V. J., Irani, K., and Finkel, T. (1995). Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270, 296-299. Svegliati S., Cancello R., Sambo P., Luchetti M., Paroncini P., Orlandini G., Discepoli G., Paterno R., Santillo M., Cuozzo C., Cassano S., Avvedimento E.V., Gabrielli A. (2005). Platelet-derived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts. J Biol Chem. 280, 36474-82. Tabet, F., Schiffrin, E. L., Callera, G. E., He, Y., Yao, G., Ostman, A., Kappert, K., Tonks, N. K., and Touyz, R. M. (2008). Redox-sensitive signaling by angiotensin II involves oxidative inactivation and blunted phosphorylation of protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells from SHR. Circ Res 103, 149158. Tamura, M., Shiozaki, I., Ono, S., Miyano, K., Kunihiro, S., and Sasaki, T. (2007). p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox. FEBS Lett 581, 4533-4538. Taylor, V., Wong, M., Brandts, C., Reilly, L., Dean, N. M., Cowsert, L. M., Moodie, S., and Stokoe, D. (2000). 5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells. Mol Cell Biol 20, 6860-6871. Testa, J. R., and Tsichlis, P. N. (2005). AKT signaling in normal and malignant cells. Oncogene 24, 7391-7393. Tew, D. G. (1993). Inhibition of cytochrome P450 reductase by the diphenyliodonium cation. Kinetic analysis and covalent modifications. Biochemistry 32, 10209-10215. Thannickal, V. J., Day, R. M., Klinz, S. G., Bastien, M. C., Larios, J. M., and Fanburg, B. L. (2000). Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1. FASEB J 14, 1741-1748. Toker, A., and Newton, A. C. (2000). Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site. J Biol Chem 275, 8271-8274. Tokunaga, E., Oki, E., Egashira, A., Sadanaga, N., Morita, M., Kakeji, Y., and Maehara, Y. (2008). Deregulation of the Akt pathway in human cancer. Curr Cancer Drug Targets 8, 27-36. Toyokuni, S., Okamoto, K., Yodoi, J., and Hiai, H. (1995). Persistent oxidative stress in cancer. FEBS Lett 358, 1-3. Ueyama, T., Tatsuno, T., Kawasaki, T., Tsujibe, S., Shirai, Y., Sumimoto, H., Leto, T. L., and Saito, N. (2007). A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox. Mol Biol Cell 18, 441-454. Ushio-Fukai, M., Alexander, R. W., Akers, M., and Griendling, K. K. (1998). p38 226 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem 273, 15022-15029. Ushio-Fukai, M., Alexander, R. W., Akers, M., Yin, Q., Fujio, Y., Walsh, K., and Griendling, K. K. (1999). Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem 274, 22699-22704. Ushio-Fukai, M., Zafari, A. M., Fukui, T., Ishizaka, N., and Griendling, K. K. (1996). p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem 271, 23317-23321. Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., and Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39, 44-84. Vanhaesebroeck B., Guillermet-Guibert J., Graupera M., Bilanges B.(2010). The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol. 11, 329-41. Van Kanegan, M. J., Adams, D. G., Wadzinski, B. E., and Strack, S. (2005). Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt. J Biol Chem 280, 36029-36036. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269, 5241-5248. Wagner, B., Ricono, J. M., Gorin, Y., Block, K., Arar, M., Riley, D., Choudhury, G. G., and Abboud, H. E. (2007). Mitogenic signaling via platelet-derived growth factor beta in metanephric mesenchymal cells. J Am Soc Nephrol 18, 2903-2911. Wang, Y., and Lou, M. F. (2009). The regulation of NADPH oxidase and its association with cell proliferation in human lens epithelial cells. Invest Ophthalmol Vis Sci 50, 2291-2300. Weigert, C., Hennige, A. M., Brodbeck, K., Häring, H. U., and Schleicher, E. D. (2005). Interleukin-6 acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by phosphorylation of Ser473 of Akt. Am J Physiol Endocrinol Metab 289, E251-E257. Weydert, C. J., Waugh, T. A., Ritchie, J. M., Iyer, K. S., Smith, J. L., Li, L., Spitz, D. R., and Oberley, L. W. (2006). Overexpression of manganese or copper-zinc superoxide dismutase inhibits breast cancer growth. Free Radic Biol Med 41, 226-237. Williams, M. R., Arthur, J. S., Balendran, A., van der Kaay, J., Poli, V., Cohen, P., and Alessi, D. R. (2000). The role of 3-phosphoinositide-dependent protein kinase in activating AGC kinases defined in embryonic stem cells. Curr Biol 10, 439-448. 227 Wu, K. L., Khan, S., Lakhe-Reddy, S., Jarad, G., Mukherjee, A., Obejero-Paz, C. A., Konieczkowski, M., Sedor, J. R., and Schelling, J. R. (2004). The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival. J Biol Chem 279, 26280-26286. Wu, M., Katta, A., Gadde, M. K., Liu, H., Kakarla, S. K., Fannin, J., Paturi, S., Arvapalli, R. K., Rice, K. M., and Wang, Y., et al. (2009). Aging-associated dysfunction of Akt/protein kinase B: S-nitrosylation and acetaminophen intervention. PloS one 4, e6430. Wu, S., Gao, J., Ohlemeyer, C., Roos, D., Niessen, H., Köttgen, E., and Gessner, R. (2005). Activation of AP-1 through reactive oxygen species by angiotensin II in rat cardiomyocytes. Free Radic Biol Med 39, 1601-1610. Wu, Y., Zu, K., Warren, M. A., Wallace, P. K., and Ip, C. (2006). Delineating the mechanism by which selenium deactivates Akt in prostate cancer cells. Mol Cancer Ther 5, 246-252. Xue, X., Piao, J. H., Nakajima, A., Sakon-Komazawa, S., Kojima, Y., Mori, K., Yagita, H., Okumura, K., Harding, H., and Nakano, H. (2005). Tumor necrosis factor alpha (TNFalpha) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha. J Biol Chem 280, 33917-33925. Yamaura, M., Mitsushita, J., Furuta, S., Kiniwa, Y., Ashida, A., Goto, Y., Shang, W. H., Kubodera, M., Kato, M., and Takata, M., et al. (2009). NADPH oxidase contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression. Cancer Res 69, 2647-2654. Yang, J. Q., Buettner, G. R., Domann, F. E., Li, Q., Engelhardt, J. F., Weydert, C. D., and Oberley, L. W. (2002). v-Ha-ras mitogenic signaling through superoxide and derived reactive oxygen species. Mol Carcinog 33, 206-218. Yang, Q., Inoki, K., Ikenoue, T., and Guan, K. L. (2006). Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes Dev 20, 2820-2832. Yasukawa, T., Tokunaga, E., Ota, H., Sugita, H., Martyn, J. A. J., and Kaneki, M. (2005). S-nitrosylation-dependent inactivation of Akt/protein kinase B in insulin resistance. J Biol Chem 280, 7511-7518. Yin, C. C., Lin, T. K., and Huang, K. T. (2007). Superoxide counteracts low-density lipoprotein-induced human aortic smooth muscle cell proliferation. J Biosci Bioeng 104, 157-162. Yin, K. J., Hsu, C. Y., Hu, X. Y., Chen, H., Chen, S. W., Xu, J., and Lee, J. M. (2006). Protein phosphatase 2A regulates bim expression via the Akt/FKHRL1 signaling pathway in amyloid-beta peptide-induced cerebrovascular endothelial cell death. J Neurosci 26, 2290-2299. 228 Yu, C. X., Li, S., and Whorton, A. R. (2005). Redox regulation of PTEN by Snitrosothiols. Mol Pharmacol 68, 847-854. Zhang, B., and Xia, C. (2009). The expression of protein kinase B in gastric cancer cell apoptosis induced by 12-O-tetradecanoylphorbol-1, 3-acetate. Cell Mol Biol Lett 14, 466-480. Zhang, J., Liu, Z., Rasschaert, J., Blero, D., Deneubourg, L., Schurmans, S., Erneux, C., and Pesesse, X. (2007). SHIP2 controls PtdIns(3,4,5)P(3) levels and PKB activity in response to oxidative stress. Cell Signal 19, 2194-2200. Zheng, M., Aslund, F., and Storz, G. (1998). Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279, 1718-1721. 229 PUBLICATION AND PRESENTATION Publication Luo Le and Marie-Véronique Clément Redox control of cytosolic Akt phosphorylation is required for the maintenance of the hyper-activation of the kinase in PTEN null cells. (In preparation) Poster presentation Luo Le and Marie-Véronique Clément Increase in the intracellular level of superoxide: a new pathway involved in the maintenance of AKT phosphorylation in absence of growth factor in PTEN knock-out mouse embryonic fibroblasts. Poster presented at the 1st Biochemistry Student Symposium, held at the Clinical Research Centre, National University of Singapore (2008). Won the best poster award for poster presentation. Luo Le and Marie-Véronique Clément Regulation of AKT phosphorylation by diphenyleneiodonium (DPI) in the PTEN-/MEFs. Poster presented at The XIV Biennial Meeting of the Society for Free Radical Research International (14th SFRR), held at Beijing, China (2008). Luo Le and Marie-Véronique Clément Redox regulation of AKT phosphorylation in mouse embryonic fibroblasts in absence of PTEN. Poster presented at The Society for Free Radical Biology and Medicine's (SFRBM) 16th Annual Meeting, held at San Francisco, CA, USA (2009). Oral presentation Luo Le and Marie-Véronique Clément Regulation of Akt phosphorylation in PTEN knock-out mouse embryonic fibroblasts. Presented at Department of Biochemistry “Research in Progress” Seminar for postgraduates. National University of Singapore (2009). 230 [...]... between the three isoforms is more than 75% as well (76% between Akt2 and Akt3 ; 82% between Akt1 and Akt2 or Akt1 and Akt3 ) All three isoforms of Akt contain three important domains, the N-terminal pleckstrin homology (PH) domain, the kinase domain and the C-terminal hydrophobic motif The PH domain is involved in the interaction with the membrane D3-phosphorylated phosphoinositides For Akt, interaction with... Chapter 1: Introduction 18 1.3.1 Structure There are three Akt isoforms in mammals, Akt1 , Akt 2 and Akt3 (or PKBα, PKBβ, and PKBγ) The sequence similarity between rat, mouse and human is greater than 95% for all the three isoforms In humans, Akt1 , Akt2 and Akt3 share a sequence similarity of more than 75% (77% between Akt2 and Akt3 ; 81% between Akt1 and Akt2 ; 82% between Akt1 and Akt3 ) In mice, the... to Akt dephosphorylation 152 xi Figure 44: NHE1 expression but not activity is involved in regulation of Akt phosphorylation 154 Figure 45: siNHE1 attenuates the effect of FBS stimulation on Akt phosphorylation 155 Figure 46: NHE1 -Akt interaction in MEFPTEN-/- cells 158 Figure 47: Akt dephosphorylation by LY294002 is delayed in MEFPTEN-/- cells OverexpressingNHE1... 1.3 Akt Akt (also known as protein kinase B) is a serine/threonine kinase with large varieties of substrates It was first identified as an oncogene within the transforming retrovirus, AKT8 The AKT8 murine retrovirus was isolated from an AKR thymoma cell line in 1977 (Staal et al., 1977) Ten years later, Staal successfully cloned the akt oncogene and the human homologues of the akt gene, AKT1 and AKT2 ... inhibited by wortmannin (Bellacosa et al., 1998) However, the PH domain mutant of Akt did not translocate to the membrane following PDGF stimulation Moreover, mutations in the predicted phospholipid binding residues resulted in a decrease in the kinase activity of Akt (Bellacosa et al., 1998) Membrane localization of Akt was reported to be essential for the activation of the Akt kinase Chapter 1: Introduction... EGF, insulin and bFGF, was abolished by the PI3K inhibitor wortmannin Expression of mutated PDGF receptor or mutated p85 subunit of PI3K provided further evidence for the essential role of PI3K in Akt activation (Burgering and Coffer, 1995) Additionally, Akt constructs with mutations in the PH domain failed to respond to PDGF stimulation, indicating a critical role for the PH domain in PDGF-mediated Akt. .. dependent Akt activation induced by Angiotensin II or arachidonic acid (AA) (Gorin et al., 2003) Regulation of Akt activation by Nox4 was further evidenced in unstimulated pancreatic cancer PANC-1 cells, in which Chapter 1: Introduction 17 suppression of ROS production by siRNA-mediated Nox4 downregulation resulted in a reduction in Akt phosphorylation (Mochizuki et al., 2006) A more general mode of Akt regulation. .. the AGC kinases and is required for their full activation (Hanada et al., 2004) 1.3.2 Activation process of Akt Full activation of Akt requires phosphorylation at two important sites, the threonine308 (Thr308) residue in the activation loop and the serine473 (Ser473) residue in the hydrophobic motif in Akt1 The corresponding phosphorylation sites are Thr309/Ser474 in Akt2 and Thr305/Ser472 in Akt3 Throughout... Nox4 in MEF cells .86 Figure 14: Intracellular level of O2.- is reduced by siNox4 88 Figure 15: Reduction of intracellular O2.- level by siNox4 results in Akt dephosphorylation 91 Figure 16: Cell cycle analysis after siNox4 92 Figure 17: Simplified diagram illustrating the two direction regulation of Akt phosphorylation 93 Figure 18: Confocal analysis of cellular... pathways of Akt phosphorylation 195 Figure C: Topology of NHE-1 and its regulatory elements .198 Figure D: A summary of the key findings in this project in the context of Akt activation process .202 xii LIST OF ABBREVIATIONS Akt Protein kinase B AMS 4-Acetamido-4-maleimidylstilbene-2,2-disulfonic acid Ang II Angiotensin II bFGF basic fibroblast growth factor CA Calyculin A DDC . Akt interacting proteins 29 1.4 Redox regulation of Akt 30 1.4.1 PI3K related redox regulation of Akt 30 1.4.2 Akt as the direct target for redox regulation 34 1.5 Rationale of the project. critical for the maintenance of the hyperphosphorylated Akt observed in MEF PTEN- /- cells. In the current project, the PTEN- independent pathways involved in the regulation of Akt phosphorylation. reduction of intracellular level of O 2 in MEF PTEN- /- cells results in a decrease in the phosphorylation level of the otherwise hyperphosphorylated Akt kinase. In investigating how O 2