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Reversible inactivation of the 90 kDa ribosomal S6 kinase by H2O2
Abstracts / Nitric Oxide 27 (2012) S2–S50
products. We found that cupric ions can efficiently catalyze tyrosine nitration in the presence of nitric oxide (NO), oxygen and H2O2, or in the presence of nitrite and H2O2. With mechanism study, we showed that cupric ions and peptide-cupric ion complexes are excellent Fenton-catalysts, even better than Fe (III) or heme, for the formation of hydroxyl radicals (OH) and/or copper (II)-bound OH from hydrogen peroxide. These radicals were efficiently scavenged by tyrosine to form tyrosine radicals and by nitrite to form NO2, leading to tyrosine nitration in polypeptides. This study shows that copper has a polyvalent role in the processes of tyrosine nitration.
References [1] L. Qiao et al. JACS (2011) 19823–19831.
S17
may be regulated by reversible reduction/oxidation involving cellular oxidants such as hydrogen peroxide (H2O2). Here, we show that oxidation of RSK1 results in a reversible inability to phosphorylate S6 peptide (KRRRLASLR) and nNOS (neuronal nitric oxide synthase) at Ser847 in vitro. A number of protein kinases are regulated by a redox-sensitive thiol modification within their kinase domains. RSK1 has two catalytic domains, in that Cys223 is in the N-terminal kinase domain and Cys575 is in the C-terminal kinase domain. Mutated RSK1 (C223S), but not C575S was refractory to oxidation-induced inability of enzyme activity. Furthermore, dithiothreitol was able to enhance the activity of wild type and C575S RSK1 but not of the C223S mutant. This is suggested that oxidation of the enzyme at Cys223 readily occurs during the assay procedure when the reducing agent is absent. We propose that oxidation stress might regulate cellular function via a decreased ability of RSK1 through Cys223 oxidation.
http://dx.doi.org/10.1016/j.niox.2012.04.059 http://dx.doi.org/10.1016/j.niox.2012.04.061
P-2
P-4
Reversible inactivation of Ca2+/calmodulin-dependent protein kinase IV by oxidation of the active-site cysteine residue Yukihiro Tsuchiya, Jun Kimura, Yasuo Watanabe Department of Pharmacology, Showa Pharmaceutical University, Tokyo, Japan
Chronic inhibition of nitric oxide synthase activity by NG-nitro-Larginine induces nitric oxide synthase expression in the developing rat cerebellum Zoltán Serfozo a, Beáta Lontay b, Zoltán Kukor c, Ferenc Erdodi b a Department of Experimental Zoology, Centre for Ecological Research, Balaton Limnological Research Institute, Hungarian Academy of Sciences, Tihany, Hungary, b Department of Medical Chemistry, Cell Signaling Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary, c Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is serine/ threonine-specific protein kinase that is involved in many signaling cascades, and plays a key role in memory. CaMKIV is activated via binding to Ca2+/CaM complex and phosphorylation by upstream kinase, CaM kinase. In the present study, we investigate direct mechanism of inactivation of CaMKIV by oxidative stress. We show that CaMKIV is directly inhibited by its sulfenic acid formation at the Cys198. In situ studies demonstrated that treatment of CaMKIV with hydrogen peroxide (H2O2) results in inactivation of the enzyme, with a concomitant sulfenic acid formation of CaMKIV at Cys198. The methodology of detection of sulfenic acid formation is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Mutagenesis studies confirmed that sulfenic acid formation of Cys198 is both necessary and sufficient for the inhibition of CaMKIV by H2O2. In transfected cells expressing CaMKIV, treatment with H2O2 caused a reversible decrease in CaMKIV activity. Cells expressing mutant CaMKIV (C198V) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKIV via its sulfenic acid formation at Cys198 is an important mechanism in processing calcium signal transduction in cells.
http://dx.doi.org/10.1016/j.niox.2012.04.060
P-3 Reversible inactivation of the 90 kDa ribosomal S6 kinase by H2O2 Tsuyoshi Takata, Yukihiro Tsuchiya, Yasuo Watanabe Laboratory of Pharmacology, Showa Pharmaceutical University, Machida, Japan Many intracellular signaling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. The 90-kDa ribosomal S6 kinase (RSK) is a family of serine/threonine kinases and is involved in cell survival. This study explores the proposal that RSK1
Studies on chronic inhibition of NO synthase (NOS) in the CNS suggest a plastic change in NO synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor NG-nitro-L-arginine (L-NNA) was examined on NO production, NOsensitive cGMP levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5–200 mg/kg) to 21-day old rats reduced NOS activity and NO concentration dose dependently by 70–90% and the tissue cGMP level by 60–80%. By contrast, chronic application of L-NNA between postnatal days 4–21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10–50%. Chronic treatment of 10 mg/kg L-NNA induced nNOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50 mg/kg) of L-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced iNOS expression and activity in glial cells. Treatments did not affect eNOS expression. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced chronic inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.
http://dx.doi.org/10.1016/j.niox.2012.04.062
products. We found that cupric ions can efficiently catalyze tyrosine nitration in the presence of nitric oxide (NO), oxygen and H2O2, or in the presence of nitrite and H2O2. With mechanism study, we showed that cupric ions and peptide-cupric ion complexes are excellent Fenton-catalysts, even better than Fe (III) or heme, for the formation of hydroxyl radicals (OH) and/or copper (II)-bound OH from hydrogen peroxide. These radicals were efficiently scavenged by tyrosine to form tyrosine radicals and by nitrite to form NO2, leading to tyrosine nitration in polypeptides. This study shows that copper has a polyvalent role in the processes of tyrosine nitration.
References [1] L. Qiao et al. JACS (2011) 19823–19831.
S17
may be regulated by reversible reduction/oxidation involving cellular oxidants such as hydrogen peroxide (H2O2). Here, we show that oxidation of RSK1 results in a reversible inability to phosphorylate S6 peptide (KRRRLASLR) and nNOS (neuronal nitric oxide synthase) at Ser847 in vitro. A number of protein kinases are regulated by a redox-sensitive thiol modification within their kinase domains. RSK1 has two catalytic domains, in that Cys223 is in the N-terminal kinase domain and Cys575 is in the C-terminal kinase domain. Mutated RSK1 (C223S), but not C575S was refractory to oxidation-induced inability of enzyme activity. Furthermore, dithiothreitol was able to enhance the activity of wild type and C575S RSK1 but not of the C223S mutant. This is suggested that oxidation of the enzyme at Cys223 readily occurs during the assay procedure when the reducing agent is absent. We propose that oxidation stress might regulate cellular function via a decreased ability of RSK1 through Cys223 oxidation.
http://dx.doi.org/10.1016/j.niox.2012.04.059 http://dx.doi.org/10.1016/j.niox.2012.04.061
P-2
P-4
Reversible inactivation of Ca2+/calmodulin-dependent protein kinase IV by oxidation of the active-site cysteine residue Yukihiro Tsuchiya, Jun Kimura, Yasuo Watanabe Department of Pharmacology, Showa Pharmaceutical University, Tokyo, Japan
Chronic inhibition of nitric oxide synthase activity by NG-nitro-Larginine induces nitric oxide synthase expression in the developing rat cerebellum Zoltán Serfozo a, Beáta Lontay b, Zoltán Kukor c, Ferenc Erdodi b a Department of Experimental Zoology, Centre for Ecological Research, Balaton Limnological Research Institute, Hungarian Academy of Sciences, Tihany, Hungary, b Department of Medical Chemistry, Cell Signaling Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary, c Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is serine/ threonine-specific protein kinase that is involved in many signaling cascades, and plays a key role in memory. CaMKIV is activated via binding to Ca2+/CaM complex and phosphorylation by upstream kinase, CaM kinase. In the present study, we investigate direct mechanism of inactivation of CaMKIV by oxidative stress. We show that CaMKIV is directly inhibited by its sulfenic acid formation at the Cys198. In situ studies demonstrated that treatment of CaMKIV with hydrogen peroxide (H2O2) results in inactivation of the enzyme, with a concomitant sulfenic acid formation of CaMKIV at Cys198. The methodology of detection of sulfenic acid formation is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Mutagenesis studies confirmed that sulfenic acid formation of Cys198 is both necessary and sufficient for the inhibition of CaMKIV by H2O2. In transfected cells expressing CaMKIV, treatment with H2O2 caused a reversible decrease in CaMKIV activity. Cells expressing mutant CaMKIV (C198V) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKIV via its sulfenic acid formation at Cys198 is an important mechanism in processing calcium signal transduction in cells.
http://dx.doi.org/10.1016/j.niox.2012.04.060
P-3 Reversible inactivation of the 90 kDa ribosomal S6 kinase by H2O2 Tsuyoshi Takata, Yukihiro Tsuchiya, Yasuo Watanabe Laboratory of Pharmacology, Showa Pharmaceutical University, Machida, Japan Many intracellular signaling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. The 90-kDa ribosomal S6 kinase (RSK) is a family of serine/threonine kinases and is involved in cell survival. This study explores the proposal that RSK1
Studies on chronic inhibition of NO synthase (NOS) in the CNS suggest a plastic change in NO synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor NG-nitro-L-arginine (L-NNA) was examined on NO production, NOsensitive cGMP levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5–200 mg/kg) to 21-day old rats reduced NOS activity and NO concentration dose dependently by 70–90% and the tissue cGMP level by 60–80%. By contrast, chronic application of L-NNA between postnatal days 4–21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10–50%. Chronic treatment of 10 mg/kg L-NNA induced nNOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50 mg/kg) of L-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced iNOS expression and activity in glial cells. Treatments did not affect eNOS expression. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced chronic inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.
http://dx.doi.org/10.1016/j.niox.2012.04.062