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Title: S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

Abstract

It is well known that the reactive oxygen species, nitric oxide (NO), can trigger cell death in plants, but the underlying molecular mechanisms are not well understood. Here, we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicon) through inhibiting the phosphoinositide-dependent kinase 1 (PDK1) kinase activity via S-nitrosylation. Biotin-switch assays and LC-MS/MS analyses demonstrated that SlPDK1 was a target of S-nitrosylation modification, which primarily occurred on the cysteine residue at position 128 (Cys128). Accordingly, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione (GSNO) both in vitro and in vivo in a concentration-dependent manner, indicating that SlPDK1 activity is regulated by S-nitrosylation. The inhibition of SlPDK1 kinase activity by GSNO was reversible in the presence of a reducing agent but synergistically enhanced by hydrogen peroxide (H2O2). Mutation of Cys128 to serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for the inhibition of the kinase activity of SlPDK1. In sum, our results established a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1, a conserved negative regulator of cell death in yeasts, mammals and plants. Nitric oxide (NO) potentiates the induction of hypersensitive cell death inmore » soybean cells by reactive oxygen species (ROS) (1). However, the molecular mechanism of the NO-induced cell death remains an enigma. One potential mechanism is that the activity of proteins that control cell death may be altered by a post-translational modification, S-nitrosylation. S-nitrosylation is the addition of the NO moiety to thiol groups, including cysteine (Cys) residues in proteins, to form S-nitrosothiols (SNOs). S-nitrosylation is an enzyme-independent post-translational and labile modification that can function as an on/off switch of protein activity (2- 4). Thousands of diverse classes of proteins, both in plants and in mammals, have been identified as targets of S-nitrosylation (5-9). In plants, proteins with diverse functions are S-nitrosylated at specific Cys residue(s) and their functions are either inhibited or enhanced by this modification (10-25). 3-Phosphoinositide-dependent protein kinase-1 (PDK1) and its downstream target, protein kinase B (PKB; also known as Akt), are central regulators of mammalian apoptosis (26-28). PKB is a member of the AGC family of protein kinases, which is activated by second messengers such as phospholipids and Ca2+ through PDK1. Mammalian PDK1 phosphorylates PKB to promote its function in suppressing programmed cell death (PCD) (27-30). PKB negatively regulates apoptosis by phosphorylation and inactivation of pro-apoptotic factors such as BAD and activation of anti-apoptotic factors such as CREB and IKK (27-29; and 31). Deficiency of the PDK1 gene(s) in Drosophila (32), mice (33), yeast (34-35) and tomato (36), respectively, results in lethality or severe apoptosis. PKB knockout mice display spontaneous apoptosis in several different tissues (37). In tomato, the PKB/Akt homolog, Adi3 (AvrPto-dependent Pto-interacting protein 3), physically interacts with and is phosphorylated by SlPDK1 (36). Silencing both SlPDK1 and Adi3 or treatment with a PDK1 inhibitor results in MAPKKK -dependent cell death, indicating that Adi3 functions analogously to the mammalian PKB/Akt by negatively regulating cell death via PDK1 phosphorylation (36). Yasukawa et al (38) showed that NO donors induced S-nitrosylation and inactivation of Akt/PKB kinase activity in vitro and in vivo and the mutant Akt1/PKB (C224S) was resistant to S-nitrosylation by NO and its kinase inactivation (38). Although the NO and PDK1-PKB/Akt pathways are both key regulators of cell death, the link between these two pathways has not been firmly established in plants. Here we show that the kinase activity of tomato SlPDK1 was inhibited by GSNO in a conce« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1430439
Report Number(s):
PNNL-SA-129673
Journal ID: ISSN 0021-9258; KP1501021
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 292; Journal Issue: 48; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English

Citation Formats

Liu, Jian-Zhong, Duan, Jicheng, Ni, Min, Liu, Zhen, Qiu, Wen-Li, Whitham, Steven A., and Qian, Wei-Jun. S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1). United States: N. p., 2017. Web. doi:10.1074/jbc.M117.803882.
Liu, Jian-Zhong, Duan, Jicheng, Ni, Min, Liu, Zhen, Qiu, Wen-Li, Whitham, Steven A., & Qian, Wei-Jun. S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1). United States. doi:10.1074/jbc.M117.803882.
Liu, Jian-Zhong, Duan, Jicheng, Ni, Min, Liu, Zhen, Qiu, Wen-Li, Whitham, Steven A., and Qian, Wei-Jun. Fri . "S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)". United States. doi:10.1074/jbc.M117.803882.
@article{osti_1430439,
title = {S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)},
author = {Liu, Jian-Zhong and Duan, Jicheng and Ni, Min and Liu, Zhen and Qiu, Wen-Li and Whitham, Steven A. and Qian, Wei-Jun},
abstractNote = {It is well known that the reactive oxygen species, nitric oxide (NO), can trigger cell death in plants, but the underlying molecular mechanisms are not well understood. Here, we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicon) through inhibiting the phosphoinositide-dependent kinase 1 (PDK1) kinase activity via S-nitrosylation. Biotin-switch assays and LC-MS/MS analyses demonstrated that SlPDK1 was a target of S-nitrosylation modification, which primarily occurred on the cysteine residue at position 128 (Cys128). Accordingly, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione (GSNO) both in vitro and in vivo in a concentration-dependent manner, indicating that SlPDK1 activity is regulated by S-nitrosylation. The inhibition of SlPDK1 kinase activity by GSNO was reversible in the presence of a reducing agent but synergistically enhanced by hydrogen peroxide (H2O2). Mutation of Cys128 to serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for the inhibition of the kinase activity of SlPDK1. In sum, our results established a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1, a conserved negative regulator of cell death in yeasts, mammals and plants. Nitric oxide (NO) potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen species (ROS) (1). However, the molecular mechanism of the NO-induced cell death remains an enigma. One potential mechanism is that the activity of proteins that control cell death may be altered by a post-translational modification, S-nitrosylation. S-nitrosylation is the addition of the NO moiety to thiol groups, including cysteine (Cys) residues in proteins, to form S-nitrosothiols (SNOs). S-nitrosylation is an enzyme-independent post-translational and labile modification that can function as an on/off switch of protein activity (2- 4). Thousands of diverse classes of proteins, both in plants and in mammals, have been identified as targets of S-nitrosylation (5-9). In plants, proteins with diverse functions are S-nitrosylated at specific Cys residue(s) and their functions are either inhibited or enhanced by this modification (10-25). 3-Phosphoinositide-dependent protein kinase-1 (PDK1) and its downstream target, protein kinase B (PKB; also known as Akt), are central regulators of mammalian apoptosis (26-28). PKB is a member of the AGC family of protein kinases, which is activated by second messengers such as phospholipids and Ca2+ through PDK1. Mammalian PDK1 phosphorylates PKB to promote its function in suppressing programmed cell death (PCD) (27-30). PKB negatively regulates apoptosis by phosphorylation and inactivation of pro-apoptotic factors such as BAD and activation of anti-apoptotic factors such as CREB and IKK (27-29; and 31). Deficiency of the PDK1 gene(s) in Drosophila (32), mice (33), yeast (34-35) and tomato (36), respectively, results in lethality or severe apoptosis. PKB knockout mice display spontaneous apoptosis in several different tissues (37). In tomato, the PKB/Akt homolog, Adi3 (AvrPto-dependent Pto-interacting protein 3), physically interacts with and is phosphorylated by SlPDK1 (36). Silencing both SlPDK1 and Adi3 or treatment with a PDK1 inhibitor results in MAPKKK -dependent cell death, indicating that Adi3 functions analogously to the mammalian PKB/Akt by negatively regulating cell death via PDK1 phosphorylation (36). Yasukawa et al (38) showed that NO donors induced S-nitrosylation and inactivation of Akt/PKB kinase activity in vitro and in vivo and the mutant Akt1/PKB (C224S) was resistant to S-nitrosylation by NO and its kinase inactivation (38). Although the NO and PDK1-PKB/Akt pathways are both key regulators of cell death, the link between these two pathways has not been firmly established in plants. Here we show that the kinase activity of tomato SlPDK1 was inhibited by GSNO in a conce},
doi = {10.1074/jbc.M117.803882},
journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 48,
volume = 292,
place = {United States},
year = {2017},
month = {9}
}

Works referenced in this record:

S-nitrosylation triggers ABI5 degradation to promote seed germination and seedling growth
journal, October 2015

  • Albertos, Pablo; Romero-Puertas, María C.; Tatematsu, Kiyoshi
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms9669

Unbiased identification of cysteine S-nitrosylation sites on proteins
journal, June 2007

  • Derakhshan, Behrad; Wille, Pamela C.; Gross, Steven S.
  • Nature Protocols, Vol. 2, Issue 7
  • DOI: 10.1038/nprot.2007.210

dbSNO: a database of cysteine S-nitrosylation
journal, July 2012


Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1
journal, December 2014

  • Wang, Pengcheng; Du, Yanyan; Hou, Yueh-Ju
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 2
  • DOI: 10.1073/pnas.1423481112

S -Nitrosylation Positively Regulates Ascorbate Peroxidase Activity during Plant Stress Responses
journal, February 2015


Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines
journal, January 2017

  • Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun
  • Molecular BioSystems, Vol. 13, Issue 5
  • DOI: 10.1039/C6MB00861E

Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain
journal, May 1999


From the Cyclooxygenase-2 Inhibitor Celecoxib to a Novel Class of 3-Phosphoinositide-Dependent Protein Kinase-1 Inhibitors
journal, June 2004


Cross Talk between Reactive Nitrogen and Oxygen Species during the Hypersensitive Disease Resistance Response
journal, June 2006

  • Zaninotto, Federica; Camera, Sylvain La; Polverari, Annalisa
  • Plant Physiology, Vol. 141, Issue 2
  • DOI: 10.1104/pp.106.078857

S-Nitrosothiols: cellular formation and transport
journal, April 2005


The Arabidopsis PARAQUAT RESISTANT2 gene encodes an S-nitrosoglutathione reductase that is a key regulator of cell death
journal, October 2009

  • Chen, Ruiqiang; Sun, Shulan; Wang, Chun
  • Cell Research, Vol. 19, Issue 12
  • DOI: 10.1038/cr.2009.117

Redox Regulation of the NPR1-TGA1 System of Arabidopsis thaliana by Nitric Oxide
journal, August 2010

  • Lindermayr, Christian; Sell, Simone; Müller, Bernd
  • The Plant Cell, Vol. 22, Issue 8
  • DOI: 10.1105/tpc.109.066464

Site-Specific Nitrosoproteomic Identification of Endogenously S -Nitrosylated Proteins in Arabidopsis
journal, February 2015

  • Hu, Jiliang; Huang, Xiahe; Chen, Lichao
  • Plant Physiology, Vol. 167, Issue 4
  • DOI: 10.1104/pp.15.00026

S-Nitrosylation of Peroxiredoxin II E Promotes Peroxynitrite-Mediated Tyrosine Nitration
journal, December 2007

  • Romero-Puertas, Maria C.; Laxa, Miriam; Mattè, Alessandro
  • The Plant Cell, Vol. 19, Issue 12
  • DOI: 10.1105/tpc.107.055061

S-nitrosylation of NADPH oxidase regulates cell death in plant immunity
journal, October 2011

  • Yun, Byung-Wook; Feechan, Angela; Yin, Minghui
  • Nature, Vol. 478, Issue 7368
  • DOI: 10.1038/nature10427

The life of a cell: apoptosis regulation by the PI3K/PKB pathway
journal, October 2008


A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans
journal, March 2001

  • Liu, Limin; Hausladen, Alfred; Zeng, Ming
  • Nature, Vol. 410, Issue 6827
  • DOI: 10.1038/35068596

A central role for S-nitrosothiols in plant disease resistance
journal, May 2005

  • Feechan, A.; Kwon, E.; Yun, B. -W.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 22
  • DOI: 10.1073/pnas.0501456102

Protein S -Nitrosylation: Potential Targets and Roles in Signal Transduction
journal, July 2007


Essential Roles of S-Nitrosothiols in Vascular Homeostasis and Endotoxic Shock
journal, February 2004


Mechanisms and consequences of activation of protein kinase B/Akt
journal, April 1998


Proteomic Identification of S -Nitrosylated Proteins in Arabidopsis
journal, February 2005

  • Lindermayr, Christian; Saalbach, Gerhard; Durner, Jörg
  • Plant Physiology, Vol. 137, Issue 3
  • DOI: 10.1104/pp.104.058719

Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast
journal, February 1999


Redox signaling: Nitrosylation and related target interactions of nitric oxide
journal, September 1994


PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development
journal, March 2013

  • Venigalla, Ram K. C.; McGuire, Victoria A.; Clarke, Rosemary
  • The EMBO Journal, Vol. 32, Issue 7
  • DOI: 10.1038/emboj.2013.40

Virus-induced gene silencing in tomato
journal, September 2002


Metacaspase Activity of Arabidopsis thaliana Is Regulated by S -Nitrosylation of a Critical Cysteine Residue
journal, November 2006

  • Belenghi, Beatrice; Romero-Puertas, Maria C.; Vercammen, Dominique
  • Journal of Biological Chemistry, Vol. 282, Issue 2
  • DOI: 10.1074/jbc.M608931200

SNObase, a database for S-nitrosation modification
journal, November 2012


Nitric Oxide Modulates Histone Acetylation at Stress Genes by Inhibition of Histone Deacetylases
journal, December 2016

  • Mengel, Alexander; Ageeva, Alexandra; Georgii, Elisabeth
  • Plant Physiology, Vol. 173, Issue 2
  • DOI: 10.1104/pp.16.01734

PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway
journal, June 2011

  • Desai, S.; Pillai, P.; Win-Piazza, H.
  • Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1813, Issue 6
  • DOI: 10.1016/j.bbamcr.2011.03.007

S -Nitrosylation of AtSABP3 Antagonizes the Expression of Plant Immunity
journal, November 2008

  • Wang, Yi-Qin; Feechan, Angela; Yun, Byung-Wook
  • Journal of Biological Chemistry, Vol. 284, Issue 4
  • DOI: 10.1074/jbc.M806782200

Nitric Oxide as a Pro-apoptotic as well as Anti-apoptotic Modulator
journal, January 2002


S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding
journal, June 2005

  • Hara, Makoto R.; Agrawal, Nishant; Kim, Sangwon F.
  • Nature Cell Biology, Vol. 7, Issue 7
  • DOI: 10.1038/ncb1268

Regulation by S -Nitrosylation of Protein Post-translational Modification
journal, December 2011

  • Hess, Douglas T.; Stamler, Jonathan S.
  • Journal of Biological Chemistry, Vol. 287, Issue 7
  • DOI: 10.1074/jbc.R111.285742

S-Nitrosylation of Ascorbate Peroxidase Is Part of Programmed Cell Death Signaling in Tobacco Bright Yellow-2 Cells
journal, October 2013


S -Nitrosylation-dependent Inactivation of Akt/Protein Kinase B in Insulin Resistance
journal, January 2005

  • Yasukawa, Takashi; Tokunaga, Eriko; Ota, Hidetaka
  • Journal of Biological Chemistry, Vol. 280, Issue 9
  • DOI: 10.1074/jbc.M411871200

Site-Specific Proteomics Approach for Study Protein S-Nitrosylation
journal, September 2010

  • Liu, Miao; Hou, Jinxuan; Huang, Lin
  • Analytical Chemistry, Vol. 82, Issue 17
  • DOI: 10.1021/ac100569d

SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures
journal, January 2006

  • Hao, G.; Derakhshan, B.; Shi, L.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 4
  • DOI: 10.1073/pnas.0508412103

Adi3 is a Pdk1-interacting AGC kinase that negatively regulates plant cell death
journal, December 2005

  • Devarenne, Timothy P.; Ekengren, Sophia K.; Pedley, Kerry F.
  • The EMBO Journal, Vol. 25, Issue 1
  • DOI: 10.1038/sj.emboj.7600910

Nitric Oxide-Dependent Posttranslational Modification in Plants: An Update
journal, November 2012

  • Astier, Jeremy; Lindermayr, Christian
  • International Journal of Molecular Sciences, Vol. 13, Issue 12
  • DOI: 10.3390/ijms131115193

Multiple Enzymatic Digestion for Enhanced Sequence Coverage of Proteins in Complex Proteomic Mixtures Using Capillary LC with Ion Trap MS/MS
journal, February 2003

  • Choudhary, Gargi; Wu, Shiaw-Lin; Shieh, Paul
  • Journal of Proteome Research, Vol. 2, Issue 1
  • DOI: 10.1021/pr025557n

Plant Immunity Requires Conformational Charges of NPR1 via S-Nitrosylation and Thioredoxins
journal, August 2008


Nitric oxide increases the enzymatic activity of three ascorbate peroxidase isoforms in soybean root nodules
journal, July 2011

  • Keyster, Marshall; Klein, Ashwil; Egbich, Ifeanyi
  • Plant Signaling & Behavior, Vol. 6, Issue 7
  • DOI: 10.4161/psb.6.7.14879

The phosphatidylinositol 3-Kinase–AKT pathway in human cancer
journal, July 2002

  • Vivanco, Igor; Sawyers, Charles L.
  • Nature Reviews Cancer, Vol. 2, Issue 7
  • DOI: 10.1038/nrc839

Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics
journal, March 2010

  • Swaney, Danielle L.; Wenger, Craig D.; Coon, Joshua J.
  • Journal of Proteome Research, Vol. 9, Issue 3
  • DOI: 10.1021/pr900863u

Growth retardation and increased apoptosis in mice with homozygous disruption of the akt1 gene
journal, September 2001


Small changes huge impact: the role of thioredoxin 1 in the regulation of apoptosis by S-nitrosylation
journal, December 2012

  • Li, H.; Wan, A.; Xu, G.
  • Acta Biochimica et Biophysica Sinica, Vol. 45, Issue 3
  • DOI: 10.1093/abbs/gms103

Site-specific detection ofS-nitrosylated PKB α/Akt1 from rat soleus muscle using CapLC-Q-TOFmicro mass spectrometry
journal, September 2005

  • Lu, Xiao-Ming; Lu, Mary; Tompkins, Ronald G.
  • Journal of Mass Spectrometry, Vol. 40, Issue 9
  • DOI: 10.1002/jms.885

Nitric Oxide and Protein S -Nitrosylation Are Integral to Hydrogen Peroxide-Induced Leaf Cell Death in Rice
journal, November 2011


Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S -nitrosylation
journal, November 2013

  • Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira
  • Journal of Experimental Botany, Vol. 65, Issue 2
  • DOI: 10.1093/jxb/ert396

Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose
journal, August 1998

  • Durner, J.; Wendehenne, D.; Klessig, D. F.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 17
  • DOI: 10.1073/pnas.95.17.10328

Nitric oxide functions as a signal in plant disease resistance
journal, August 1998

  • Delledonne, Massimo; Xia, Yiji; Dixon, Richard A.
  • Nature, Vol. 394, Issue 6693
  • DOI: 10.1038/29087

PI3K/Akt signalling pathway and cancer
journal, April 2004

  • Vara, Juan Ángel Fresno; Casado, Enrique; de Castro, Javier
  • Cancer Treatment Reviews, Vol. 30, Issue 2
  • DOI: 10.1016/j.ctrv.2003.07.007

Regulation of apoptosis by protein S-nitrosylation
journal, November 2006