Cargando…
Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography
Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the tr...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Biochemistry and Molecular Biology
2013
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567860/ https://www.ncbi.nlm.nih.gov/pubmed/23172892 http://dx.doi.org/10.1074/mcp.M112.020560 |
_version_ | 1782258725397987328 |
---|---|
author | Hoehenwarter, Wolfgang Thomas, Martin Nukarinen, Ella Egelhofer, Volker Röhrig, Horst Weckwerth, Wolfram Conrath, Uwe Beckers, Gerold J. M. |
author_facet | Hoehenwarter, Wolfgang Thomas, Martin Nukarinen, Ella Egelhofer, Volker Röhrig, Horst Weckwerth, Wolfram Conrath, Uwe Beckers, Gerold J. M. |
author_sort | Hoehenwarter, Wolfgang |
collection | PubMed |
description | Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)(3)-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO(2)-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment. |
format | Online Article Text |
id | pubmed-3567860 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | The American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-35678602013-02-11 Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography Hoehenwarter, Wolfgang Thomas, Martin Nukarinen, Ella Egelhofer, Volker Röhrig, Horst Weckwerth, Wolfram Conrath, Uwe Beckers, Gerold J. M. Mol Cell Proteomics Research Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)(3)-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO(2)-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment. The American Society for Biochemistry and Molecular Biology 2013-02 2012-11-20 /pmc/articles/PMC3567860/ /pubmed/23172892 http://dx.doi.org/10.1074/mcp.M112.020560 Text en © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles |
spellingShingle | Research Hoehenwarter, Wolfgang Thomas, Martin Nukarinen, Ella Egelhofer, Volker Röhrig, Horst Weckwerth, Wolfram Conrath, Uwe Beckers, Gerold J. M. Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography |
title | Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography
|
title_full | Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography
|
title_fullStr | Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography
|
title_full_unstemmed | Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography
|
title_short | Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography
|
title_sort | identification of novel in vivo map kinase substrates in arabidopsis thaliana through use of tandem metal oxide affinity chromatography |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567860/ https://www.ncbi.nlm.nih.gov/pubmed/23172892 http://dx.doi.org/10.1074/mcp.M112.020560 |
work_keys_str_mv | AT hoehenwarterwolfgang identificationofnovelinvivomapkinasesubstratesinarabidopsisthalianathroughuseoftandemmetaloxideaffinitychromatography AT thomasmartin identificationofnovelinvivomapkinasesubstratesinarabidopsisthalianathroughuseoftandemmetaloxideaffinitychromatography AT nukarinenella identificationofnovelinvivomapkinasesubstratesinarabidopsisthalianathroughuseoftandemmetaloxideaffinitychromatography AT egelhofervolker identificationofnovelinvivomapkinasesubstratesinarabidopsisthalianathroughuseoftandemmetaloxideaffinitychromatography AT rohrighorst identificationofnovelinvivomapkinasesubstratesinarabidopsisthalianathroughuseoftandemmetaloxideaffinitychromatography AT weckwerthwolfram identificationofnovelinvivomapkinasesubstratesinarabidopsisthalianathroughuseoftandemmetaloxideaffinitychromatography AT conrathuwe identificationofnovelinvivomapkinasesubstratesinarabidopsisthalianathroughuseoftandemmetaloxideaffinitychromatography AT beckersgeroldjm identificationofnovelinvivomapkinasesubstratesinarabidopsisthalianathroughuseoftandemmetaloxideaffinitychromatography |