The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.

We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically asses...

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Autores principales:	Anna Schönberg, Elena Bergner, Stefan Helm, Birgit Agne, Beatrix Dünschede, Danja Schünemann, Mike Schutkowski, Sacha Baginsky
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7393be6b1f054a9fbc5a435987b1de3a
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spelling	oai:doaj.org-article:7393be6b1f054a9fbc5a435987b1de3a2021-11-25T05:57:30ZThe peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.1932-620310.1371/journal.pone.0108344https://doaj.org/article/7393be6b1f054a9fbc5a435987b1de3a2014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0108344https://doaj.org/toc/1932-6203We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.Anna SchönbergElena BergnerStefan HelmBirgit AgneBeatrix DünschedeDanja SchünemannMike SchutkowskiSacha BaginskyPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 10, p e108344 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anna Schönberg Elena Bergner Stefan Helm Birgit Agne Beatrix Dünschede Danja Schünemann Mike Schutkowski Sacha Baginsky The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.
description	We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.
format	article
author	Anna Schönberg Elena Bergner Stefan Helm Birgit Agne Beatrix Dünschede Danja Schünemann Mike Schutkowski Sacha Baginsky
author_facet	Anna Schönberg Elena Bergner Stefan Helm Birgit Agne Beatrix Dünschede Danja Schünemann Mike Schutkowski Sacha Baginsky
author_sort	Anna Schönberg
title	The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.
title_short	The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.
title_full	The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.
title_fullStr	The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.
title_full_unstemmed	The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.
title_sort	peptide microarray "chlorophos1.0" identifies new phosphorylation targets of plastid casein kinase ii (pckii) in arabidopsis thaliana.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/7393be6b1f054a9fbc5a435987b1de3a
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The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.

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