Details
| Original language | English |
|---|---|
| Pages (from-to) | 546-558 |
| Number of pages | 13 |
| Journal | Plant Journal |
| Volume | 104 |
| Issue number | 2 |
| Early online date | 3 Aug 2020 |
| Publication status | Published - Oct 2020 |
| Externally published | Yes |
Abstract
Casein kinase 2 is a ubiquitous protein kinase that has puzzled researchers for several decades because of its pleiotropic activity. Here, we set out to identify the in vivo targets of plastid casein kinase 2 (pCK2) in Arabidopsis thaliana. Survey phosphoproteome analyses were combined with targeted analyses with wild-type and pck2 knockdown mutants to identify potential pCK2 targets by their decreased phosphorylation state in the mutant. To validate potential substrates, we complemented the pck2 knockdown line with tandem affinity tag (TAP)-tagged pCK2 and found it to restore growth parameters, as well as many, but not all, putative pCK2-dependent phosphorylation events. We further performed a targeted analysis at the end-of-night to increase the specificity of target protein identification. This analysis confirmed light-independent phosphorylation of several pCK2 target proteins. Based on the aforementioned data, we define a set of in vivo pCK2-targets that span different chloroplast functions, such as metabolism, transcription, translation and photosynthesis. The pleiotropy of pCK2 functions is also manifested by altered state transition kinetics during short-term acclimation and significant alterations in the mutant metabolism, supporting its function in photosynthetic regulation. Thus, our data expand our understanding on chloroplast phosphorylation networks and provide insights into kinase networks in the regulation of chloroplast functions.
Keywords
- Arabidopsis/genetics, Arabidopsis Proteins/genetics, Casein Kinase II/genetics, Chloroplast Proteins/metabolism, Darkness, Gene Knockdown Techniques, Light, Mutation, Phosphorylation, Plastids/metabolism, Protein Interaction Maps, Proteomics/methods
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In: Plant Journal, Vol. 104, No. 2, 10.2020, p. 546-558.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Working day and night
T2 - plastid casein kinase 2 catalyses phosphorylation of proteins with diverse functions in light- and dark-adapted plastids
AU - Rödiger, Anja
AU - Galonska, Johann
AU - Bergner, Elena
AU - Agne, Birgit
AU - Helm, Stefan
AU - Alseekh, Saleh
AU - Fernie, Alisdair R
AU - Thieme, Domenika
AU - Hoehenwarter, Wolfgang
AU - Hause, Gerd
AU - Pfannschmidt, Thomas
AU - Baginsky, Sacha
N1 - Funding Information: We are grateful for financial support from the DFG, grant number ?BA 1902/2-2? and the European Regional Development Fund of the European Commission grant W21004490 via Land Sachsen-Anhalt to SB. SB gratefully acknowledges DFG support for the acquisition of a Synapt G2-S mass spectrometer (INST 271/283-1 FUGG). We thank Monika Grycko for her help in the state transition experiments and Jessica Fostvedt for mutant characterization. Open access funding enabled and organized by Projekt DEAL.
PY - 2020/10
Y1 - 2020/10
N2 - Casein kinase 2 is a ubiquitous protein kinase that has puzzled researchers for several decades because of its pleiotropic activity. Here, we set out to identify the in vivo targets of plastid casein kinase 2 (pCK2) in Arabidopsis thaliana. Survey phosphoproteome analyses were combined with targeted analyses with wild-type and pck2 knockdown mutants to identify potential pCK2 targets by their decreased phosphorylation state in the mutant. To validate potential substrates, we complemented the pck2 knockdown line with tandem affinity tag (TAP)-tagged pCK2 and found it to restore growth parameters, as well as many, but not all, putative pCK2-dependent phosphorylation events. We further performed a targeted analysis at the end-of-night to increase the specificity of target protein identification. This analysis confirmed light-independent phosphorylation of several pCK2 target proteins. Based on the aforementioned data, we define a set of in vivo pCK2-targets that span different chloroplast functions, such as metabolism, transcription, translation and photosynthesis. The pleiotropy of pCK2 functions is also manifested by altered state transition kinetics during short-term acclimation and significant alterations in the mutant metabolism, supporting its function in photosynthetic regulation. Thus, our data expand our understanding on chloroplast phosphorylation networks and provide insights into kinase networks in the regulation of chloroplast functions.
AB - Casein kinase 2 is a ubiquitous protein kinase that has puzzled researchers for several decades because of its pleiotropic activity. Here, we set out to identify the in vivo targets of plastid casein kinase 2 (pCK2) in Arabidopsis thaliana. Survey phosphoproteome analyses were combined with targeted analyses with wild-type and pck2 knockdown mutants to identify potential pCK2 targets by their decreased phosphorylation state in the mutant. To validate potential substrates, we complemented the pck2 knockdown line with tandem affinity tag (TAP)-tagged pCK2 and found it to restore growth parameters, as well as many, but not all, putative pCK2-dependent phosphorylation events. We further performed a targeted analysis at the end-of-night to increase the specificity of target protein identification. This analysis confirmed light-independent phosphorylation of several pCK2 target proteins. Based on the aforementioned data, we define a set of in vivo pCK2-targets that span different chloroplast functions, such as metabolism, transcription, translation and photosynthesis. The pleiotropy of pCK2 functions is also manifested by altered state transition kinetics during short-term acclimation and significant alterations in the mutant metabolism, supporting its function in photosynthetic regulation. Thus, our data expand our understanding on chloroplast phosphorylation networks and provide insights into kinase networks in the regulation of chloroplast functions.
KW - Arabidopsis/genetics
KW - Arabidopsis Proteins/genetics
KW - Casein Kinase II/genetics
KW - Chloroplast Proteins/metabolism
KW - Darkness
KW - Gene Knockdown Techniques
KW - Light
KW - Mutation
KW - Phosphorylation
KW - Plastids/metabolism
KW - Protein Interaction Maps
KW - Proteomics/methods
UR - http://www.scopus.com/inward/record.url?scp=85089692977&partnerID=8YFLogxK
U2 - 10.1111/tpj.14944
DO - 10.1111/tpj.14944
M3 - Article
C2 - 32745315
VL - 104
SP - 546
EP - 558
JO - Plant Journal
JF - Plant Journal
SN - 0960-7412
IS - 2
ER -