Substrate profiling of the Arabidopsis Ca 2+ -dependent protein kinase AtCPK4 and its Ricinus communis ortholog RcCDPK1

AtCPK4 and AtCPK11 are Arabidopsis thaliana Ca -dependent protein kinase (CDPK) paralogs that have been reported to positively regulate abscisic acid (ABA) signal transduction by phosphorylating ABA-responsive transcription factor-4 (AtABF4). By contrast, RcCDPK1, their closest Ricinus communis orth...

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Published in:Plant science (Limerick) 2023-06, Vol.331, p.111675
Main Authors: Kilburn, Ryan, Fedosejevs, Eric T, Mehta, Devang, Soleimani, Faranak, Ghahremani, Mina, Monaghan, Jacqueline, Thelen, Jay J, Uhrig, R Glen, Snedden, Wayne A, Plaxton, William C
Format: Article
Language:English
Subjects:
Abscisic Acid - metabolism
Arabidopsis - metabolism
Arabidopsis Proteins - metabolism
Calcium - metabolism
Chromatography, Liquid
Gene Expression Regulation, Plant
Germination - genetics
Protein Kinases - genetics
Protein Kinases - metabolism
Ricinus - genetics
Ricinus - metabolism
Tandem Mass Spectrometry
Transcription Factors - metabolism
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Summary:AtCPK4 and AtCPK11 are Arabidopsis thaliana Ca -dependent protein kinase (CDPK) paralogs that have been reported to positively regulate abscisic acid (ABA) signal transduction by phosphorylating ABA-responsive transcription factor-4 (AtABF4). By contrast, RcCDPK1, their closest Ricinus communis ortholog, participates in the control of anaplerotic carbon flux in developing castor oil seeds by catalyzing inhibitory phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser451. LC-MS/MS revealed that AtCPK4 and RcCDPK1 transphosphorylated several common, conserved residues of AtABF4 and its castor ortholog, TRANSCRIPTION FACTOR RESPONSIBLE FOR ABA REGULATON. Arabidopsis atcpk4/atcpk11 mutants displayed an ABA-insensitive phenotype that corroborated the involvement of AtCPK4/11 in ABA signaling. A kinase-client assay was employed to identify additional AtCPK4/RcCDPK1 targets. Both CDPKs were separately incubated with a library of 2095 peptides representative of Arabidopsis protein phosphosites; five overlapping targets were identified including PLANT INTRACELLULAR RAS-GROUP-RELATED LEUCINE-RICH REPEAT PROTEIN-9 (AtPIRL9) and the E3-ubiquitin ligase ARABIDOPSIS TOXICOS EN LEVADURA 6 (AtATL6). AtPIRL9 and AtATL6 residues phosphorylated by AtCPK4/RcCDPK1 conformed to a CDPK recognition motif that was conserved amongst their respective orthologs. Collectively, this study provides evidence for novel AtCPK4/RcCDPK1 substrates, which may help to expand regulatory networks linked to Ca - and ABA-signaling, immune responses, and central carbon metabolism.
ISSN:1873-2259