Untersuchung der Beziehung zwischen Konservierung und Phosphorylierung von Serinen in der basischen Domäne pflanzlicher bZIP-Transkriptionsfaktoren

01.10.2020 bis 30.09.2023

Phosphorylation-dependent post-translational regulation of bZIP transcription factors has been demonstrated in numerous organisms including fungi, animals and plants. Different groups of plant bZIP factors possess distinct pattern of potentially phosphorylatable residues in their highly conserved basic region, ranging from one (groups D, F, H) till three (groups C, G, S) residues. We have shown that by the substitution of the two most conserved serine residues (Ser15 and Ser19 within the basic region), which are in the direct contact with the phospho-diester bonds of the DNA backbone, to phospho-mimicking aspartate, the interaction of bZIP factors with their cognate DNA is completely abolished, while their other molecular features remain unaltered. Furthermore, we identified calcium-dependent protein kinase 3 (CPK3) as the upstream kinase able to interact with the C-group factors inside the nucleus and to phosphorylate in vitro all three conserved serine residues of the C-group member bZIP63. Bearing in mind that Ser11 within the basic region of bZIP63 can be phosphorylated in vivo, we now propose to study the upstream mechanisms governing this phosphorylation, and the impact of such modification on bZIP63 protein function. Loss-of-function cpk3 mutant and transgenic lines expressing constitutively active CPK3 form will be used to find out, which residues within the conserved bZIP domain of bZIP63 are in vivo CPK3 target sites and how does their phospho-status respond to different signals. Further on, the effects of this phosphorylation on bZIP63 interaction with genomic DNA and with proteins, including dimer formation, as well as on the activation of bZIP63 target genes will be investigated. Overall, the obtained data will help to understand how signals, transduced by a specific kinase to bZIP63, can be integrated in the complex network of functional responses governed by the interconnected cluster of bZIP transcription factors. Besides, the scientific community will benefit from the produced large-scale datasets.