Funktionelle Charakterisierung von AtCPK1 und AtCPK2 aus Arabidopsis thaliana unter Verwendung einer chemisch-genetischen Methode

Calcium-dependent protein kinases (CDPKs) play a biological role in a variety of cellular processes, as has been shown by ectopic expression and silencing analyses. However, analysis of CDPK mutant lines has not revealed any phenotypic differences, probably due to functional redundancy and plant adaptation mechanisms. Only in the case of NtCDPK2 from tobacco could a biological function be identified. In this work a chemical-genetic method for the isoform-specific analysis of CDPKs was established. The nucleotide binding properties of CDPKs were changed by mutating the ATP-binding pocket such that the enzymes could be inhibited by the kinase inhibitor 1-NA-PP1. Biochemical characterization of NtCDPK2 as a model enzyme shows that this mutation is functionally silent and has no influence on kinase activity and substrate specificity. The isoforms AtCPK1 and AtCPK2 have been chosen for functional characterization based on microarray expression data. The use of classical genetic methods, such as the analysis of promoter-GUS fusion lines, shows that AtCPK1 is ubiquitously expressed. AtCPK2 on the other hand shows a specific localization in root tips and in cells exhibiting tip growth, for example pollen. Analysis of pollen tube growth shows a significant reduction of the pollen tube length in the cpk2 mutant line. For the reverse chemical-genetic screen, a T-DNA insertion line of AtCPK1 has been complemented with an ATP-binding pocket variant of this isoform. Phosphoproteomic analysis revealed five proteins that show a differential phosphorylation pattern after chemical inhibition. Metabolomic analysis revealed differentially regulated metabolites. In this work clues to the biological function of AtCPK1 and AtCPK2 could be identified using mutant analysis and a new chemical-genetic method for the analysis of CDPKs. This shows that the chemical-genetic screen can complement classical genetic methods and often provides the only method for isoform-specific analysis. In a second part of this work phosphoregulation of CDPKs was analyzed. After transient expression in N. benthamiana a stimulus-dependent phosphorylation site in AtCPK2 could be mapped. In NtCDPK2, a potential function for a phosphorylation site in the ATP-binding pocket was postulated. This phosphorylation site could be the key position in a new regulatory mechanism that bears similarities to that of cyclin-dependent protein kinases.