A chemical-genetic approach to elucidate protein kinase function in planta

Abstract

The major objective in protein kinase research is the identification of the biological process, in which an individual enzyme is integrated. Protein kinase-mediated signalling is thereby often addressed by single knock-out mutation-or co-suppression-based reverse genetics approaches. If a protein kinase of interest is a member of a multi gene family, however, no obvious phenotypic alteration in the morphology or in biochemical parameters may become evident because mutant phenotypes may be compensated by functional redundancy or homeostasis. Here we establish a chemical-genetic screen combining ATP-analogue sensitive (as) kinase variants and molecular fingerprinting techniques to study members of the plant calcium-dependent protein kinase (CDPK) family in vivo. CDPKs have been implicated in fast signalling responses upon external abiotic and biotic stress stimuli. CDPKs carrying the as-mutation did not show altered phosphorylation kinetics with ATP as substrate, but were able to use ATP analogues as phosphate donors or as kinase inhibitors. For functional characterization in planta, we have substituted an Arabidopsis thaliana mutant line of AtCPK1 with the respective as-variant under the native CPK1 promoter. Seedlings of Arabidopsis wild type and AtCPK1 as-lines were treated with the ATP analogue inhibitor 1-NA-PP1 and exposed to cold stress conditions. Rapid cold-induced changes in the phosphoproteome were analysed by 2D-gel-electrophoresis and phosphoprotein staining. The comparison between wild type and AtCPK1 as-plants before and after inhibitor treatment revealed differential CPK1-dependent and cold-stress-induced phosphoprotein signals. In this study, we established the chemical-genetic approach as a tool, which allows the investigation of plant-specific classes of protein kinases in planta and which facilitates the identification of rapid changes of molecular biomarkers in kinase-mediated signalling networks.

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress

“…In previous studies, a number of experimental approaches had been employed to identify CDPK-interacting proteins (Patharkar and Cushman, 2000;Lee et al, 2003;Shao and Harmon, 2003;Rodriguez Milla et al, 2006;Böhmer and Romeis, 2007;Vlad et al, 2008;Uno et al, 2009). Among these different approaches, yeast two-hybrid screening with baits containing only the kinase domain of the protein had been demonstrated to be successful for identification of CDPK substrates, such as for ice plant (Mesembryanthemum crystallinum) CPK1 (Patharkar and Cushman, 2000) and tobacco (Nicotiana tabacum) CDPK1 (Lee et al, 2003).…”

CDPKs in immune and stress signaling

“…Unlike in salt or drought signaling, OsCPK13 (OsCDPK7) confers cold resistance without affecting transcriptional regulation [71]. Similarly, AtCPK1 does not affect CBF induction but regulates the phosphoproteome after cold treatment [90]. A membrane-bound rice CDPK is activated by cold after 18–24h, suggesting a role in the adaptive process rather than early responses [91].…”

Arabidopsis Calcium-Dependent Protein Kinase CPK10 Functions in Abscisic Acid- and Ca2+-Mediated Stomatal Regulation in Response to Drought Stress

“…Obviously, to identify potential targets of CDPKs is becoming an important task for further understanding of the CDPK-involved plant signaling mechanisms. Various approaches have been employed to screen CDPK-interacting proteins, such as a phage display library screening (Shao and Harmon, 2003), yeast twohybrid screening (Patharkar and Cushman, 2000;Lee et al, 2003;Choi et al, 2005;Rodriguez Milla et al, 2006;Uno et al, 2009), and a chemical-genetic approach (Bö hmer and Romeis, 2007).…”