Arabidopsis AMP-activated protein kinases in poteasomal complexes and their role in cell signalling

AMP-activated protein kinases (AMPK) are structurally and functionally highly conserved in all eukaryotes. The most profoundly studied yeast AMPK ortholog, Snf1, plays a role in the sensing and response to low energy and nutrient levels. There is much less information available about the function of plant Snf1 orthologs, termed Snf1-related protein kinases (SnRKs) and their regulatory roles in plant specific nutrient regulated mechanism, such as photosynthesis, plant hormone signaling and metabolism. Genetic studies of plant SnRK1 kinases are impeded by the lack of T-DNA insertion mutants in the SnRK1 catalytic subunits and the lack of male transmission of mutations affecting the SnRKγ subunit. The present work describes the isolation of T-DNA insertion mutations in SnRK1 β subunit genes and characterization of these mutants. Single and double mutant plants did not show any observable developmental or sugar signaling phenotype. Triple SnRK1β mutant could not be generated because no mutant allele is available for the third beta subunit. In our experiments overexpression of GFP-tagged SnRK1 beta subunits did not show subcellular relocalization in response to nutritional stress, a mechanism which was described in yeast. Instead, rapid proteasomal degradation of Arabidopsis SnRK1 subunits was observed in response to sugars and light. Proteasomal degradation of SnRK1 subunits prevented their efficient overexpression for biochemical studies. Former in vitro and in vivo studies found SnRK1 kinases in proteasomal complexes in Arabidopsis. This observation correlates with the fact that numerous nutrient status regulated metabolic enzymes are proteasomal substrates in Arabidopsis as in yeast and their degradation is controlled by phosphorylation and sugar availability. In order to identify SnRK1 interacting partners in vivo, it was attempted to isolate protein complexes from Arabidopsis through tagged SnRK1 subunits. In contrast to a former and mostly unsuccessful immunoaffinity purification approach, this work applies a rapid tandem affinity chromatography-based protein purification method. In addition to developing a biochemical approach for identification of SnRK1 interacting partners, we further explored the proteasomal connection of SnRK1 kinases and searched for their potential substrates using in vitro phosphorylation studies. Several sugar-regulated proteasomal substrates and F-box proteins were overexpressed in E. coli and purified to near homogeneity along with similar purification of SnRK1 kinase catalytic subunits. The purified proteins were subjected to in vitro kinase reactions using radioactive γ32P-ATP and phosphorylation was detected in more cases. Some of the phosphorylation sites were successfully identified by mass spectrometric analysis. Site-directed mutagenesis of the phosphorylation site on IAA6 and overexpression of the mutant construct indicates the importance of phosphorylation site in vivo. In order to provide further evidence for interaction of SnRK1 kinases with their substrates in vivo, an assay system for testing the stability of kinase substrates was developed.