Identification of components and substrates of Snf1-related kinase 1 (SnRK1) complexes in <em>Arabidopsis thaliana</em>

Abstract

Similarly to yeast Sucrose nonfermenting 1 (Snf1) and animal AMP-activated protein kinases (AMPKs), plant Snf1-related (SnRK1) kinases play a central role in the regulation of cellular energy homeostasis and responses to carbon source availability. Members of the Snf1/SnRK1/AMPK family are differentially activated by carbon source depletion and increasing AMP/ATP ratio to confer down-regulation of energy consuming anabolic pathways and parallel activation of energy producing processes by phosphorylation of key metabolic enzymes and transcription factors. Inhibition of photosynthetic CO₂ fixation and ATP production stimulates plant SnRK1 activation in leaves, which provide sucrose as main transported sugar for developing sink organs. Sensing of sucrose availability by conversion of its metabolic products to trehalose-6-phosphate (T6P) is reported to inhibit SnRK1 through a yet unknown protein factor. As Snf1 and AMPKs, Arabidopsis SnRK1 enzymes form trimeric complexes with activating γ/SNF4 and substrate targeting β1/2/3-subunits. SnRK1 activity is stimulated by T-loop phosphorylation of catalytic α-subunits AKIN10/11 by upstream activating kinases and inhibited through dephosphorylation by PP2C protein phosphatases acting in ABA/sugar signaling. Our current knowledge on plant SnRK1 kinases is largely based on protein-protein interaction assays in heterologous systems, and transcriptomics and phosphoproteomics studies using antisense inhibition and overexpression of SnRK1 catalytic subunits in leaf protoplasts and seedlings carrying various mutations in metabolic and hormonal pathways.<br /> A major goal of this Ph.D. work was to use precisely modified native gene constructs for expression of SnRK1 subunits in fusion with suitable tags, such as green and red fluorescent proteins (GFP and mCherry) in plants, and exploit this technology for purification of SnRK1 complexes and identification of their interacting partners. By enlarging the ATP-binding pocket of SnRK1α1 subunit AKIN10, an analog-sensitive AS-kinase carrying a combined affinity tag (GFPPIPL) was constructed by recombineering-based site-directed mutagenesis and expressed in plants. Unlike other kinases in Arabidopsis, the AS-AKIN10 kinase can catalyze phosphorylation of substrates with bulky N⁶-substituted thioATP derivatives, which can be specifically detected, enriched and identified by mass spectrometry.<br /> Our study demonstrates that exchange of the phosphorylated T-loop Thr175 residue of AKIN10 to A and D/E residues results only in partial inactivation and limited stimulation of substrate phosphorylation activity of SnRK1 <em>in vitro</em> and, upon ectopic expression of cDNA constructs by a CaMV35S promoter <em>in vivo</em>, respectively. Ectopic expression of wild type and T-loop mutant versions of AKIN10 resulted only in minor developmental changes, including earlier flowering on short day, and enhanced root and hypocotyl elongation in the case of T175D T-loop AKIN10 derivative. Expression of AKIN10-GFP/PIPL and SNF4-YFP constructs by native genes provided suitable materials for affinity purification of SnRK1 complexes and identification of their interacting partners by mass spectrometry. In addition to previously described two-hybrid interacting partners, such as the HSPRO2 and DUF581 domain proteins, these studies confirmed reciprocal co-immunoprecipitation and association of class II trehalose synthase/ phosphatase (TPS) enzymes with SnRK1. Dimerization of trimeric SnRK1 enzymes was detected in cytoplasmic complexes. Association of SnRK1 with TPS partners, such as TPS8, was found to confer UDP-glucose and T6P mediated inhibition of SnRK1. This indicated that class II TPS enzymes might serve as metabolic sensors, which negatively regulate SnRK1 in response to the availability of sucrose-derived metabolic signals. However, using a nuclear protein purification approach optimized for the isolation of NTC spliceosome-activating complex and associated spliceosome components, we failed to identify novel interacting partners of SnRK1. Optimization of <em>in situ</em> kinase reactions in isolated nuclei and nuclear extracts, as well as enrichment of thiophosphorylated substrates of the analog-sensitive AS-AKIN10 kinase, nevertheless resulted in the identification of several novel candidate SnRK1 substrates. One of these, the nuclear NAP57/CBF5/DYSKERIN pseudouridine synthase involved in the regulation of telomere length and ribosome biogenesis was found to be phosphorylated in its catalytic domain by SnRK1. Further analysis of AS-kinase substrates and components of nuclear kinase complexes is expected to provide deeper insight into transcription targets and regulatory roles of plant SnRK1.