Functional analysis of plant Snf1-related protein kinase signaling complexes using proteomics and reverse genetics in Arabidopsis

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Conserved members of Snf1/AMPK protein kinase family function as cellular sugar/energy sensors controlling key metabolic and signaling pathways in eukaryotes. In the trimeric core of these kinases the catalytic Snf1/a and activating Snf4/g subunits are anchored by one of the different substrate targeting Gal83/Sip/ß subunits. Arabidopsis encodes three a, two ß and two isomorphic g subunits, AtSNF4 and AKINßg. Through a KIS-domain present normally only in ß subunits, AKINßg may recruit alone the a subunits to perform plant specific signaling functions. We found that different Arabidopsis Snf1-related kinases (SnRKs), formed by combinatorial assembly of the subunits in vivo, occur in a spliceosomal complex of kinase inhibitor PRL1 protein, PAM1 protein methylase and spliceosome assembly factor RSP33; in proteasome-SCF ubiquitin ligase complexes; and bound to SnRK- and PRL1-interacting kinases and No Apical Meristem class of signaling factors. The proposed project aims to systematically characterize the components of SnRK signaling kinases by monitoring the spatial and temporal regulation of corresponding genes and proteins; immunoaffinity purification and mass spectrometry analysis of protein complexes; and functional analysis of their regulatory subunits by determining the effects of knockout mutations on the SnRK-controlled signaling pathways.