AbstractSNF1-related kinases (SnRK1s) play central roles in coordinating energy balance and nutrient metabolism in plants. SNF1 and AMPK, the SnRK1 homologs in budding yeast (Saccharomyces cerevisiae) and mammals, are activated by phosphorylation of conserved threonine residues in their activation loops. Arabidopsis (Arabidopsis thaliana) GRIK1 and GRIK2, which were first characterized as geminivirus Rep interacting kinases, are phylogenetically related to SNF1 and AMPK activating kinases. In this study, we used recombinant proteins produced in bacteria to show that both GRIKs specifically bind to the SnRK1 catalytic subunit and phosphorylate the equivalent threonine residue in its activation loop in vitro. GRIK-mediated phosphorylation increased SnRK1 kinase activity in autophosphorylation and peptide substrate assays. These data, together with earlier observations that GRIKs could complement yeast mutants lacking SNF1 activation activities, established that the GRIKs are SnRK1 activating kinases. Given that the GRIK proteins only accumulate in young tissues and geminivirus-infected mature leaves, the GRIK-SnRK1 cascade may function in a developmentally regulated fashion and coordinate the unique metabolic requirements of rapidly growing cells and geminivirus-infected cells that have been induced to reenter the cell cycle.