Arabidopsis Protein Kinases GRIK1 and GRIK2 Specifically Activate SnRK1 by Phosphorylating Its Activation Loop

SNF1-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.Protein kinases play central roles in signal transduction and regulatory pathways in eukaryotes. They often function as cascades in which upstream kinases activate downstream kinases by phosphorylating a Ser, Thr, or Tyr residue in the activation loop of the kinase domain. Phosphorylation induces a conformational change that moves the activation loop and allows access to the kinase active site. This mechanism is highly conserved for protein kinases, as exemplified by the well-characterized cyclin-dependent kinase and mitogen-activated protein kinase cascades. More recently, sucrose nonfermenting-1 (SNF1), a kinase that modulates sugar metabolism in budding yeast (Saccharomyces cerevisiae), has been shown to be activated by three partially redundant kinases (for review, see Hardie, 2007). In animals, the SNF1 homolog, AMP-activated protein kinase (AMPK), is activated by two kinases that are phylogenetically related to the yeast SNF1 activating kinases (Hardie, 2007). In plants, DNA sequence analysis and yeast complementation assays have implicated the GRIKs, which were originally identified as geminivirus Rep interacting kinases (Kong and Hanley-Bowdoin, 2002), as the upstream activators of SNF1-related kinases (SnRK1; Shen and Hanley-Bowdoin, 2006; Hey et al., 2007). An analysis of the relationship between the GRIKs and SnRK1 activation represents an important first step in understanding the role of this putative protein kinase cascade in plants.SnRK1, SNF1, and AMPK belong to a conserved family of protein kinases consisting of an α-catalytic subunit and regulatory β- and γ-subunits (for review, see Polge and Thomas, 2007). These kinases play central roles in regulating and coordinating carbon metabolism and energy balance in eukaryotes (for review, see Hardie et al., 1998; Halford et al., 2003, 2004; Hardie, 2007; Polge and Thomas, 2007; Baena-Gonzalez and Sheen, 2008). Metabolic stresses, such as sugar starvation and lack of light, stimulate SnRK1 activity (Baena-Gonzalez et al., 2007). Suc-P synthase (SPS), 3-hydroxy-3-methylglutaryl-CoA reductase, nitrate reductase, and trehalose-6-P synthase are negatively regulated by SnRK1 phosphorylation (McMichael et al., 1995; Barker et al., 1996; Sugden et al., 1999b; Harthill et al., 2006), suggesting that SnRK1 modulates metabolism by phosphorylating key metabolic enzymes. However, SnRK1 is also thought to act as a master regulator of global gene expression in plants grown under starvation and energy deprivation conditions (Baena-Gonzalez et al., 2007). The global expression profile resulting from SnRK1 overexpression positively correlates with treatments that limit energy and is inversely related to those associated with high-energy conditions (Baena-Gonzalez et al., 2007). Many SnRK1-regulated genes are involved in plant primary and secondary metabolism, and catabolic pathways are generally up-regulated, while biosynthetic pathways are down-regulated (Baena-Gonzalez et al., 2007).SnRK1 also impacts metabolic processes during development and disease. Studies in grains, legumes, and tuberous plants showed that loss of SnRK1 alters seed maturation, longevity, and germination, retards root growth, and reduces starch accumulation (Zhang et al., 2001; Lovas et al., 2003; Radchuk et al., 2006; Lu et al., 2007; Rosnoblet et al., 2007), while SnRK1 overexpression increases starch accumulation (McKibbin et al., 2006). Reduced expression of the SnRK1 β-subunit is responsible for sugar reallocation to roots during herbivore attack (Schwachtje et al., 2006). SnRK1 has also been implicated in resistance to geminivirus infection (Hao et al., 2003). However, there is evidence that SnRK1 has additional functions beyond modulating metabolism. Studies in Arabidopsis (Arabidopsis thaliana) showed that SnRK1 is essential for viability and that plants silenced for both SnRK1.1 and SnRK1.2 are severely stunted and impaired for flowering (Baena-Gonzalez et al., 2007). Importantly, unlike Physcomitrella patens SnRK1 mutants (Thelander et al., 2004), these plants are not rescued by energy-surplus conditions, such as continuous light or growth medium containing 1% Suc. A number of SnRK1-responsive genes are associated with the cell division cycle and/or development, and some encode transcription factors and chromatin assembly/modifying factors (Baena-Gonzalez et al., 2007).Phosphorylation of a conserved Thr residue in the activation loop of the kinase domain is an essential step during activation of SNF1 and AMPK. In budding yeast, three related and functionally redundant kinases, SAK1, TOS3, and ELM1, activate SNF1 (Hong et al., 2003; Nath et al., 2003; Sutherland et al., 2003). Sugars do not regulate these upstream kinases in yeast; instead, Glc promotes dephosphorylation of the activation loop by making it available to the protein phosphatase, Glc7-Reg1 (Rubenstein et al., 2008). In mammals, AMPK is activated by two upstream kinases, LKB1 and CaMKKβ (Hawley et al., 2003, 2005; Woods et al., 2003a, 2003b, 2005; Hurley et al., 2005). LKB1, which activates 12 AMPK-related kinases, is constitutively active (Lizcano et al., 2004), while CaMKKβ expression is associated with Ca²⁺ surges primarily in neural cells (Hawley et al., 2005). The LKB1-AMPK cascade plays roles in cell division, establishment of cell polarity, and senescence (for review, see Koh and Chung, 2007; Williams and Brenman, 2008). LKB1 has also been associated with cancer in humans (Alessi et al., 2006). A mitogen-activated protein kinase kinase kinase can also functionally complement a yeast sak1Δ tos3Δ elm1Δ mutant and may activate AMPK in animals (Momcilovic et al., 2006).GRIK1 and GRIK2 are the sole members of their kinase family in Arabidopsis, with the number of GRIK homologs ranging from one to three in other plant species (Kong and Hanley-Bowdoin, 2002; Shen and Hanley-Bowdoin, 2006). In Arabidopsis, GRIK transcript levels vary minimally across different tissues and developmental stages, while GRIK proteins are detected exclusively in young tissues undergoing DNA synthesis and cell division, such as shoot apical meristems (SAMs), flower buds, and developing siliques (Shen and Hanley-Bowdoin, 2006). The GRIK proteins also accumulate in geminivirus-infected cells that support DNA replication but not in healthy cells of mature leaves (Shen and Hanley-Bowdoin, 2006). Studies using the proteasome inhibitor MG132 showed that the GRIK proteins are subject to proteasome-mediated degradation (Shen and Hanley-Bowdoin, 2006).The existence of a plant SnRK1 activating kinase was proposed several years ago (Sugden et al., 1999a). The Arabidopsis GRIK1 and GRIK2 proteins (also called SNAK2 and SNAK1, respectively) are phylogenetically related to yeast SAK1, TOS3, and ELM1 and mammalian LKB1 and CaMKKβ (Wang et al., 2003; Shen and Hanley-Bowdoin, 2006; Hey et al., 2007). Each GRIK can complement a yeast sak1Δ tos3Δ elm1Δ triple mutant (Shen and Hanley-Bowdoin, 2006; Hey et al., 2007). Together, these observations suggested that the GRIKs are upstream activators of SnRK1 in plants. To test this hypothesis, we investigated whether GRIK1 and GRIK2 can phosphorylate and activate Arabidopsis SnRK1.