Regulation of a Plant SNF1-Related Protein Kinase by Glucose-6-Phosphate

Abstract

One of the major protein kinases (PK III ) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, we have developed a fluorescence-based continuous assay for measurement of PK III activity. Using the continuous assay, along with the fixed-time-point 32 P-incorporation assay, we demonstrate that PK III activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8.5 to 5.5 and by reducing the concentration of Mg 2ϩ in the assay from 10 to 2 mm. Under likely physiological conditions (pH 7.0 and 2 mm Mg 2ϩ ), 10 mm Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PK III in the following order: Glc-6-P Ͼ mannose-6-P, fructose-1,6P 2 Ͼ ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PK III activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.Suc-P synthase (SPS) is a highly regulated enzyme that catalyzes the penultimate reaction in Suc synthesis in plants. One of the mechanisms for the regulation of SPS activity involves reversible protein phosphorylation. SPS is known to be phosphorylated on multiple seryl residues, with three of the sites potentially of regulatory significance (Huber and Huber, 1996; Toroser and Huber, 1997). The first site to be identified was Ser-158 of spinach SPS, which is the major site responsible for light/dark modulation of SPS activity (McMichael et al., 1993; Toroser et al., 1999). Ser-158 is potentially phosphorylated by several leaf protein kinases, one of which (the PK III enzyme) (McMichael et al., 1995; Toroser and Huber, 1997) is now known to be a SNF1-related protein kinase (SnRK1) (Douglas et al., 1997; Sugden et al., 1999).The control of the phosphorylation status of specific sites on SPS continues to be an area of considerable interest. With respect to Ser-158 and light/ dark modulation of SPS activity, changes in leaf metabolite pools are thought to play at least some role. Inorganic phosphate (Pi) inhibits the PP2A that dephosphorylates and activates phospho-SPS-158 (Weiner et al., 1992(Weiner et al., , 1993. Thus, the decrease in cytosolic [Pi] that may occur in response to induction of photosynthetic metabolism would increase PP2A activity in vivo and therefore promote SPS activation. As cytosolic [Pi] decreases, the concentration of P-esters would increase proportionally. This could also contribute to SPS activation, as Glc-6-P has been shown to inhibit the phosphorylation/inactivation of SPS in lea...

Sugar Sensing and Signaling

“…New findings now show that the evolutionarily conserved Arabidopsis protein kinases KIN10/11, catalytic subunits of the plant SnRK1 (Snf1-related protein kinase) heterotrimeric kinase complex, are master regulators of transcription networks and activate catabolism and repress anabolism under energy deprivation conditions (Baena-González et al, 2007;Baena-González and Sheen, 2008). Consistent with a role in energy depleting conditions, sugar phosphates (especially glucose-6-phosphate) have been shown to inhibit SnRK1 activity (Toroser et al, 2000). Genome-wide microarray analyses of KIN10 overexpressing cells and the characterization of kin10/11 double mutant plants reveal that the energy sensors KIN10/11 control transcriptional responses to darkness, sugar and stress conditions (BaenaGonzález et al, 2007;Baena-González and Sheen, 2008) (Fig.…”

The Trehalose 6-Phosphate/SnRK1 Signaling Pathway Primes Growth Recovery following Relief of Sink Limitation

“…S1, A and B). G6P and G1P inhibit SnRK1 (Toroser et al, 2000;Nunes et al, 2013), although far less potently than T6P. This milder effect on gene expression and smaller stimulation of growth compared with Suc control could be due to regulation of SnRK1 at least in part by these metabolites.…”

“…The changes in gene expression occurred in spite of no change in T6P content. It is known that Glc 6-P and Glc 1-P also inhibit SnRK1 (Toroser et al, 2000;Nunes et al, 2013). We measured these metabolites to determine if they could contribute to changes in SnRK1-induced gene expression in Glc-fed seedlings.…”

Acclimation of metabolism to light in Arabidopsis thaliana: the glucose 6‐phosphate/phosphate translocator GPT2 directs metabolic acclimation

“…G6P also increases the activity of SPS via regulation of the PKIII protein kinase (Huber & Huber 1996; Toroser et al . 2000). SnRK1, which activates sucrose synthase, is inhibited by G6P (Smeekens et al .…”