Nonphosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase Is Phosphorylated in Wheat Endosperm at Serine-404 by an SNF1-Related Protein Kinase Allosterically Inhibited by Ribose-5-Phosphate    

Abstract

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg 2+ or Mn 2+(being Ca 2+ independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.

Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Is Phosphorylated during Seed Development

“…Searching to gain knowledge in the subject, we sought to find a condition under which NAD-GAPDH could be phosphorylated in vitro , following the methodology previously employed for similar studies performed with np-NADP-GAPDH (Piattoni et al, 2011). Recombinant NAD-GAPDH from wheat ( Tae NAD-GAPDH) was expressed and purified as described Piattoni et al (2013).…”

Master regulators in plant glucose signaling networks

“…Interestingly, plants appear to have evolved novel regulators of SNRK1, including myoinositol polyphosphate 5-phosphatase and cyclin-dependent kinase E1 as positive regulators (Ananieva et al, 2008; Ng et al, 2013), and a WD protein PRL1 (pleiotropic regulatory locus1), as well as ribose-5-phosphate, fructose-1,6-bisphosphate, 3-phosphoglycerate, glucose-6-phosphate and trehalose-6-phosphate as potential negative regulators (Bhalerao et al, 1999; Toroser et al, 2000; Zhang et al, 2009; Piattoni et al, 2011). Extensive genetic and biochemical analyses have supported a central role of trehalose-6-phosphate or trehalose metabolism in embryogenic, vegetative and inflorescence growth and branching, as well as the regulation of flowering (van Dijken et al, 2004; Schluepmann et al, 2004; Satoh-Nagasawa et al, 2006; Paul et al, 2007; Ramon et al, 2007; Chary et al, 2008; Zhang et al, 2009; Gomez et al, 2010; Delatte et al, 2011; Wahl et al, 2013).…”

Cloning and characterization of TaSnRK2.3, a novel SnRK2 gene in common wheat

“…In previous studies, we observed dynamic expression of TaSnRK2.4 and TaSnRK2.7–8 under various abiotic stresses; their overexpression resulted in enhanced tolerance to multi-environmental stresses (Mao et al ., 2010; Zhang et al ., 2010, 2011). Thus, solid evidence shows that the SnRK family of protein kinases is involved in multi-environmental stress responses and all have potential use for improvement of abiotic stress tolerance and yield enhancement (Piattoni et al ., 2011). …”