PHOSPHORYLATION OF NON-PHOSPHORYLATING GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM WHEAT

PIATTONI, C.V; BUSTOS, DM; GUERRERO, SA; IGLESIAS, AA

Mar del Plata

Congreso; SAIB; 2007

Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular

Non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (EC: 1.2.1.9; NP-GAPDHase) is modified by phosphorylation in heterotrophic plant cells. Phosphorylation is a prerequisite for interaction with regulatory 14-3-3 proteins, followed by a decrease in activity. We heterologously expressed the wheat NP-GAPDHase in Escherichia coli. The recombinant enzyme was purified and utilized to study its phosphorylation by wheat endosperm extracts, under reaction conditions specific for SnRK, GSK-3, and MAPK kinases. NP-GAPDHase was better phosphorylated by a SnRK kinase. The latter was partially purified and characterized. The kinase required Mn2+ or Mg2+ for activity, with optimal conditions reached at 2.5 mM or 5 mM of the respective essential divalent cation. Ca2+ was ineffective. Interestingly, as previously reported for plant SnRK kinase, it was inhibited by glucose-6-P. NP-GAPDHase has two putative phosphorylation sites, Ser404 and Ser447. Studies with single mutants S404A and S447A, showed that Ser404 is critical for phosphorylation of the enzyme. These results are consistent with the Ser404 being located in a protein domain characteristic of a site for phosphorylation by SnRK kinase. Also, it is suggested that NP-GAPDHase could be regulated in vivo, together with other enzymes, in agreement with a system for the control of carbon and energy metabolism.