Phosphorylation-dependent interactions between enzymes of plant metabolismand 14-3-3 proteins

Far-Western overlays of soluble extracts of cauliflower revealed many prote ins that bound to digoxygenin (DIG)labelled 14-3-3 proteins. Binding to DIG -14-3-3s was prevented by prior dephosphorylation of the extract proteins o r by competition with 14-3-3-binding phosphopeptides, indicating that the 1 4-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 an tibodies, demonstrating that they were bound to endogenous plant 14-3-3 pro teins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatog raphy on immobilised 14-3-3 proteins and specific elution with a 14-3-3-bin ding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phos phate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a pr otein (LIM17) that has been implicated in early floral development, an appr oximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-depen dent protein kinase that was capable of phosphorylating and rendering nitra te reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast t o the phosphorylated NR-14-3-3 complex which is activated by dissociation w ith 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase acti vity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosp hopeptide and the phosphopeptide-inhibited activity was reactivated by addi ng excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulati ng several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-bi nding status of defined target proteins change in response to extracellular stimuli.