Phosphorylation‐dependent interactions between enzymes of plant metabolism and 14‐3‐3 proteins

Summary Far‐Western overlays of soluble extracts of cauliflower revealed many proteins 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 or by competition with 14‐3‐3‐binding phosphopeptides, indicating that the 14‐3‐3 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 antibodies, demonstrating that they were bound to endogenous plant 14‐3‐3 proteins. 14‐3‐3‐binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14‐3‐3 proteins and specific elution with a 14‐3‐3‐binding phosphopeptide. Purified 14‐3‐3‐binding proteins included sucrose–phosphate synthase, trehalose‐6‐phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium‐dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14‐3‐3 proteins. In contrast to the phosphorylated NR‐14‐3‐3 complex which is activated by dissociation with 14‐3‐3‐binding phosphopeptides, the total sugar–phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14‐3‐3‐binding phosphopeptide and the phosphopeptide‐inhibited activity was reactivated by adding excess 14‐3‐3 proteins. Thus, 14‐3‐3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14‐3‐3‐binding status of defined target proteins change in response to extracellular stimuli.