Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase.
- 14-3-3 protein
- calcium-dependent protein kinase
- kinase assay
- protein–protein interaction
- tyrosine phosphorylation
Abbreviations: BGG, bovine γ-globulin; CBB, Coomassie Brilliant Blue; CID, collision-induced dissociation; CPK, calcium-dependent protein kinase; dMS, differential MS; ETD, electron transfer dissociation; IGF-1, insulin-like growth factor 1; NR, nitrate reductase; NTVD, N-terminal variable domain; PKCD, protein kinase catalytic domain; pNR, phosphorylated NR; λ-PPase, λ-phosphatase; PQD, Pro-Q Diamond; SPS, sucrose-phosphate synthase
- © The Authors Journal compilation © 2014 Biochemical Society