Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases.
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.
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
Kirby N
KN
Swatek
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
Rashaun S
RS
Wilson
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
Nagib
N
Ahsan
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
Rebecca L
RL
Tritz
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
Jay J
JJ
Thelen
D048948:14-3-3 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011897:Random Allocation; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D048948:14-3-3 Proteins; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases