Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog

Abstract

Arabidopsis GF14 omega was originally described because of its apparent association with a DNA-protein complex; it is a member of the 14-3-3 kinase regulatory protein family that is conserved throughout eukaryotes. Here, we demonstrated that recombinant GF14 omega is expressed in Escherichia coli as a dimer. Blot binding and electrophoretic mobility shift analyses indicated that GF14 omega binds calcium. Equilibrium dialysis further demonstrated that GF14 omega binds an equimolar amount of calcium with an apparent binding constant of 5.5 x 10(4) M-1 under physiological conditions. The C-terminal domain, which contains a potential EF hand motif, is responsible for the calcium binding. The C-terminal domain also cross-reacted with the anti-GF14 omega monoclonal antibody. In addition, GF14 omega is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro. Therefore, GF14 omega protein has biochemical properties consistent with potential signaling roles in plants. The presence of a potential EF hand-like motif in the highly conserved C terminus of 14-3-3 proteins together with the calcium-dependent multiple functions attributed to the 14-3-3 proteins indicate that the C terminus EF hand is a common functional element of this family of proteins.