14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H(+)ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions.

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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.

In most higher eukaryotes, the predominantly phosphoprotein-binding 14-3-3 proteins are the products of a multigene family, with many organisms having 10 or more family members. However, current models for 14-3-3/phosphopeptide interactions suggest that there is little specificity among 14-3-3s for diverse phosphopeptide clients. Therefore, the existence of sequence diversity among 14-3-3s within a single organism begs questions regarding the in vivo specificities of the interactions between the various 14-3-3s and their clients. Chief among those questions is, Do the different 14-3-3 isoforms interact with different clients within the same cell? Although the members of the Arabidopsis 14-3-3 family of proteins typically contain highly conserved regions of sequence, they also display distinctive variability with deep evolutionary roots. In the current study, a survey of several Arabidopsis 14-3-3/GFP fusions revealed that 14-3-3s demonstrate distinct and differential patterns of subcellular distribution, by using trichomes and stomate guard cells as in vivo experimental cellular contexts. The effects of client interaction on 14-3-3 localization were further analyzed by disrupting the partnering with peptide and chemical agents. Results indicate that 14-3-3 localization is both isoform specific and highly dependent upon interaction with cellular clients.

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