The 14-3-3 protein family - Conserved structure, diverse functions

Abstract: While once abstruse, 14-3-3 proteins are now well known as players in several cellular processes. These processes range from apoptosis and mitosis in mammals and yeast, to regulation of primary metabolism in plants. To date, 104 proteins have been shown to interact with 14-3-3s of which 85 are found in mammals and yeast, and 21 are found in plants. Their consensus action is to bind to phosphorylated motifs within target proteins resulting in activation, inhibition, stabilization, or translocation. Although 14-3-3s have been crystallized, are ubiquitous in all Eukaryotes, and extremely well studied, several questions regarding their mechanism of interaction with other proteins remain. In plants, 14-3-3s inhibit enzymes in primary metabolism such as nitrate reductase and sucrose phosphate synthase, leading to accumulation of substrates, whilst the plasma membrane (PM) H+ATPase is activated. At present, the PM H+ATPase 14-3-3 interaction is the best characterized in plants. The identification of the deviating 14-3-3 binding motif in the PM H+ATPase allowed examination of the possibility of isoform specificity. Indeed, differences were observed in binding specificity between nine different Arabidopsis thaliana14-3-3 isoforms and a phosphopeptide corresponding to the binding motif in the A. thaliana PM H+ATPase isoform AHA2. Examinations of the three-dimensional structure of 14-3-3s have been made in an attempt to identify residues responsible for functional specificity. With the presumption that evolutionary insight may allow more exact extrapolation of observations made in unicellular organisms to multicellular organisms, we data-mined public databases, identifying all 14-3-3 isoforms present. Phylogenetic analysis showed kingdom-wise clustering. Within Metazoa, isoforms grouped between species, whilst isoform groupings observed within Viridiplantae occurred within species. When examining only full-length plant 14-3-3s approximately 42% of all residues are conserved, at a threshold of 98%, with an apparently higher percentage within each cluster. This large divergence, resulting from numerous gene duplications, negates the possibility of functional extrapolation outside of families of species in plants. The inability of all nine tested A. thaliana 14-3-3 isoforms to bind to a phosphopeptide corresponding to the motif in the A. thaliana PM H+ATPase isoform AHA9 suggested the existence of additional 14-3-3 isoforms in A. thaliana. The completion of the Arabidopsis genome sequencing allowed identification of all 14-3-3 genes, authentic and putative. In addition to the 10 previously cloned 14-3-3s in A. thaliana, five novel genes were identified. Subsequent expression analysis showed that two out of the five were transcribed, bringing the total number of expressed isoforms to twelve.