Evolution of the 14-3-3 protein family: Does the large number of isoforms in multicellular organisms reflect functional specificity?

14-3-3 proteins constitute a family of eukaryotic proteins that an key regu lators 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 prot eins. 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 p roteins have become mon frequent. An examination of 14-3-3 entries in the p ublic 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 Ara bidopsis thaliana. A phylogenetic analysis reveals that there are four majo r evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Meta zoa (animals). A close examination of the aligned amino acid sequences iden tifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear exp ort function. Given the fact that 53% of the protein is conserved, includin g all amino acid residues in the target binding groove of the 14-3-3 monome r, one might expect little to no isoform specificity for target protein bin ding. 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 r eason for the large number of isoforms found in multicellular organisms is isoform-specific functions.