Phosphomimetic mutation of a conserved serine residue in Arabidopsis thaliana 14-3-3ω suggests a regulatory role of phosphorylation in dimerization and target interactions.

14-3-3s are evolutionarily conserved eukaryotic regulatory proteins that are involved in diverse biological processes. The common mode of action for the 14-3-3 proteins is through the binding of phosphorylated target proteins. In many species, multiple 14-3-3 isoforms exist and these different isoforms can exhibit distinct ranges of target interactions. The dimerization of 14-3-3s is central to their function. 14-3-3 isoforms can form different combinations of homo- and heterodimers, which contribute to the broad functional diversity of the family. In this study, we showed that phosphomimetic mutation of a conserved serine residue in the dimerization interface of 14-3-3 isoforms, Ser-62, not only affects the ability of Arabidopsis 14-3-3ω to form homodimers, but alters the range of 14-3-3 family members with which it can form heterodimers. Furthermore, we demonstrated that the phosphorylation status of Ser-62 can regulate the binding of 14-3-3ω to target proteins, suggesting that Ser-62 might be a conserved key element to modulate target binding in both plants and animals.

Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors.

Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs). The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood. In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A. thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.