The results showed that V5-tagged PP2c (but not S744A mutant protein which has Ala instead of Ser in the mode 3 motif), directly bound to GST-14-3-3 with high affinity and to GST-II +I (heterodimerized recombinant proteins) with lower affinity (Figure 2A upper panel). far lower affinity to those peptides containing RSxis phosphoserine) than human 14-3-3 proteins, demonstrating the atypical target recognition by 14-3-3 proteins. We found that the putative protein phosphatase 2C (PP2c) binds to 14-3-3 proteins utilizing its mode 3 motif (Cgenome database, and tested their binding. As a result, 14-3-3 proteins interacted with three out of eight chimeric proteins including two with high affinity. Importantly, 14-3-3 proteins co-immunoprecipitated with an uncharacterized full-length protein containing identified high-affinity mode 3 motif, suggesting that both proteins form a complex 14-3-3 proteins with high affinity. Conclusion/Significance Because of the atypical target recognition of 14-3-3 proteins, no 14-3-3-binding proteins have been successfully identified in until now whereas over 200 human 14-3-3-binding proteins have been identified. This report describes the first discovery of the 14-3-3-binding proteins and their binding motifs. Veralipride The high-affinity phosphopeptide will be a powerful tool to identify novel 14-3-3-binding proteins. Introduction is the causative agent of sleeping sickness in man and nagana disease in cattle and one of the most divergent eukaryotes from mammals. The disease is spread by RAC1 the tsetse fly, in which the procyclic forms (PCF) proliferate and differentiate into bloodstream forms (BSF), the life stage that then proliferates in the mammalian host. The disease is fatal if left untreated and no effective drug is currently available for treatment of the late stage of the disease (i.e., involvement of the central nervous system). The 14-3-3 proteins are highly conserved dimeric acidic proteins acting as phosphoserine/phosphothreonine-dependent chaperones [1], [2]. Homologues of 14-3-3 proteins have been Veralipride found in all eukaryotes [3], [4]. Every organism expresses at least one 14-3-3 protein that binds to phosphopeptides containing consensus motifs (mode 1 and/or mode 2) with high affinity (nanomolar levels). The motifs include both RSxis phosphoserine Veralipride [5], and the recently identified C14-3-3I and II proteins play important roles in cell motility, cytokinesis and the cell cycle [14], phosphoserine-dependent 14-3-3-interacting proteins have not been found until now in spite of extensive efforts. Therefore, we examined the differences between human 14-3-3 isoforms and 14-3-3 isoforms with respect to affinities to various ligands. Here we provide several lines of evidence that the 14-3-3I, and especially the II, isoforms bind far less efficiently to the conventional consensus motifs (modes 1 and 2). In addition, heterodimerized 14-3-3I and II, the major existing forms in vivo ([14] and unpublished data), showed detectable affinities to the chimeric proteins containing the mode 3 motif, leading us to identify the 14-3-3 binding proteins. The overall data highlight the scarcity of 14-3-3 target proteins with high affinity in the cells and may indicate the divergent roles of 14-3-3 proteins. The newly identified phosphopeptide that binds to 14-3-3 proteins may be utilized in isolating a novel class of 14-3-3 binding proteins, since over 200 human 14-3-3-binding proteins can be purified from HeLa cell extracts by a competitive elution from 14-3-3 affinity columns with alternative mode 1 phosphopeptide or high-affinity peptide antagonist of 14-3-3 proteins [13], [15]. Results and Discussion 14-3-3 proteins only weakly bind with c-Raf and conventional consensus phosphopeptides Amino acid sequences of 14-3-3 proteins responsible for monomer stabilization, dimer formation and serine/threonine-phosphorylated motif binding are well conserved throughout the eukaryotes [8], [9], [10], [11]. The critical amino acid residues, with the exception of those responsible for dimer formation [9],.
The results showed that V5-tagged PP2c (but not S744A mutant protein which has Ala instead of Ser in the mode 3 motif), directly bound to GST-14-3-3 with high affinity and to GST-II +I (heterodimerized recombinant proteins) with lower affinity (Figure 2A upper panel)