Cell signaling regulates several physiological processes by receiving, processing, and transmitting signals between the extracellular and intracellular environments. In signal transduction, phosphorylation is a crucial effector as the most common posttranslational modification. Selectively recognizing specific phosphorylated motifs of target proteins and modulating their functions through binding interactions, the yeast 14-3-3 proteins Bmh1 and Bmh2 are involved in catabolite repression, carbon metabolism, endocytosis, and mitochondrial retrograde signaling, among other key cellular processes. These conserved scaffolding molecules also mediate crosstalk between ubiquitination and phosphorylation, the spatiotemporal control of meiosis, and the activity of ion transporters Trk1 and Nha1. In humans, deregulation of analogous processes triggers the development of serious diseases, such as diabetes, cancer, viral infections, microbial conditions and neuronal and age-related diseases. Accordingly, the aim of this review article is to provide a brief overview of the latest findings on the functions of yeast 14-3-3 proteins, focusing on their role in modulating the aforementioned processes.

• Keywords
• 14-3-3 proteins, adaptor protein, molecular mechanism, phosphorylation, protein-protein interaction, scaffolding, yeast,
• Publication type
• Journal Article MeSH
• Review MeSH

Potassium is an essential intracellular ion, and a sufficient intracellular concentration of it is crucial for many processes; therefore it is fundamental for cells to precisely regulate K+ uptake and efflux through the plasma membrane. The uniporter Trk1 is a key player in K+ acquisition in yeasts. The TRK1 gene is expressed at a low and stable level; thus the activity of the transporter needs to be regulated at a posttranslational level. S. cerevisiae Trk1 changes its activity and affinity for potassium ion quickly and according to both internal and external concentrations of K+, as well as the membrane potential. The molecular basis of these changes has not been elucidated, though phosphorylation is thought to play an important role. In this study, we examined the role of the second, short, and highly conserved intracellular hydrophilic loop of Trk1 (IL2), and identified two phosphorylable residues (Ser882 and Thr900) as very important for 1) the structure of the loop and consequently for the targeting of Trk1 to the plasma membrane, and 2) the upregulation of the transporter's activity reaching maximal affinity under low external K+ conditions. Moreover, we identified three residues (Thr155, Ser414, and Thr900) within the Trk1 protein as strong candidates for interaction with 14-3-3 regulatory proteins, and showed, in an in vitro experiment, that phosphorylated Thr900 of the IL2 indeed binds to both isoforms of yeast 14-3-3 proteins, Bmh1 and Bmh2.

• Keywords
• 14–3–3 proteins, Phosphorylation, Potassium ion uptake, Saccharomyces cerevisiae, Trk1,
• Publication type
• Journal Article MeSH

Signal transduction cascades efficiently transmit chemical and/or physical signals from the extracellular environment to intracellular compartments, thereby eliciting an appropriate cellular response. Most often, these signaling processes are mediated by specific protein-protein interactions involving hundreds of different receptors, enzymes, transcription factors, and signaling, adaptor and scaffolding proteins. Among them, 14-3-3 proteins are a family of highly conserved scaffolding molecules expressed in all eukaryotes, where they modulate the function of other proteins, primarily in a phosphorylation-dependent manner. Through these binding interactions, 14-3-3 proteins participate in key cellular processes, such as cell-cycle control, apoptosis, signal transduction, energy metabolism, and protein trafficking. To date, several hundreds of 14-3-3 binding partners have been identified, including protein kinases, phosphatases, receptors and transcription factors, which have been implicated in the onset of various diseases. As such, 14-3-3 proteins are promising targets for pharmaceutical interventions. However, despite intensive research into their protein-protein interactions, our understanding of the molecular mechanisms whereby 14-3-3 proteins regulate the functions of their binding partners remains insufficient. This review article provides an overview of the current state of the art of the molecular mechanisms whereby 14-3-3 proteins regulate their binding partners, focusing on recent structural studies of 14-3-3 protein complexes.

• Keywords
• 14-3-3 proteins, adaptor protein, molecular mechanism, phosphorylation, protein-protein interactions, scaffolding,
• Publication type
• Journal Article MeSH
• Review MeSH

The 14-3-3 proteins, a family of highly conserved scaffolding proteins ubiquitously expressed in all eukaryotic cells, interact with and regulate the function of several hundreds of partner proteins. Yeast neutral trehalases (Nth), enzymes responsible for the hydrolysis of trehalose to glucose, compared with trehalases from other organisms, possess distinct structure and regulation involving phosphorylation at multiple sites followed by binding to the 14-3-3 protein. Here we report the crystal structures of yeast Nth1 and its complex with Bmh1 (yeast 14-3-3 isoform), which, together with mutational and fluorescence studies, indicate that the binding of Nth1 by 14-3-3 triggers Nth1's activity by enabling the proper 3D configuration of Nth1's catalytic and calcium-binding domains relative to each other, thus stabilizing the flexible part of the active site required for catalysis. The presented structure of the Bmh1:Nth1 complex highlights the ability of 14-3-3 to modulate the structure of a multidomain binding partner and to function as an allosteric effector. Furthermore, comparison of the Bmh1:Nth1 complex structure with those of 14-3-3:serotonin N-acetyltransferase and 14-3-3:heat shock protein beta-6 complexes revealed similarities in the 3D structures of bound partner proteins, suggesting the highly conserved nature of 14-3-3 affects the structures of many client proteins.

• Keywords
• 14-3-3 protein, allostery, crystal structure, enzyme, trehalase,
• MeSH
• Arylalkylamine N-Acetyltransferase metabolism   MeSH
• Databases, Chemical * MeSH
• Phosphorylation MeSH
• Glucose metabolism   MeSH
• Catalytic Domain MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Protein Domains MeSH
• 14-3-3 Proteins genetics   metabolism   MeSH
• Heat-Shock Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae enzymology   genetics   metabolism   MeSH
• Trehalase chemistry   metabolism   MeSH
• Trehalose metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arylalkylamine N-Acetyltransferase MeSH
• Glucose MeSH
• NTH1 protein, S cerevisiae MeSH Browser
• 14-3-3 Proteins MeSH
• Heat-Shock Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Trehalase MeSH
• Trehalose MeSH

Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.

• Keywords
• 14–3-3, Bmh, Calcium, Enzyme Mechanisms, H/D Exchange, Mass Spectrometry (MS), Neutral Trehalase, Protein Cross-linking, Protein Structure, SAXS,
• MeSH
• Enzyme Activation MeSH
• Catalytic Domain MeSH
• Models, Molecular MeSH
• EF Hand Motifs * MeSH
• 14-3-3 Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae enzymology   MeSH
• Amino Acid Sequence MeSH
• Trehalase chemistry   metabolism   MeSH
• Calcium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BMH1 protein, S cerevisiae MeSH Browser
• 14-3-3 Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Trehalase MeSH
• Calcium MeSH

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the G(t)βγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function.

• MeSH
• Spectrometry, Fluorescence MeSH
• Phosphatidylcholines MeSH
• Phosphoproteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Rats MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Eye Proteins chemistry   metabolism   MeSH
• 14-3-3 Proteins metabolism   MeSH
• GTP-Binding Protein Regulators chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Serine metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Tryptophan MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-myristoyl-2-(12-((5-dimethylamino-1-naphthalenesulfonyl)amino)dodecanoyl)-sn-glycero-3-phosphocholine MeSH Browser
• Phosphatidylcholines MeSH
• Phosphoproteins MeSH
• Eye Proteins MeSH
• phosducin MeSH Browser
• 14-3-3 Proteins MeSH
• GTP-Binding Protein Regulators MeSH
• Serine MeSH
• Tryptophan MeSH
• YWHAZ protein, human MeSH Browser

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.

• MeSH
• Circular Dichroism MeSH
• Phosphorylation MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Scattering, Small Angle MeSH
• 14-3-3 Proteins chemistry   genetics   metabolism   MeSH
• GTPase-Activating Proteins chemistry   genetics   metabolism   MeSH
• RGS Proteins MeSH
• GTP-Binding Proteins chemistry   genetics   metabolism   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Protein Structure, Secondary MeSH
• Signal Transduction MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 14-3-3 Proteins MeSH
• GTPase-Activating Proteins MeSH
• RGS Proteins MeSH
• GTP-Binding Proteins MeSH
• RGS3 protein, human MeSH Browser