Enzyme activity is regulated by several mechanisms, including phosphorylation. Phosphorylation is a key signal transduction process in all eukaryotic cells and is thus crucial for virtually all cellular processes. In addition to its direct effect on protein structure, phosphorylation also affects protein-protein interactions, such as binding to scaffolding 14-3-3 proteins, which selectively recognize phosphorylated motifs. These interactions then modulate the catalytic activity, cellular localisation and interactions of phosphorylated enzymes through different mechanisms. The aim of this mini-review is to highlight several examples of 14-3-3 protein-dependent mechanisms of enzyme regulation previously studied in our laboratory over the past decade. More specifically, we address here the regulation of the human enzymes ubiquitin ligase Nedd4-2, procaspase-2, calcium-calmodulin dependent kinases CaMKK1/2, and death-associated protein kinase 2 (DAPK2) and yeast neutral trehalase Nth1.

• MeSH
• Phosphorylation MeSH
• Humans MeSH
• 14-3-3 Proteins * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• 14-3-3 Proteins * MeSH

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

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.

• Keywords
• 14-3-3 protein, Estrogen Receptor, Nuclear receptors, PPI stabilization, protein–protein interactions,
• MeSH
• Estrogen Receptor alpha * genetics   metabolism   MeSH
• Estrogen Antagonists pharmacology   MeSH
• Humans MeSH
• Ligands MeSH
• Drug Discovery MeSH
• 14-3-3 Proteins * genetics   metabolism   MeSH
• Tamoxifen pharmacology   MeSH
• Protein Binding drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• afimoxifene MeSH Browser
• Estrogen Receptor alpha * MeSH
• Estrogen Antagonists MeSH
• Ligands MeSH
• 14-3-3 Proteins * MeSH
• Tamoxifen 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

Expansion of the polyglutamine tract in the N terminus of Ataxin-1 is the main cause of the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1). However, the C-terminal part of the protein - including its AXH domain and a phosphorylation on residue serine 776 - also plays a crucial role in disease development. This phosphorylation event is known to be crucial for the interaction of Ataxin-1 with the 14-3-3 adaptor proteins and has been shown to indirectly contribute to Ataxin-1 stability. Here we show that 14-3-3 also has a direct anti-aggregation or "chaperone" effect on Ataxin-1. Furthermore, we provide structural and biophysical information revealing how phosphorylated S776 in the intrinsically disordered C terminus of Ataxin-1 mediates the cytoplasmic interaction with 14-3-3 proteins. Based on these findings, we propose that 14-3-3 exerts the observed chaperone effect by interfering with Ataxin-1 dimerization through its AXH domain, reducing further self-association. The chaperone effect is particularly important in the context of SCA1, as it was previously shown that a soluble form of mutant Ataxin-1 is the major driver of pathology.

• Keywords
• HDX-MS, SAXS, crystal structure, neurodegeneration, protein aggregation,
• MeSH
• Ataxin-1 chemistry   metabolism   MeSH
• Cell Line MeSH
• Cytoplasm metabolism   MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Protein Domains MeSH
• 14-3-3 Proteins metabolism   MeSH
• Protein Stability MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ataxin-1 MeSH
• ATXN1 protein, human MeSH Browser
• 14-3-3 Proteins MeSH

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

• Keywords
• 14-3-3 protein, 70-kDa heat shock protein (Hsp70), HSP70, Hsp70, Tomm34, dimerization, hydrogen-deuterium exchange, molecular chaperone, phosphorylation, protein folding, protein import, protein kinase A (PKA), protein-nucleic acid interaction, protein–protein interaction, translocase of outer mitochondrial membrane 34 (TOMM34),
• MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Phosphorylation physiology   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Molecular Chaperones metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• 14-3-3 Proteins metabolism   MeSH
• HSP70 Heat-Shock Proteins metabolism   MeSH
• HSP72 Heat-Shock Proteins metabolism   MeSH
• HSP90 Heat-Shock Proteins metabolism   MeSH
• Signal Transduction MeSH
• Transcription Factors genetics   metabolism   MeSH
• Mitochondrial Membrane Transport Proteins genetics   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BCL2-associated athanogene 1 protein MeSH Browser
• DNA-Binding Proteins MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Proteins MeSH
• Molecular Chaperones MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• 14-3-3 Proteins MeSH
• HSP70 Heat-Shock Proteins MeSH
• HSP72 Heat-Shock Proteins MeSH
• HSP90 Heat-Shock Proteins MeSH
• TOMM34 protein, human MeSH Browser
• Transcription Factors MeSH
• Mitochondrial Membrane Transport Proteins 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

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.

• Keywords
• 14-3-3 protein, apoptosis signal-regulating kinase 1 (ASK1), fluorescence, nuclear magnetic resonance (NMR), protein cross-linking, small-angle x-ray scattering (SAXS),
• MeSH
• X-Ray Diffraction MeSH
• Phosphorylation MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Scattering, Small Angle MeSH
• MAP Kinase Kinase Kinase 5 antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• 14-3-3 Proteins chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MAP Kinase Kinase Kinase 5 MeSH
• MAP3K5 protein, human MeSH Browser
• 14-3-3 Proteins MeSH
• YWHAE protein, human MeSH Browser

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.

• Keywords
• 14-3-3 protein, fluorescence, hydrogen-deuterium exchange, phosducin, protein complex, protein phosphorylation, small-angle x-ray scattering (SAXS),
• MeSH
• Phosphoproteins chemistry   genetics   metabolism   MeSH
• Phosphorylation MeSH
• Rats MeSH
• Humans MeSH
• Models, Molecular MeSH
• Eye Proteins chemistry   genetics   metabolism   MeSH
• 14-3-3 Proteins chemistry   genetics   metabolism   MeSH
• GTP-Binding Protein Regulators chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Binding Sites 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
• Phosphoproteins MeSH
• Eye Proteins MeSH
• phosducin MeSH Browser
• 14-3-3 Proteins MeSH
• GTP-Binding Protein Regulators MeSH
• YWHAZ protein, human MeSH Browser

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