The yeast 14-3-3 proteins Bmh1 and Bmh2 regulate key signaling pathways

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

Modulators of 14-3-3 Protein-Protein Interactions

Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein-protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as 'undruggable' targets, the last two decades have seen an increasing number of successful examples of PPI modulators resulting in a growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This Perspective focuses on the hub protein 14-3-3, which has several hundred identified protein interaction partners and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide-mimetics and natural products

A Structural Study of the Cytoplasmic Chaperone Effect of 14-3-3 Proteins on Ataxin-1

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

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

Structural Insights Support Targeting ASK1 Kinase for Therapeutic Interventions

Apoptosis signal-regulating kinase (ASK) 1, a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, modulates diverse responses to oxidative and endoplasmic reticulum (ER) stress and calcium influx. As a crucial cellular stress sensor, ASK1 activates c-Jun N-terminal kinases (JNKs) and p38 MAPKs. Their excessive and sustained activation leads to cell death, inflammation and fibrosis in various tissues and is implicated in the development of many neurological disorders, such as Alzheimer’s, Parkinson’s and Huntington disease and amyotrophic lateral sclerosis, in addition to cardiovascular diseases, diabetes and cancer. However, currently available inhibitors of JNK and p38 kinases either lack efficacy or have undesirable side effects. Therefore, targeted inhibition of their upstream activator, ASK1, stands out as a promising therapeutic strategy for treating such severe pathological conditions. This review summarizes recent structural findings on ASK1 regulation and its role in various diseases, highlighting prospects for ASK1 inhibition in the treatment of these pathologies

14-3-3-protein regulates Nedd4-2 by modulating interactions between HECT and WW domains

Neural precursor cell expressed developmentally down-regulated 4 ligase (Nedd4-2) is an E3 ubiquitin ligase that targets proteins for ubiquitination and endocytosis, thereby regulating numerous ion channels, membrane receptors and tumor suppressors. Nedd4-2 activity is regulated by autoinhibition, calcium binding, oxidative stress, substrate binding, phosphorylation and 14-3-3 protein binding. However, the structural basis of 14-3-3-mediated Nedd4-2 regulation remains poorly understood. Here, we combined several techniques of integrative structural biology to characterize Nedd4-2 and its complex with 14-3-3. We demonstrate that phosphorylated Ser$^{342}$ and Ser$^{448}$ are the key residues that facilitate 14-3-3 protein binding to Nedd4-2 and that 14-3-3 protein binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Overall, our findings provide the structural glimpse into the 14-3-3-mediated Nedd4-2 regulation and highlight the potential of the Nedd4-2:14-3-3 complex as a pharmacological target for Nedd4-2-associated diseases such as hypertension, epilepsy, kidney disease and cancer

14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory phosphosites

Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. However, the structural basis of 14-3-3-mediated DAPK2 regulation remains unclear. Here, we structurally and biochemically characterize the full-length human DAPK2:14-3-3 complex by combining several biophysical techniques. The results from our X-ray crystallographic analysis revealed that Thr369 phosphorylation at the DAPK2 C terminus creates a high-affinity canonical mode III 14-3-3-binding motif, further enhanced by the diterpene glycoside Fusicoccin A. Moreover, concentration-dependent DAPK2 dimerization is disrupted by Ca$^{2+}$/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca$^{2+}$/CaM binding. Overall, our findings provide mechanistic insights into 14-3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti‐inflammatory therapies

CaMKK2 kinase domain interacts with the autoinhibitory region through the N-terminal lobe including the RP insert

alcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a member of the Ca$^{⁠2+}$/calmodulin-dependent kinase (CaMK) family, functions as an upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase. Thus, CaMKK2 is involved in the regulation of several key physiological and pathophysiological processes. Previous studies have suggested that Ca$^{⁠2+}$/CaM binding may cause unique conformational changes in the CaMKKs compared with other CaMKs. However, the underlying mechanistic details remain unclear.MethodsIn this study, hydrogen-deuterium exchange coupled to mass spectrometry, time-resolved fluorescence spectroscopy, small-angle x-ray scattering and chemical cross-linking were used to characterize Ca$^{⁠2+}$/CaM binding-induced structural changes in CaMKK2.ResultsOur data suggest that: (i) the CaMKK2 kinase domain interacts with the autoinhibitory region (AID) through the N-terminal lobe of the kinase domain including the RP insert, a segment important for targeting downstream substrate kinases; (ii) Ca$^{⁠2+}$/CaM binding affects the structure of several regions surrounding the ATP-binding pocket, including the activation segment; (iii) although the CaMKK2:Ca$^{⁠2+}$/CaM complex shows high conformational flexibility, most of its molecules are rather compact; and (iv) AID-bound Ca$^{⁠2+}$/CaM transiently interacts with the CaMKK2 kinase domain.ConclusionsInteractions between the CaMKK2 kinase domain and the AID differ from those of other CaMKs. In the absence of Ca$^{⁠2+}$/CaM binding the autoinhibitory region inhibits CaMKK2 by both blocking access to the RP insert and by affecting the structure of the ATP-binding pocket.General significanceOur results corroborate the hypothesis that Ca$^{⁠2+}$/CaM binding causes unique conformational changes in the CaMKKs relative to other CaMKs