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

Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating

Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating

14-3-3 Proteins Interact with a Hybrid Prenyl-Phosphorylation Motif to Inhibit G Proteins

Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins

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

Molecular Evolution of Multiple Arylalkylamine N-Acetyltransferase (AANAT) in Fish

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to arylalkylamines, including indolethylamines and phenylethylamines. Multiple aanats are present in teleost fish as a result of whole genome and gene duplications. Fish aanat1a and aanat2 paralogs display different patterns of tissue expression and encode proteins with different substrate preference: AANAT1a is expressed in the retina, and acetylates both indolethylamines and phenylethylamines; while AANAT2 is expressed in the pineal gland, and preferentially acetylates indolethylamines. The two enzymes are therefore thought to serve different roles. Here, the molecular changes that led to their specialization were studied by investigating the structure-function relationships of AANATs in the gilthead seabream (sb, Sperus aurata). Acetylation activity of reciprocal mutated enzymes pointed to specific residues that contribute to substrate specificity of the enzymes. Inhibition tests followed by complementary analyses of the predicted three-dimensional models of the enzymes, suggested that both phenylethylamines and indolethylamines bind to the catalytic pocket of both enzymes. These results suggest that substrate selectivity of AANAT1a and AANAT2 is determined by the positioning of the substrate within the catalytic pocket, and its accessibility to catalysis. This illustrates the evolutionary process by which enzymes encoded by duplicated genes acquire different activities and play different biological roles

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

Chimeric 14-3-3 proteins for unraveling interactions with intrinsically disordered partners

In eukaryotes, several "hub" proteins integrate signals from different interacting partners that bind through intrinsically disordered regions. The 14-3-3 protein hub, which plays wide-ranging roles in cellular processes, has been linked to numerous human disorders and is a promising target for therapeutic intervention. Partner proteins usually bind via insertion of a phosphopeptide into an amphipathic groove of 14-3-3. Structural plasticity in the groove generates promiscuity allowing accommodation of hundreds of different partners. So far, accurate structural information has been derived for only a few 14-3-3 complexes with phosphopeptide-containing proteins and a variety of complexes with short synthetic peptides. To further advance structural studies, here we propose a novel approach based on fusing 14-3-3 proteins with the target partner peptide sequences. Such chimeric proteins are easy to design, express, purify and crystallize. Peptide attachment to the C terminus of 14-3-3 via an optimal linker allows its phosphorylation by protein kinase A during bacterial co-expression and subsequent binding at the amphipathic groove. Crystal structures of 14-3-3 chimeras with three different peptides provide detailed structural information on peptide-14-3-3 interactions. This simple but powerful approach, employing chimeric proteins, can reinvigorate studies of 14-3-3/phosphoprotein assemblies, including those with challenging low-affinity partners, and may facilitate the design of novel biosensors

Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

Sox9 is a transcription factor that is required for tissue development in mammals. In general, such transcription factors require co-regulators for precise temporal and spatial control of the activation and inactivation of the numerous genes necessary for precise development during embryogenesis. Here we identify a new Sox9 co-regulator: Using affinity chromatography with immobilized Sox9 protein, we identified exportin 4 (Exp4) as an interacting protein of Sox9 in human cultured cells. Interaction between endogenous Exp4 and Sox9 proteins was confirmed in the human osteosarcoma U2OS cells by immunoprecipitation experiments using anti-Sox9 antibody. siRNA depletion of Exp4 enhanced transcription of Sox9 target genes in U2OS cells, but did not affect nuclear localization of Sox9. These results suggest that Exp4 regulates Sox9 activity in the nucleus. Furthermore we found that the HMG box of Sox9 was responsible for binding to Exp4, and the HMG box was required for suppression of Sox9-mediated transcription. This contrasts with the known Sox9 co-regulators which bind to its transcriptional activation domain. Chromatin immunoprecipitation analyses revealed that Exp4 prevents Sox9 binding to the enhancers of its target genes. These results demonstrate that Exp4 acts as a Sox9 co-regulator that directly regulates binding of Sox9 to its target genes

Cross-Regulation between Oncogenic BRAFV600E Kinase and the MST1 Pathway in Papillary Thyroid Carcinoma

BACKGROUND:The BRAF(V600E) mutation leading to constitutive signaling of MEK-ERK pathways causes papillary thyroid cancer (PTC). Ras association domain family 1A (RASSF1A), which is an important regulator of MST1 tumor suppressor pathways, is inactivated by hypermethylation of its promoter region in 20 to 32% of PTC. However, in PTC without RASSF1A methylation, the regulatory mechanisms of RASSF1A-MST1 pathways remain to be elucidated, and the functional cooperation or cross regulation between BRAF(V600E) and MST1,which activates Foxo3,has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS:The negative regulators of the cell cycle, p21 and p27, are strongly induced by transcriptional activation of FoxO3 in BRAF(V600E) positive thyroid cancer cells. The FoxO3 transactivation is augmented by RASSF1A and the MST1 signaling pathway. Interestingly, introduction of BRAF(V600E)markedly abolished FoxO3 transactivation and resulted in the suppression of p21 and p27 expression. The suppression of FoxO3 transactivation by BRAF(V600E)is strongly increased by coexpression of MST1 but it is not observed in the cells in which MST1, but not MST2,is silenced. Mechanistically, BRAF(V600E)was able to bind to the C-terminal region of MST1 and resulted in the suppression of MST1 kinase activities. The induction of the G1-checkpoint CDK inhibitors, p21 and p27,by the RASSF1A-MST1-FoxO3 pathway facilitates cellular apoptosis, whereas addition of BRAF(V600E) inhibits the apoptotic processes through the inactivation of MST1. Transgenic induction of BRAF(V600E)in the thyroid gland results in cancers resembling human papillary thyroid cancers. The development of BRAF(V600E)transgenic mice with the MST1 knockout background showed that these mice had abundant foci of poorly differentiated carcinomas and large areas without follicular architecture or colloid formation. CONCLUSIONS/SIGNIFICANCE:The results of this study revealed that the oncogenic effect of BRAF(V600E) is associated with the inhibition of MST1 tumor suppressor pathways, and that the activity of RASSF1A-MST1-FoxO3 pathways determines the phenotypes of BRAF(V600E) tumors