Structural characterization of a unique interface between carbohydrate response element binding protein (ChREBP) and 14-3-3β
ABSTRACT Carbohydrate response element binding protein (ChREBP) is an insulin independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrate to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose responsive ChREBP nuclear import and export. Here we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4Åresolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well-defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β:ChREBP α2 complex.
SourceAvailable from: Nikolai Sluchanko[Show abstract] [Hide abstract]
ABSTRACT: Molecular mechanisms governing selective binding of a huge number of various phosphorylated protein partners to 14-3-3 remain obscure. Phosphate can bind to 14-3-3 and therefore being present at high intracellular concentration, which undergoes significant changes under physiological conditions, phosphate can theoretically regulate interaction of 14-3-3 with phosphorylated targets. In order to check this hypothesis we analyzed effect of phosphate and other natural abundant anions on interaction of 14-3-3 with phosphorylated human small heat shock protein HspB6 (Hsp20) participating in regulation of different intracellular processes. Inorganic phosphate, glycerol-1-phosphate and glycerol-2-phosphate at physiologically relevant concentrations (5-15 mM) significantly destabilized complexes formed by 14-3-3ζ and phosphorylated HspB6 (pHspB6), presumably, via direct interaction with the substrate-binding site of 14-3-3. Phosphate also destabilized complexes between pHspB6 and 14-3-3γ or the monomeric mutant form of 14-3-3ζ. Inorganic sulfate and pyrophosphate were less effective in modulation of 14-3-3 interaction with its target protein. The inhibitory effect of all anions on pHspB6/14-3-3 interaction was concentration-dependent. It is hypothesized that physiological changes in phosphate anions concentration can modulate affinity and specificity of interaction of 14-3-3 with its multiple targets and therefore the actual phosphointeractome of 14-3-3.PLoS ONE 08/2013; 8(8):e72597. DOI:10.1371/journal.pone.0072597 · 3.53 Impact Factor
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ABSTRACT: The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in converting excess carbohydrate to storage fat in liver. In response to changing glucose levels, ChREBP activity is regulated by nucleo-cytoplasmic shuttling of ChREBP via interactions with 14-3-3 proteins and importins. The nuclear/ cytosol trafficking is regulated partly by phosphorylation/dephosphorylation of serine-196 mediated by cAMP-dependent protein kinase and protein phosphatase. We show here that protein-free extracts of starved and high fat-fed livers contain metabolites that activate interaction of ChREBP/14-3-3 and inhibit ChREBP-importin α interaction, resulting in cytosolic localization. These metabolites were identified as β-hydroxybutyrate (βHB) and acetoacetate (AcAc). Nuclear localization of GFP-ChREBP is rapidly inhibited in hepatocytes incubated in βHB or fatty acids, and the observed inhibition is closely correlated with the production of ketone bodies. These observations show that ketone bodies play an important role in regulation of ChREBP activity by restricting ChREBP localization to the cytoplasm, thus inhibiting fat synthesis during periods of ketosis.Journal of Biological Chemistry 08/2013; DOI:10.1074/jbc.M113.498550 · 4.60 Impact Factor
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ABSTRACT: The tyrosine 3-monooxygenase-tryptophan-5- monooxygenase activation proteins, commonly referred to as the 14-3-3 proteins (14-3-3s), are a highly conserved family of molecular chaperones. The 14-3-3s family consists of seven distinct isoforms (β, γ, ε, ζ, η, θ, and σ), each with its own structure, function and expression [1-3]. 14-3-3s work by interacting with hundreds of different target proteins and are involved in various cell processes such as cell cycle, transcriptional control, signal transduction, intracellular trafficking/targeting, stress response and malignant transformation . 14-3-3s are tightly regulated in normal cells but several studies have reported their altered expression in a broad spectrum of diseases [5, 6]. 14-3-3s have been investigated in several types of human cancers, such as: glioma , renal carcinoma , primary nervous system , meningioma , lung  and astrocytoma , where 14-3-3s isoform-specific expression was reported. This article is protected by copyright. All rights reserved.European Journal of Clinical Investigation 01/2014; DOI:10.1111/eci.12241 · 2.83 Impact Factor