Look for the Scaffold: Multifaceted Regulation of Enzyme Activity by 14-3-3 Proteins.

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.

Allosteric activation of yeast enzyme neutral trehalase by calcium and 14-3-3 protein.

Neutral trehalase 1 (Nth1) from Saccharomyces cerevisiae catalyzes disaccharide trehalose hydrolysis and helps yeast to survive adverse conditions, such as heat shock, starvation or oxidative stress. 14-3-3 proteins, master regulators of hundreds of partner proteins, participate in many key cellular processes. Nth1 is activated by phosphorylation followed by 14-3-3 protein (Bmh) binding. The activation mechanism is also potentiated by Ca(2+) binding within the EF-hand-like motif. This review summarizes the current knowledge about trehalases and the molecular and structural basis of Nth1 activation. The crystal structure of fully active Nth1 bound to 14-3-3 protein provided the first high-resolution view of a trehalase from a eukaryotic organism and showed 14-3-3 proteins as structural modulators and allosteric effectors of multi-domain binding partners.

Mechanisms of the 14-3-3 protein function: regulation of protein function through conformational modulation.

Many aspects of protein function regulation require specific protein-protein interactions to carry out the exact biochemical and cellular functions. The highly conserved members of the 14-3-3 protein family mediate such interactions and through binding to hundreds of other proteins provide multitude of regulatory functions, thus playing key roles in many cellular processes. The 14-3-3 protein binding can affect the function of the target protein in many ways including the modulation of its enzyme activity, its subcellular localization, its structure and stability, or its molecular interactions. In this minireview, we focus on mechanisms of the 14-3-3 protein-dependent regulation of three important 14-3-3 binding partners: yeast neutral trehalase Nth1, regulator of G-protein signaling 3 (RGS3), and phosducin.

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: a family of versatile molecular regulators.

The 14-3-3 proteins are a family of acidic regulatory molecules found in all eukaryotes. 14-3-3 proteins function as molecular scaffolds by modulating the conformation of their binding partners. Through the functional modulation of a wide range of binding partners, 14-3-3 proteins are involved in many processes, including cell cycle regulation, metabolism control, apoptosis, and control of gene transcription. This minireview includes a short overview of 14-3-3 proteins and then focuses on their role in the regulation of two important binding partners: FOXO forkhead transcription factors and an enzyme tyrosine hydroxylase.

Structure/function relationships underlying regulation of FOXO transcription factors.

The FOXO subgroup of forkhead transcription factors plays a central role in cell-cycle control, differentiation, metabolism control, stress response and apoptosis. Therefore, the function of these important molecules is tightly controlled by a wide range of protein-protein interactions and posttranslational modifications including phosphorylation, acetylation and ubiquitination. The mechanisms by which these processes regulate FOXO activity are mostly elusive. This review focuses on recent advances in structural studies of forkhead transcription factors and the insights they provide into the mechanism of DNA recognition. On the basis of these data, we discuss structural aspects of protein-protein interactions and posttranslational modifications that target the forkhead domain and the nuclear localization signal of FOXO proteins.

Protein modeling combined with spectroscopic techniques: an attractive quick alternative to obtain structural information.

Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys501, Gly502 and Arg544, also Glu446, Phe475 and Gln482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth beta-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.

14-3-3 proteins in pineal photoneuroendocrine transduction: how many roles?

Recent studies suggest that a common theme links the diverse elements of pineal photoneuroendocrine transduction--regulation via binding to 14-3-3 proteins. The elements include photoreception, neurotransmission, signal transduction and the synthesis of melatonin from tryptophan. We review general aspects of 14-3-3 proteins and their biological function as binding partners, and also focus on their roles in pineal photoneuroendocrine transduction.

14-3-3 Proteins and photoneuroendocrine transduction: role in controlling the daily rhythm in melatonin.

This paper describes the role 14-3-3 proteins play in vertebrate photoneuroendocrine transduction. 14-3-3 proteins form a complex with arylalkylamine N-acetyltransferase (AANAT), the enzyme which turns melatonin production on during the day and off at night. Complex formation is triggered at night by cAMP-dependent phosphorylation of the enzyme, and results in activation and protection against proteolysis. This enhances melatonin production >10-fold. Light exposure results in dephosphorylation of the enzyme and disassociation from 14-3-3, leading to destruction and a rapid drop in melatonin production and release and circulating levels.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN --> RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K(m) for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme regulation.

Serotonin N-acetyltransferase (AANAT) controls the daily rhythm in melatonin synthesis. When isolated from tissue, AANAT copurifies with isoforms epsilon and zeta of 14-3-3. We have determined the structure of AANAT bound to 14-3-3zeta, an association that is phosphorylation dependent. AANAT is bound in the central channel of the 14-3-3zeta dimer, and is held in place by extensive interactions both with the amphipathic phosphopeptide binding groove of 14-3-3zeta and with other parts of the central channel. Thermodynamic and activity measurements, together with crystallographic analysis, indicate that binding of AANAT by 14-3-3zeta modulates AANAT's activity and affinity for its substrates by stabilizing a region of AANAT involved in substrate binding.

Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase.

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.

Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na(+)/K(+)-ATPase.

The secondary structure of Na(+)/K(+)-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na(+)/K(+)-ATPase additionally to alpha-helix also beta-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na(+)/K(+)-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na(+)/K(+)-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H(2)O)(4)ATP and Co(NH(3))(4)ATP, on the other hand, affected the secondary structure of Na(+)/K(+)-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.

Different cation binding to the I domains of alpha1 and alpha2 integrins: implication of the binding site structure.

In the present work, we studied the interactions of recombinant alpha1 and alpha2 integrin I domains with cations Tb(3+), Mn(2+), Mg(2+) and Ca(2+). We observed that alpha1 and alpha2 I domains bind these cations with significantly different characteristics. The binding of Mg(2+) by the alpha1 I domain was accompanied by significant changes of tryptophan fluorescence which could be interpreted as a conformational change. Comparison of the alpha1 integrin I domain structure obtained by comparative modeling with a known structure of the alpha2 integrin I domain shows distinct differences in the metal ion binding sites which could explain the differences in cation binding.

The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts.

Hypericin (HY) and Hypocrellin-A (HA) photosensitization induce rapid depolarization of plasma membrane in 3T3 cells as revealed by confocal microspectrofluorimetry using diO-C5(3) fluorescent probe. HY and HA are also able to rigidify the lipid membrane of DMPC liposomes as indicated by the decrease of pyrene excimer fluorescence used as a marker of the lipid membrane fluidity. We have also observed a nonspecific inhibition of Na+,K+-ATPase activity due to the HY and HA photosensitization. The described effects are concentration- and light dose-dependent and generally more pronounced for HA than for HY. All these observations suggest that the lipid membranes can play an important role in the photosensitization process induced by HY and HA at the cellular level. It can be hypothesized that for HA and HY the secondary mechanism following type I or type II photosensitization process can be the peroxidation of membrane lipids as well, and thus intracellular membranes seem to be one of the most important targets of these photosensitizers.

Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic.

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.

Effect of aminophospholipid glycation on order parameter and hydration of phospholipid bilayer.

The effect of aminophospholipid glycation on lipid order and lipid bilayer hydration was investigated using time-resolved fluorescence spectroscopy. The changes of lipid bilayer hydration were estimated both from its effect on the fluorescence lifetime of The 1-[4-(trimethylammonium)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and 1,6-diphenylhexa-1,3,5-triene (DPH) and using solvatochromic shift studies with 1-anilinonaphthalene-8-sulfonic acid. The head-group and acyl chain order were determined from time-resolved fluorescence anisotropy measurements of the TMA-DPH and DPH. The suspensions of small unilamellar vesicles (with phosphatidylethanolamine/phosphatidylcholine molar ratio 1:2.33) were incubated with glyceraldehyde and it was found that aminophospholipids react with glyceraldehyde to form products with the absorbance and the fluorescence properties typical for protein advanced glycation end products. The lipid glycation was accompanied by the progressive oxidative modification of unsaturated fatty acid residues. It was found that aminophospholipid glycation increased the head-group hydration and lipid order in both regions of the membrane. The lipid oxidation accompanying the lipid glycation affected mainly the lipid order, while the effect on the lipid hydration was small. The increase in the lipid order was presumably the result of two effects: (1) the modification of head-groups of phosphatidylethanolamine by glycation; and (2) the degradation of unsaturated fatty acid residues by oxidation.

The isolated H4-H5 cytoplasmic loop of Na,K-ATPase overexpressed in Escherichia coli retains its ability to bind ATP.

The H4-H5 loop of the alpha-subunit of mouse brain Na,K-ATPase was expressed and isolated from E.coli cells. Using fluorescence analogues of ATP, this loop was shown to retain its capability to bind ATP. Isolation of a soluble H4-H5 loop with the native ATP binding site is a critical step for detailed studies of the molecular mechanism of ATP binding and utilisation.

Molecular distance measurements reveal an (alpha beta)2 dimeric structure of Na+/K+-ATPase. High affinity ATP binding site and K+-activated phosphatase reside on different alpha-subunits.

ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic alpha-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by Förster energy transfer after affinity labeling of the E1ATP site by fluorescein 5'-isothiocyanate (FITC). Erythrosin 5'-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/alpha-subunit of 0.6 and of ErITC/alpha-subunit of 0.48 indicate 2 ATP sites per (alpha beta)2 diprotomer. Measurements of Förster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 +/- 0.64 nm. This distance excludes 2 ATP sites per alpha-subunit since the diameter of alpha is 4-5 nm. Förster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 +/- 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (alpha beta)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).

The isolated H4-H5 cytoplasmic loop of Na,K-ATPase overexpressed in Escherichia coli retains its ability to bind ATP.

The H4-H5 loop of the alpha-subunit of mouse brain Na,K-ATPase was expressed and isolated from Escherichia coli cells. Using fluorescence analogues of ATP, this loop was shown to retain its capability to bind ATP. Isolation of a soluble H4-H5 loop with the native ATP binding site is a crucial step for detailed studies of the molecular mechanism of ATP binding and utilisation.