14-3-3 protein inhibits CaMKK1 by blocking the kinase active site with its last two C-terminal helices.

Ca2+ /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca2+ /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca2+ /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.

Two-photon lifetime-based photoconversion of EGFP for 3D-photostimulation in FLIM.

Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and biomedical applications. Surprisingly, some interesting photochemical properties of EGFP have remained unexplored. Here we report on two-photon-induced photoconversion of EGFP, which can be permanently converted by intense IR irradiation to a form with a short fluorescence lifetime and spectrally conserved emission. Photoconverted EGFP thus can be distinguished from the unconverted tag by the time-resolved detection. Nonlinear dependence of the two-photon photoconversion efficiency on the light intensity allows for an accurate 3D localization of the photoconverted volume within cellular structures, which is especially useful for kinetic FLIM applications. For illustration, we used the two photon photoconversion of EGFP for measurements of redistribution kinetics of nucleophosmin and histone H2B in nuclei of live cells. Measurements revealed high mobility of fluorescently tagged histone H2B in the nucleoplasm and their redistribution between spatially separated nucleoli.

Cytoplasmic localization of Mdm2 in cells expressing mutated NPM is mediated by p53.

Specific C-terminal nucleophosmin (NPM) mutations are related to the acute myeloid leukaemia and cause mistargeting of mutated NPM (NPMmut) to the cytoplasm. Consequently, multiple NPM-interacting partners, e.g., the tumour suppressor p53, become also mislocalized. We found that ubiquitin ligase Mdm2 mislocalizes to the cytoplasm in the presence of NPMmut as well. Since p53 interacts with Mdm2, we searched for the NPMmut-p53-Mdm2 complex and interactions of its constituents in live cells and cell lysates using fluorescently tagged proteins, fluorescence lifetime imaging and immunoprecipitation. We proved existence of the ternary complex, which likely adopts a chain-like configuration. Interaction between Mdm2 and NPMmut was not detected, even under conditions of upregulated Mdm2 and p53 induced by Actinomycin D. We assume that p53 serves in the complex as a bridging link between Mdm2 and NPMmut. This conclusion was supported by disruption of the Mdm2-p53 interaction by Nutlin-3A, which resulted in relocalization of Mdm2 to the nucleus, while both NPMmut and p53 remained in the cytoplasm. Importantly, silencing of p53 also prevented mislocalization of Mdm2 in the presence of NPMmut.

Nedd4-2 binding to 14-3-3 modulates the accessibility of its catalytic site and WW domains.

Neural precursor cells expressed developmentally downregulated protein 4-2 (Nedd4-2), a homologous to the E6-AP carboxyl terminus (HECT) ubiquitin ligase, triggers the endocytosis and degradation of its downstream target molecules by regulating signal transduction through interactions with other targets, including 14-3-3 proteins. In our previous study, we found that 14-3-3 binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Here, we used time-resolved fluorescence intensity and anisotropy decay measurements, together with fluorescence quenching and mass spectrometry, to further characterize interactions between Nedd4-2 and 14-3-3 proteins. The results showed that 14-3-3 binding affects the emission properties of AEDANS-labeled WW3, WW4, and, to a lesser extent, WW2 domains, and reduces their mobility, but not those of the WW1 domain, which remains mobile. In contrast, 14-3-3 binding has the opposite effect on the active site of the HECT domain, which is more solvent exposed and mobile in the complexed form than in the apo form of Nedd4-2. Overall, our results suggest that steric hindrance of the WW3 and WW4 domains combined with conformational changes in the catalytic domain may account for the 14-3-3 binding-mediated regulation of Nedd4-2.

TRPM5 Channel Binds Calcium-Binding Proteins Calmodulin and S100A1.

Melastatin transient receptor potential (TRPM) channels belong to one of the most significant subgroups of the transient receptor potential (TRP) channel family. Here, we studied the TRPM5 member, the receptor exposed to calcium-mediated activation, resulting in taste transduction. It is known that most TRP channels are highly modulated through interactions with extracellular and intracellular agents. The binding sites for these ligands are usually located at the intracellular N- and C-termini of the TRP channels, and they can demonstrate the character of an intrinsically disordered protein (IDP), which allows such a region to bind various types of molecules. We explored the N-termini of TRPM5 and found the intracellular regions for calcium-binding proteins (CBPs) the calmodulin (CaM) and calcium-binding protein S1 (S100A1) by in vitro binding assays. Furthermore, molecular docking and molecular dynamics simulations (MDs) of the discovered complexes confirmed their known common binding interface patterns and the uniqueness of the basic residues present in the TRPM binding regions for CaM/S100A1.

TRPM7 N-terminal region forms complexes with calcium binding proteins CaM and S100A1.

Transient receptor potential melastatin 7 (TRPM7) represents melastatin TRP channel with two significant functions, cation permeability and kinase activity. TRPM7 is widely expressed among tissues and is therefore involved in a variety of cellular functions representing mainly Mg2+ homeostasis, cellular Ca2+ flickering, and the regulation of DNA transcription by a cleaved kinase domain translocated to the nucleus. TRPM7 participates in several important biological processes in the nervous and cardiovascular systems. Together with the necessary function of the TRPM7 in these tissues and its recently analyzed overall structure, this channel requires further studies leading to the development of potential therapeutic targets. Here we present the first study investigating the N-termini of TRPM7 with binding regions for important intracellular modulators calmodulin (CaM) and calcium-binding protein S1 (S100A1) using in vitro and in silico approaches. Molecular simulations of the discovered complexes reveal their potential binding interfaces with common interaction patterns and the important role of basic residues present in the N-terminal binding region of TRPM.

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 Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca2+/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.

AML-Related NPM Mutations Drive p53 Delocalization into the Cytoplasm with Possible Impact on p53-Dependent Stress Response.

Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations.

NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization.

Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wild-type NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemia-derived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered.

Intrinsically disordered protein domain of human ameloblastin in synthetic fusion with calmodulin increases calmodulin stability and modulates its function.

Constantly increasing attention to bioengineered proteins has led to the rapid development of new functional targets. Here we present the biophysical and functional characteristics of the newly designed CaM/AMBN-Ct fusion protein. The two-domain artificial target consists of calmodulin (CaM) and ameloblastin C-terminus (AMBN-Ct). CaM as a well-characterized calcium ions (Ca2+) binding protein offers plenty of options in terms of Ca2+ detection in biomedicine and biotechnologies. Highly negatively charged AMBN-Ct belongs to intrinsically disordered proteins (IDPs). CaM/AMBN-Ct was designed to open new ways of communication synergies between the domains with potential functional improvement. The character and function of CaM/AMBN-Ct were explored by biophysical and molecular modelling methods. Experimental studies have revealed increased stability and preserved CaM/AMBN-Ct function. The results of molecular dynamic simulations (MDs) outlined different interface patterns between the domains with potential allosteric communication within the fusion.

Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4.

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators-calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein-protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

Interaction of an IκBα Peptide with 14-3-3.

Inflammatory responses mediated by the transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) play key roles in immunity, autoimmune diseases, and cancer. NF-κB is directly regulated through protein-protein interactions, including those with IκB and 14-3-3 proteins. These two important regulatory proteins have been reported to interact with each other, although little is known about this interaction. We analyzed the inhibitor of nuclear factor kappa B α (IκBα)/14-3-3σ interaction via a peptide/protein-based approach. Structural data were acquired via X-ray crystallography, while binding affinities were measured with fluorescence polarization assays and time-resolved tryptophan fluorescence. A high-resolution crystal structure (1.13 Å) of the uncommon 14-3-3 interaction motif of IκBα (IκBαpS63) in a complex with 14-3-3σ was evaluated. This motif harbors a tryptophan that makes this crystal structure the first one with such a residue visible in the electron density at that position. We used this tryptophan to determine the binding affinity of the unlabeled IκBα peptide to 14-3-3 via tryptophan fluorescence decay measurements.

TRPM6 N-Terminal CaM- and S100A1-Binding Domains.

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions-N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member-the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different.

A revised annotated checklist of louse flies (Diptera, Hippoboscidae) from Slovakia.

The list of all known locality and host records from the literature on louse flies from Slovakia are summarized, with the addition of new collection data. New locality data are provided for five species. Three species are added to the Slovakian list: Icostaminor (Bigot in Thomson, 1858), which was erroneously cited for Moravia instead of Slovakia in the previous checklist, and Ornithophilametallica (Schiner, 1864) and Ornithomyachloropus (Bergroth, 1901), which were overlooked from the last checklist. As a result, the louse fly fauna of Slovakia increases to 19 species: 12 autochtonous species and seven rare, non-native species only occasionally imported to Slovakia or migrating to the country with their hosts. This is by far the largest regional fauna of Hippoboscidae in Central Europe, and matches the richest southern European faunas. In total, 78 host-parasite associations concerning 46 bird-host species from eight orders and nine species of mammals, including humans, have been found from a literature review in Slovakia. Two host-parasite associations are reported from Slovakia for the first time: Ornithomyaavicularia (Linnaeus, 1758) on Prunellamodularis (Linnaeus, 1758) (Aves: Prunellidae) and Lipoptenafortisetosa Maa, 1965 on Homosapiens Linnaeus, 1758 (Mammalia: Hominidae).

Lifetime-based photoconversion of EGFP as a tool for FLIM.

BACKGROUND: EGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM. METHODS: Fluorescence lifetime imaging and spectral FLIM has been used for characterization of the EGFP photoconversion and protein tracking. RESULT: Our data suggest that EGFP can be permanently photoconverted to a short-fluorescence-lifetime form (PC-EGFP) by intense blue irradiation. PC-EGFP cannot be reverted back by 405 nm light and exhibits the same spectral emission properties with blue-shifted absorption compared to the unconverted EGFP. Fluorescence of PC-EGFP is pH-independent and the photoconversion efficiency decreases with the solvent viscosity. Utilization of the EGFP photoconversion was demonstrated by tracking of a nucleophosmin mutant in live HEK-293 T cells during its cytoplasm-nuclear relocalization induced by Leptomycin B. CONCLUSIONS: Besides potential FLIM artifacts caused by an unintended EGFP photoconversion, the controlled photoconversion turns EGFP to an excellent tool for kinetic FLIM applications. Since the photoconversion occurs in the lifetime domain, PC-EGFP can be easily distinguished from the unconverted tag by time-resolved detection while all other spectral channels stay free for multicolor labeling. GENERAL SIGNIFICANCE: The reported lifetime photoconversion lines up EGFP with other photoconvertible fluorescent proteins with special advantage for fluorescence lifetime imaging where lifetime-photoconvertible labels are scarce.

14-3-3 protein masks the nuclear localization sequence of caspase-2.

Caspase-2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase-2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14-3-3, which recognizes two phosphoserines located in the linker between the caspase recruitment domain and the p19 domains of the caspase-2 zymogen. However, the structural details of this interaction and the exact role of 14-3-3 in the regulation of caspase-2 activation remain unclear. Moreover, the caspase-2 region with both 14-3-3-binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14-3-3 binding may regulate the subcellular localization of caspase-2. Here, we report a structural analysis of the 14-3-3ζ:caspase-2 complex using a combined approach based on small angle X-ray scattering, NMR, chemical cross-linking, and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase-2 and 14-3-3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase-2 are positioned within the central channel of the 14-3-3 dimer and stabilized through interactions with the C-terminal helices of both 14-3-3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase-2 activation by dimerization, is sterically occluded by the 14-3-3 dimer, thereby likely preventing caspase-2 activation. In addition, 14-3-3 protein binding to caspase-2 masks its NLS. Therefore, our results suggest that 14-3-3 protein binding to caspase-2 may play a key role in regulating caspase-2 activation. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.ww pdb.org (PDB ID codes 6GKF and 6GKG).

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

BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a member of the Ca⁠2+/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 Ca2+/CaM binding may cause unique conformational changes in the CaMKKs compared with other CaMKs. However, the underlying mechanistic details remain unclear. METHODS: In 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 Ca2+/CaM binding-induced structural changes in CaMKK2. RESULTS: Our 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) Ca2+/CaM binding affects the structure of several regions surrounding the ATP-binding pocket, including the activation segment; (iii) although the CaMKK2:Ca2+/CaM complex shows high conformational flexibility, most of its molecules are rather compact; and (iv) AID-bound Ca2+/CaM transiently interacts with the CaMKK2 kinase domain. CONCLUSIONS: Interactions between the CaMKK2 kinase domain and the AID differ from those of other CaMKs. In the absence of Ca2+/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 SIGNIFICANCE: Our results corroborate the hypothesis that Ca2+/CaM binding causes unique conformational changes in the CaMKKs relative to other CaMKs.

Monitoring of nucleophosmin oligomerization in live cells.

Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.

14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2).

BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca2+/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown. METHODS: The interaction between phosphorylated CaMKK2 and the 14-3-3γ protein, as well as the architecture of their complex, were studied using enzyme activity measurements, small-angle x-ray scattering (SAXS), time-resolved fluorescence spectroscopy and protein crystallography. RESULTS: Our data suggest that the 14-3-3 protein binding does not inhibit the catalytic activity of phosphorylated CaMKK2 but rather slows down its dephosphorylation. Structural analysis indicated that the complex is flexible and that CaMKK2 is located outside the phosphopeptide-binding central channel of the 14-3-3γ dimer. Furthermore, 14-3-3γ appears to interact with and affect the structure of several regions of CaMKK2 outside the 14-3-3 binding motifs. In addition, the structural basis of interactions between 14-3-3 and the 14-3-3 binding motifs of CaMKK2 were elucidated by determining the crystal structures of phosphopeptides containing these motifs bound to 14-3-3. CONCLUSIONS: 14-3-3γ protein directly interacts with the kinase domain of CaMKK2 and the region containing the inhibitory phosphorylation site Thr145 within the N-terminal extension. GENERAL SIGNIFICANCE: Our results suggested that CaMKK isoforms differ in their 14-3-3-mediated regulations and that the interaction between 14-3-3 protein and the N-terminal 14-3-3-binding motif of CaMKK2 might be stabilized by small-molecule compounds.

Shared CaM- and S100A1-binding epitopes in the distal TRPM4 N terminus.

The transient receptor potential channel of melastatin 4 (TRPM4) belongs to a group of large ion receptors that are involved in countless cell signalling cascades. This unique member is ubiquitously expressed in many human tissues, especially in cardiomyocytes, where it plays an important role in cardiovascular processes. Transient receptor potential channels (TRPs) are usually constituted by intracellular N- and C- termini, which serve as mediators affecting allosteric modulation of channels, resulting in the regulation of the channel function. The TRPs tails contain a number of conserved epitopes that specifically bind the intracellular modulators. Here, we identify new binding sites for the calmodulin (CaM) and S100 calcium-binding protein A1 (S100A1), located in the very distal part of the TRPM4 N terminus. We have used chemically synthesized peptides of the TRPM4, mimicking the binding epitopes, along with fluorescence methods to determine and specify CaM- and S100A1-binding sites. We have found that the ligands binding epitopes at the TRPM4 N terminus overlap, but the interacting mechanism of both complexes is probably different. The molecular models supported by data from the fluorescence method confirmed that the complexes formations are mediated by the positively charged (R139, R140, R144) and hydrophobic (L134, L138, V143) residues present at the TRPM4 N terminus-binding epitopes. The data suggest that the molecular complexes of TRPM4/CaM and TRPM4/S100A1 would lead to the modulation of the channel functions.