Design, expression, purification and crystallization of human 14-3-3ζ protein chimera with phosphopeptide from proapoptotic protein BAD.

14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the "chimeric" approach for studies of other relevant 14-3-3 complexes.

Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth.

Targeting protein-protein interactions (PPIs) is a promising approach in the development of drugs for many indications. 14-3-3 proteins are a family of phosphoprotein-binding molecules with critical functions in dozens of cell signaling networks. 14-3-3s are abundant in the central nervous system, and the small molecule fusicoccin-A (FC-A), a tool compound that can be used to manipulate 14-3-3 PPIs, enhances neurite outgrowth in cultured neurons. New semisynthetic FC-A derivatives with improved binding affinity for 14-3-3 complexes have recently been developed. Here, we use a series of screens that identify these compounds as potent inducers of neurite outgrowth through a polypharmacological mechanism. Using proteomics and X-ray crystallography, we discover that these compounds extensively regulate the 14-3-3 interactome by stabilizing specific PPIs, while disrupting others. These results provide new insights into the development of drugs to target 14-3-3 PPIs, a potential therapeutic strategy for CNS diseases.

Molecular characterization and in vitro interaction analysis of Op14-3-3 µ protein from Opuntia ficus-indica: identification of a new client protein from shikimate pathway.

In plants, 14-3-3 proteins are important modulators of protein-protein interactions in response to environmental stresses. The aim of the present work was to characterize one Opuntia ficus-indica 14-3-3 and get information about its client proteins. To achieve this goal, O. ficus-indica 14-3-3 cDNA, named as Op14-3-3 µ, was amplified by 3'-RACE methodology. Op14-3-3 µ contains an Open Reading Frame of 786 bp encoding a 261 amino acids protein. Op14-3-3 µ cDNA was cloned into a bacterial expression system and recombinant protein was purified. Differential Scanning Fluorimetry, Dynamic Light Scattering, and Ion Mobility-Mass Spectrometry were used for Op14-3-3 µ protein characterization, and Affinity-Purification-Mass Spectrometry analysis approach was used to obtain information about their potential client proteins. Pyrophosphate-fructose 6-phosphate 1-phosphotransferase, ribulose bisphosphate carboxylase large subunit, and vacuolar-type H+-ATPase were identified. Interestingly chorismate mutase p-prephenate dehydratase was also identified. Op14-3-3 µ down-regulation was observed in Opuntia calluses when they were induced with Jasmonic Acid, while increased accumulation of Op14-3-3 µ protein was observed. The putative interaction of 14-3-3 µ with chorismate mutase, which have not been reported before, suggest that Op14-3-3 µ could be an important regulator of metabolites biosynthesis and responses to stress in Opuntia spp. SIGNIFICANCE: Opuntia species are important crops in arid and semiarid areas worldwide, but despite its relevance, little information about their tolerance mechanism to cope with harsh environmental conditions is reported. 14-3-3 proteins have gained attention due to its participation as protein-protein regulators and have been linked with primary metabolism and hormones responses. Here we present the characterization of the first Opuntia ficus-indica 14-3-3 (Op14-3-3) protein using affinity purification-mass spectrometry (AP-MS) strategy. Op14-3-3 has high homology with other 14-3-3 from Caryophyllales. A novel Op14-3-3 client protein has been identified; the chorismate mutase p-prephenate dehydratase, key enzyme that links the primary with secondary metabolism. The present results open new questions about the Opuntia spp. pathways mechanisms in response to environmental stress and the importance of 14-3-3 proteins in betalains biosynthesis.

The important functionality of 14-3-3 isoforms in rice roots revealed by affinity chromatography.

Plant 14-3-3 proteins belong to a large family of proteins involved in numerous physiological processes, and function by binding to phosphorylated client proteins to regulate their function. However, little is known about their regulatory mechanisms in rice root growth. In this study, four 14-3-3 isoforms (GF14b, GF14c, GF14e, GF14f) exhibiting prominent expression profiles in rice roots, were selected for further investigation. Through a pull-down assay using four 14-3-3 isoforms in rice roots, 87 client proteins were identified that are involved in metabolism, protein synthesis and trafficking, energy metabolism, cell structure and growth, and other cellular processes. Importantly, we found that 14-3-3 proteins may play an important role in the root stress response through interactions with proteins functioning in oxidative stress, pathogenesis and secondary metabolism. By using real-time RT-PCR, it was found that 14-3-3 proteins exhibited diverse patterns of gene expression in response to salinity and drought stresses in rice root. The results revealed the isoform-specific functions of root 14-3-3 proteins. Our current study provides insight into understanding the functional roles of 14-3-3 proteins during rice root growth. SIGNIFICANCE: Rice (Oryza sativa L.) is one of the most important food crops in the world, as it is consumed by more than 3 billion people. Regulation of root growth plays an important role in rice adaption to biotic and abiotic, stress such as rhizosphere microbes and nutrient stress, and this process is directly related to the final yield. 14-3-3 proteins form a multi-gene family regulating developmental processes in plants. However, the correlation between the 14-3-3 protein family and its role in rice root growth has very little study. We applied an affinity chromatographic approach, in combination with LC-MS/MS, to explore the client proteins of four 14-3-3 isoforms that exhibit much more prominent gene expression than other members of the 14-3-3 family in rice roots. Assessments of the identified client proteins are able to obtain novel information toward understanding the functional mechanism of 14-3-3 proteins in rice root growth as expected.

Accumulation and tolerance to cadmium heavy metal ions and induction of 14-3-3 gene expression in response to cadmium exposure in Coprinus atramentarius.

Cadmium (Cd), one of the most toxic heavy-metal pollutants, has a strong and irreversible tendency to accumulate. Bioremediation is a promising technology to remedy and control heavy metal pollutants because of its low cost and ability to recycle heavy metals. Coprinus atramentarius is recognized as being able to accumulate heavy metal ions. In this work, C. atramentarius is cultivated on a solid medium containing Cd2+ ions to analyze its ability to tolerate different concentrations of the heavy metal ion. It is found that the growth of C. atramentarius is not significantly inhibited when the concentration of Cd2+ is less than 0.6mgL-1. The accumulation capacity of C. atramentarius at different Cd2+ concentrations also was determined. The results show that 76% of the Cd2+ present can be accumulated even when the concentration of the Cd2+ is 1mgL-1. The different proteins of C. atramentarius exposed to Cd2+ were further analyzed using gel electrophoresis. A 14-3-3 protein was identified and shown to be significantly up-regulated. In a further study, a full-length 14-3-3 gene was cloned containing a 759bp open reading frame encoding a polypeptide consisting of 252 amino acids and 3 introns. The gene expression work also showed that the 14-3-3 was significantly induced, and showed coordinated patterns of expression, with Cd2+ exposure.

Elution profile analysis of SDS-induced subcomplexes by quantitative mass spectrometry.

Analyzing the molecular architecture of native multiprotein complexes via biochemical methods has so far been difficult and error prone. Protein complex isolation by affinity purification can define the protein repertoire of a given complex, yet, it remains difficult to gain knowledge of its substructure or modular composition. Here, we introduce SDS concentration gradient induced decomposition of protein complexes coupled to quantitative mass spectrometry and in silico elution profile distance analysis. By applying this new method to a cellular transport module, the IFT/lebercilin complex, we demonstrate its ability to determine modular composition as well as sensitively detect known and novel complex components. We show that the IFT/lebercilin complex can be separated into at least five submodules, the IFT complex A, the IFT complex B, the 14-3-3 protein complex and the CTLH complex, as well as the dynein light chain complex. Furthermore, we identify the protein TULP3 as a potential new member of the IFT complex A and showed that several proteins, classified as IFT complex B-associated, are integral parts of this complex. To further demonstrate EPASIS general applicability, we analyzed the modular substructure of two additional complexes, that of B-RAF and of 14-3-3-ε. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the substructure of large multiprotein complexes in a highly time- as well as cost-effective manner.

The combination of hydrogen/deuterium exchange or chemical cross-linking techniques with mass spectrometry: mapping of human 14-3-3ζ homodimer interface.

Hydrogen/deuterium (H/D) exchange or chemical cross-linking by soluble carbodiimide (EDC) was employed in combination with high-resolution mass spectrometry (MS) to extend our knowledge about contact surface regions involved in the well-characterized model of interaction between two molecules of human 14-3-3ζ regulatory protein. The H/D exchange experiment provided low resolution mapping of interaction in the homodimeric 14-3-3ζ complex. A lower level of deuteration, suggesting structural protection, of two sequential segments has been demonstrated for dimeric 14-3-3ζ wild type relative to the monomeric mutant 14-3-3ζ S58D. The N-terminal sequence (the first 27 residues) from one subunit interacts with region αC'and αD'-helices (residues 45-98) of the other molecule across the dimer interface. To identify interacting amino acid residues within the studied complex, a chemical cross-linking reaction was carried out to produce the covalent homodimer, which was detected by SDS-PAGE. The MS analysis (following tryptic in-gel digestion) employing both high resolution and tandem mass spectrometry revealed cross-linked amino acid residues. Two alternative salt bridges between Glu81 and either Lys9 or the N-terminal amino group have been found to participate in transient interactions of the 14-3-3ζ isotype homodimerization. The data obtained, which have never previously been reported, were used to modify the published 14-3-3 crystal structure using molecular modeling. Based on our findings, utilization of this combination of experimental approaches, which preserve protein native structures, is suitable for mapping the contact between two proteins and also allows for the description of transient interactions or of regions with flexible structure in the studied protein complexes.

Identification and molecular characterization of a novel signaling molecule 14-3-3 epsilon in Clonorchis sinensis excretory/secretory products.

Increasing evidence shows that 14-3-3 proteins are involved in many biology events in addition to signal transduction. Extensive investigations on structural and biochemical features of these signaling molecules have implied their importance in the biological process. In the present study, we have identified and characterized the 14-3-3 epsilon (Cs14-3-3) in Clonorchis sinensis that causes human clonorchiasis. Recombinant protein was expressed in Escherichia coli (E. coli) and identified by MALDI-TOF/TOF. Immunoblot results revealed that Cs14-3-3 was a component of excretory/secretory products. Ligand blot assay indicated that 14-3-3 epsilon could bind C. sinensis MAPKAPK 2 in a nonphosphorylation-dependent manner. This protein could be detected at four stages of the life cycle by RT-PCR experiments and immunolocalization showed that Cs14-3-3 was extensively distributed in C. sinensis, especially at the outer surface and the sucker of adult worm and cyst wall of metacercaria. Taken together, 14-3-3 epsilon might play some roles in the development of the parasites. In addition, Cs14-3-3 epsilon should be addressed for the diagnostic value in C. sinensis infection in consideration of high sensitivity and specificity. As an immune stimulus, C. sinensis 14-3-3 epsilon was found to provoke a Th1/Th2 balanced immune response by inducing high levels of both IgG1 and IgG2a. Recombinant Cs14-3-3 conferred effective protection both in worm reduction rate and egg reduction rate, suggesting that the signaling molecule Cs14-3-3 was a promising vaccine candidate against C. sinensis infection.

The identification of antigenic proteins: 14-3-3 protein and propionyl-CoA carboxylase in Clonorchis sinensis.

Clonorchis sinensis, the causative agent of clonorchiasis, is widespread in East and Southeast Asia, including China, Vietnam and the Republic of Korea. We identified antigenic proteins from adult C. sinensis liver flukes using immunoproteomic analysis. In this study, we found 23 candidate antigenic proteins with a pI in the range of 5.4-6.2 in total lysates of C. sinensis. The antigenic protein spots reacted against sera from clonorchiasis patients and were identified as cysteine proteases, glutathione transferases, gelsolin, propionyl-CoA carboxylase (PCC), prohibitin and 14-3-3 protein (14-3-3) using LC-coupled ESI-MS/MS and an EST database for C. sinensis. PCC and 14-3-3 were identified for the first time as serological antigens for the diagnosis of C. sinensis. To validate the antigenicity of PCC and 14-3-3, recombinant proteins were immunoblotted with sera from clonorchiasis patients. The structural, functional and immunological characteristics of the putative amino acid sequence were predicted by bioinformatics analysis. Our novel finding will contribute to the development of diagnostics for clonorchiasis. These results suggest that immunoproteomic approaches are valuable tools to identify antigens that could be used as targets for effective parasitic infection control strategies.

[Preparation and characteristics of Schistosoma japonicum signaling protein 14-3-3].

OBJECTIVE: To prepare soluble recombinant signal transduction protein 14-3-3 of Schistosoma japonicum (rSj14-3-3) and investigate its immunologic features. METHODS: The cDNA fragment of signal transduction protein 14-3-3 gene was prepared from Schistosoma japonicum adult worm mRNA, and was subcloned to the downstream of glutathione S transferase gene of expression vector pGEX-4T-3 to construct a recombinant expression plasmid 14-3-3/pGEX-4T-3. The recombinant plasmids were transferred into E. coli BL21, the transforments containing recombinant plasmid were induced by IPTG, and the expression situation of fusion protein GST-14-3-3 was observed by SDS-PAGE. The pure recombinant 14-3-3 protein was prepared by digesting the fusion protein GST-rSj14-3-3 with thrombin and affinity chromatography. The specific antibody against rSj14-3-3 protein was prepared by an immunized rabbit with rSj14-3-3 protein. The immunogenicity and immune reactivity of rSj14-3-3 protein were analyzed by Western blotting. RESULTS: The gene encoding signal transduction protein 14-3-3 of Schistosoma japonicum Jiangsu strain was cloned successfully, the homology of open read frame sequence to the registrated sequence of Sj14-3-3 protein in genbank was 99.08%. A 55 kilo Dalton fusion protein GST-rSj14-3-3 was expressed by transferring the recombinant plasmid 14-3-3/pGEX-4T-3 into E. coli BL21 and induced with IPTG. The high titer antibody against rSj14-3-3 was produced by the immunized rabbit with rSj14-3-3 ,and could recognize the nature and recombinant rSj14-3-3 proteins. CONCLUSIONS: The soluble rSj14-3-3 protein has been prepared successfully in this study, and it has good immunogenicity and reactivity.

Identification of 14-3-3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis.

The balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14-3-3 proteins were associated with ATL31, and one of these, 14-3-3χ, was selected for detailed characterization. The interaction between ATL31 and 14-3-3χ was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3χ is catalyzed by ATL31. Degradation of 14-3-3χin vivo was shown to be correlated with ATL31 activity, and to occur in a proteasome-dependent manner. Furthermore, 14-3-3 protein accumulation was induced by a shift to high-C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over-expression of 14-3-3χ leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14-3-3 proteins for degradation via the ubiquitin-proteasome system during the response to cellular C/N status.

Identification of lily pollen 14-3-3 isoforms and their subcellular and time-dependent expression profile.

14-3-3 proteins are major regulators in plant development and physiology including primary metabolism and signal transduction pathways, typically via a phosphorylation-dependent interaction with a target protein. Four full-length 14-3-3 isoforms were identified in pollen grains of Lilium longiflorum by screening of a cDNA library and RACE (rapid amplification of cDNA ends)-PCR. Mass spectrometry analysis of partially purified 14-3-3s confirmed the presence of the four isoforms but also indicated the presence of additional, less abundant 14-3-3 isoforms in lily pollen. Separation of partially purified 14-3-3 proteins by two-dimensional gel electrophoresis resulted in nine spots that mainly contained the four major 14-3-3 isoforms. In a first step to examine putative physiological roles of specific 14-3-3 isoforms, their subcellular expression profile during pollen germination and tube growth was monitored using a characterized set of antibodies against 14-3-3 proteins with distinct crossreactivity. The abundance profile of 14-3-3 proteins associated with the cytosol, endomembranes (tonoplast, endoplasmic reticulum, Golgi, mitochondria) and plasma membrane showed high spatial-temporal dynamics. This indicates different targets of 14-3-3 proteins at different organelles and time points during pollen germination and growth.

Isolation and expression analysis of a homolog of the 14-3-3 epsilon gene in the diamondback moth, Plutella xylostella.

A full-length 14-3-3 gene homolog (also referred to as the Px14-3-3 epsilon "ε" or Px14-3-3ε gene) was cloned from cDNA of the diamondback moth, Plutella xylostella. The Px14-3-3 transcript is 789 nucleotides in length, and the predicted polypeptide is 263 amino acids in length, with a calculated molecular mass of 29.6 kDa. The Px14-3-3 gene contains the typical and predicted 14-3-3 domains and motifs. The amino acid sequence of the diamondback moth 14-3-3 gene is very similar to that of other insect epsilons (ε) but not to other insect zetas (ζ). In particular, the protein sequence of the Px14-3-3 gene shows high identity to the Bombyx mori epsilon (96.2%). Western blot analysis using an antibody against Px14-3-3ε verified the expression of 14-3-3ε in the larval, pupal, and adult stages. The Px14-3-3ε expression patterns in all the different tissue types were examined in the fourth instar larvae. Px14-3-3ε was detected in every tissue examined, including the body fat, hemocytes, brain, gut, and cuticle.

Proteomic and biochemical analysis of 14-3-3-binding proteins during C2-ceramide-induced apoptosis.

14-3-3 is a family of proteins comprising several isoforms that, in many cases, promote cell survival by association with proapoptotic proteins. This study was designed to obtain further understanding of the 14-3-3 role in apoptosis regulation, by analyzing apoptosis-related protein-14-3-3 interactions. Western blot analysis of an eluted fraction from the 14-3-3-affinity chromatography column identified proapoptotic proteins as receptor-interacting protein 3 and Bcl-2-antagonist/killer as new phophorylation-dependent 14-3-3-binding proteins under physiological conditions. The apoptosis inducer C2-ceramide promoted decay of the 14-3-3-binding signal of protein cell extracts. Investigation of the role of 14-3-3 in C2-ceramide-induced apoptosis showed that depletion of the 14-3-3zeta isoform sensitized to cell death, whereas overexpression of this isoform delayed cell death. A combination of tandem affinity purification and liquid chromatography-tandem MS techniques identified 15 proteins involved in cell survival processes whose 14-3-3-binding status changed during C2-ceramide-induced apoptosis. Under physiological conditions, desmin was clearly identified as a new 14-3-3-interactor protein, and vasodilator-stimulated phosphoprotein, nucleophosmin and calmodulin, whose 14-3-3 binding was suggested by others on the basis of MS analysis, were confirmed here as phosphorylation-dependent 14-3-3-associated proteins. Interestingly, proteins related to the regulation of DNA double-strand break repair in the early stages of apoptosis, such as DNA-dependent protein kinase, or the regulation of cell shrinkage during apoptosis, such as vasodilator-stimulated phosphoprotein and death promoters like receptor-interacting protein 3, were identified as 14-3-3-associated proteins whose 14-3-3-binding status changed when apoptosis was initiated. The functional diversity of these identified proteins suggests that 14-3-3 may regulate the apoptotic process through new mechanisms, in addition to others previously characterized.

Global phosphoproteomics identifies a major role for AKT and 14-3-3 in regulating EDC3.

Insulin plays an essential role in metabolic homeostasis in mammals, and many of the underlying biochemical pathways are regulated via the canonical phosphatidylinositol 3-kinase/AKT pathway. To identify novel metabolic actions of insulin, we conducted a quantitative proteomics analysis of insulin-regulated 14-3-3-binding proteins in muscle cells. These studies revealed a novel role for insulin in the post-transcriptional regulation of mRNA expression. EDC3, a component of the mRNA decay and translation repression pathway associated with mRNA processing bodies, was shown to be phosphorylated by AKT downstream of insulin signaling. The major insulin-regulated site was mapped to Ser-161, and phosphorylation at this site led to increased 14-3-3 binding. Functional studies indicated that induction of 14-3-3 binding to EDC3 causes morphological changes in processing body structures, inhibition of microRNA-mediated mRNA post-transcriptional regulation, and alterations in the protein- protein interactions of EDC3. These data highlight an important new arm of the insulin signaling cascade in the regulation of mRNA utilization.

Identification of different isoforms of 14-3-3 protein family in human dermal and epidermal layers.

We have previously demonstrated a high level of stratifin, also known as 14-3-3 sigma in differentiated keratinocyte cell lysate and conditioned medium (CM). In this study, we asked the question of whether other 14-3-3 isoforms are expressed in human dermal fibroblasts, keratinocytes, intact dermal and epidermal layers of skin. In order to address this question, total proteins extracted from cultured cells or skin layers were subjected to western blot analysis using seven different primary antibodies specific to well-known mammalian isoforms, beta, gamma, epsilon, eta, sigma, tau, and zeta of 14-3-3 protein family. The autoradiograms corresponding to each isoform were then quantified and compared. The results revealed the presence of very high levels of all seven isoforms in cultured keratinocyte and conditioned medium. With the exception of tau isoform, other 14-3-3 isoforms were also present in intact epidermal layer of normal skin. The profile of 14-3-3 proteins in whole skin was similar to that of epidermis. In contrast, only gamma 14-3-3 isoform, was present in dermal layer obtained from the same skin sample. On the other hand, cultured fibroblasts express a high level of beta, epsilon, gamma and eta and a low level of zeta and tau, but not sigma isoform. However, the levels of 14-3-3 epsilon, gamma and eta were barely detectable in fibroblast conditioned medium. Further, we also used immunohistochemical staining to identify the 14-3-3 isoform expressing cells in human skin sections. The finding revealed different expression profile for each of these isoforms mainly in differentiated keratinocytes located within the layer of lucidum. However, fibroblasts located within the dermal layer did not show any detectable levels of these proteins. In conclusion, all members of 14-3-3 proteins are expressed by cells of epidermal but not dermal layer of skins and that these proteins are mainly expressed by differentiated keratinocytes.

14-3-3 proteins recognize a histone code at histone H3 and are required for transcriptional activation.

Interphase phosphorylation of S10 at histone H3 is linked to transcriptional activation of a specific subset of mammalian genes like HDAC1. Recently, 14-3-3 proteins have been described as detectors for this phosphorylated histone H3 form. Here, we report that 14-3-3 binding is modulated by combinatorial modifications of histone H3. S10 phosphorylation is necessary for an interaction, but additional H3K9 or H3K14 acetylation increases the affinity of 14-3-3 for histone H3. Histone H3 phosphoacetylation occurs concomitant with K9 methylation in vivo, suggesting that histone phosphorylation and acetylation can synergize to overcome repressive histone methylation. Chromatin immunoprecipitation experiments reveal recruitment of 14-3-3 proteins to the HDAC1 gene in an H3S10ph-dependent manner. Recruitment of 14-3-3 to the promoter is enhanced by additional histone H3 acetylation and correlates with dissociation of the repressive binding module HP1gamma. Finally, siRNA-mediated loss of 14-3-3 proteins abolishes the transcriptional activation of HDAC1. Together our data indicate that 14-3-3 proteins are crucial mediators of histone phosphoacetylation signals.

14-3-3 proteins: a historic overview.

This chapter includes a historic overview of 14-3-3 proteins with an emphasis on the differences between potentially cancer-relevant isoforms on the genomic, protein and functional level. The focus will therefore be on mammalian 14-3-3s although many important developments in the field have involved Drosophila 14-3-3 proteins for example and the cross-fertilisation from parallel studies on plant 14-3-3 should not be underestimated. In the major part of this review I will attempt to focus on some novel data and aspects of 14-3-3 structure and function, in particular regulation of 14-3-3 isoforms by oncogene-related protein kinase phosphorylation and aspects of 14-3-3 research with which newcomers to the field may be less familiar.