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3.3: Proteins - Biology

3.3: Proteins - Biology


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3.3: Proteins

The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development

The highly conserved 14-3-3 protein family has risen to a position of importance in cell biology owing to its involvement in vital cellular processes, such as metabolism, protein trafficking, signal transduction, apoptosis and cell-cycle regulation. The 14-3-3 proteins are phospho-serine/phospho-threonine binding proteins that interact with a diverse array of binding partners. Because many 14-3-3 interactions are phosphorylation-dependent, 14-3-3 has been tightly integrated into the core phospho-regulatory pathways that are crucial for normal growth and development and that often become dysregulated in human disease states such as cancer. This review examines the recent advances that further elucidate the role of 14-3-3 proteins as integrators of diverse signaling cues that influence cell fate decisions and tumorigenesis.

Figures

Functions of 14-3-3 in proliferative,…

Functions of 14-3-3 in proliferative, oncogenic, survival and stress signaling. (a) Under proliferative…

Functions of 14-3-3 in TORC1…

Functions of 14-3-3 in TORC1 signaling. (a) Under growth conditions, the AKT kinase…

Functions of 14-3-3 in cytokinesis.…

Functions of 14-3-3 in cytokinesis. (a) 14-3-3σ function is required for a mitotic…

Functions of 14-3-3 in tumor-suppressor…

Functions of 14-3-3 in tumor-suppressor pathways. (a) The mammalian Hippo pathway transmits signals…


14-3-3 proteins restrain the Exo1 nuclease to prevent overresection

The DNA end resection process dictates the cellular response to DNA double strand break damage and is essential for genome maintenance. Although insufficient DNA resection hinders homology-directed repair and ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint activation, overresection produces excessive single-stranded DNA that could lead to genomic instability. However, the mechanisms controlling DNA end resection are poorly understood. Here we show that the major resection nuclease Exo1 is regulated both positively and negatively by protein-protein interactions to ensure a proper level of DNA resection. We have shown previously that the sliding DNA clamp proliferating cell nuclear antigen (PCNA) associates with the C-terminal domain of Exo1 and promotes Exo1 damage association and DNA resection. In this report, we show that 14-3-3 proteins interact with a central region of Exo1 and negatively regulate Exo1 damage recruitment and subsequent resection. 14-3-3s limit Exo1 damage association, at least in part, by suppressing its association with PCNA. Disruption of the Exo1 interaction with 14-3-3 proteins results in elevated sensitivity of cells to DNA damage. Unlike Exo1, the Dna2 resection pathway is apparently not regulated by PCNA and 14-3-3s. Our results provide critical insights into the mechanism and regulation of the DNA end resection process and may have implications for cancer treatment.

Keywords: 14-3-3 protein DNA damage response DNA repair DNA resection Exo1 checkpoint control proliferating cell nuclear antigen (PCNA).

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.


14-3-3 and 14-3-3 binding proteins

The 14-3-3 family of 27- to 32-kDa acidic proteins is expressed in the cytoplasm of eukaryotic cells. 1 Seven 14-3-3 isotypes have been found in mammals (α/β, γ, τ/θ, ε, η, σ, and ζ/δ), all of which can form homodimers and heterodimers. 1 All 14-3-3 isotypes have highly conserved sequences across species and share the key feature of phosphorylation-dependent binding to serine/threonine-based peptide motifs, which are found in >200 different intracellular phosphoproteins. 2 These motifs are categorized as mode 1 (RSXpSXP), mode 2 (RXY/FXpSXP), 3 and mode 3 (pS/TX1-2COOH), 4 where X represents an interchangeable amino acid residue and lower-case p indicates phosphorylation. 5 However, amino acid sequences in some identified 14-3-3 binding sites, although homologous, do not conform precisely to these modes, and some can be homologous to >1 mode. For example, the C-terminal 14-3-3 binding sites on platelet GPIbα contain the SIRYSGHSL 610 -CO2H sequence, which conforms to mode 3, but also show homology to modes 1 and 2. 6 Additionally, some unphosphorylated peptide sequences were also shown to interact with 14-3-3. 7,8 On the basis of the known binding motifs, tools including software, 9,10 databases, 11 and Web servers, 12 such as Scansite, 9 ELM, 10 and A Nnotation and Integrated Analysis of the 14-3-3 interactome (ANIA), 11 have been developed to help predict 14-3-3 binding peptide sequences.

In target proteins, key Ser/Thr residues of the 14-3-3 binding motifs can be phosphorylated by Ser/Thr protein kinases, including AGC kinases (eg, protein kinase A), calcium/calmodulin-dependent kinases, and LIM kinase, 13-15 and dephosphorylated by phosphatases (eg, PP2A and PP1), 16-18 thereby enhancing or inhibiting, respectively, 14-3-3 binding (Figure 1). 19 In some proteins, >1 serine residue can be phosphorylated, with variable effects. Phosphorylation at both Ser residues in the GPIbα cytoplasmic RRPS 587 ALS 590 sequence seems to be required for 14-3-3 binding. In contrast, in the 14-3-3 binding sequence (RRS 216 RS 218 FT) of the G-protein regulator RGS18, phosphorylation of Ser 218 is important for high-affinity 14-3-3γ binding, but phosphorylation at the neighboring S 216 negatively regulates the interaction. 20,21 Binding of 14-3-3 to its partners can also be negatively regulated by 14-3-3 phosphorylation at Thr 233 and Ser 185 (Figure 1). 7

Phosphorylation-regulated binding between 14-3-3 and its target proteins. Each 14-3-3 monomer contains a binding site for a serine/threonine-phosphorylated 14-3-3 binding motif. Upon phosphorylation of target proteins, a 14-3-3 dimer can bind to 2 phosphorylated motifs in tandem in 1 target protein, modulating its conformation/structure. A 14-3-3 dimer can also bind to 2 separate phosphoproteins, acting as an adapter/scaffold for assembly of protein complexes. Phosphorylation of 14-3-3 at Thr 233 and Ser 185 negatively regulates its ability to interact with target proteins. These features enable 14-3-3 to regulate protein function or transmit signals in a phosphorylation-dependent manner, which can be controlled by a single protein kinase/phosphatase pair or multiple protein kinases/phosphatases.

Phosphorylation-regulated binding between 14-3-3 and its target proteins. Each 14-3-3 monomer contains a binding site for a serine/threonine-phosphorylated 14-3-3 binding motif. Upon phosphorylation of target proteins, a 14-3-3 dimer can bind to 2 phosphorylated motifs in tandem in 1 target protein, modulating its conformation/structure. A 14-3-3 dimer can also bind to 2 separate phosphoproteins, acting as an adapter/scaffold for assembly of protein complexes. Phosphorylation of 14-3-3 at Thr 233 and Ser 185 negatively regulates its ability to interact with target proteins. These features enable 14-3-3 to regulate protein function or transmit signals in a phosphorylation-dependent manner, which can be controlled by a single protein kinase/phosphatase pair or multiple protein kinases/phosphatases.

Members of the 14-3-3 protein family are involved in a variety of phosphorylation-dependent cellular processes, either physiological or pathological. The former include proliferation, 22 differentiation, 23-25 migration, 26-28 cytoskeleton reorganization, apoptosis, 29,30 and cell-cycle checkpoint control, 31-34 and the latter, cancer progression and metastasis, 33,35 although the mechanisms of action are not totally clear. The dimeric nature of 14-3-3 proteins allows association with 2 phosphorylated serine/threonine motifs in the same or in 2 different target proteins (Figure 1). 8,36 Thus, 14-3-3 dimers can act as phosphorylation-dependent adaptors/scaffolds to influence the interactions between 2 phosphoproteins, 37-39 or they may also modulate the conformation of a single polypeptide chain by binding to 2 phosphorylated sites on the same polypeptide chain. 37,38,40 Additionally, it is also possible that 14-3-3 binding inhibits the interaction of its binding partner with other molecules. 8,41,42


Contents

There are seven genes that encode seven distinct 14-3-3 proteins in most mammals (See Human genes below) and 13-15 genes in many higher plants, though typically in fungi they are present only in pairs. Protists have at least one. Eukaryotes can tolerate the loss of a single 14-3-3 gene if multiple genes are expressed, however deletion of all 14-3-3s (as experimentally determined in yeast) results in death. [ citation needed ]

14-3-3 proteins are structurally similar to the Tetratrico Peptide Repeat (TPR) superfamily, which generally have 9 or 10 alpha helices, and usually form homo- and/or hetero-dimer interactions along their amino-termini helices. These proteins contain a number of known common modification domains, including regions for divalent cation interaction, phosphorylation & acetylation, and proteolytic cleavage, among others established and predicted. [3]

14-3-3 binds to peptides. There are common recognition motifs for 14-3-3 proteins that contain a phosphorylated serine or threonine residue, although binding to non-phosphorylated ligands has also been reported. This interaction occurs along a so-called binding groove or cleft that is amphipathic in nature. To date, the crystal structures of six classes of these proteins have been resolved and deposited in the public domain. [ citation needed ]

The motif sites are way more diverse than the patterns here suggest. For an example with a modern recognizer using an artificial neural network, see the cited article. [5]

Discovery and naming Edit

14-3-3 proteins were initially found in brain tissue in 1967 and purified using chromatography and gel electrophoresis. In bovine brain samples, 14-3-3 proteins were located in the 14th fraction eluting from a DEAE-cellulose column and in position 3.3 on a starch electrophoresis gel. [6]

Function Edit

14-3-3 proteins play an isoform-specific role in class switch recombination. They are believed to interact with the protein Activation-Induced (Cytidine) Deaminase in mediating class switch recombination. [7]

Phosphorylation of Cdc25C by CDS1 and CHEK1 creates a binding site for the 14-3-3 family of phosphoserine binding proteins. Binding of 14-3-3 has little effect on Cdc25C activity, and it is believed that 14-3-3 regulates Cdc25C by sequestering it to the cytoplasm, thereby preventing the interactions with CycB-Cdk1 that are localized to the nucleus at the G2/M transition. [8]

The eta isoform is reported to be a biomarker (in synovial fluid) for rheumatoid arthritis. [9]

  • Raf-1
  • Bad – see Bcl-2
  • Bax
  • Cdc25
  • Akt
  • SOS1[10] – see RSK
  • YWHAB – "14-3-3 beta"
  • YWHAE – "14-3-3 epsilon"
  • YWHAG – "14-3-3 gamma"
  • YWHAH – "14-3-3 eta"
  • YWHAQ – "14-3-3 tau"
  • YWHAZ – "14-3-3 zeta"
  • SFN or YWHAS – "14-3-3 sigma" (Stratifin)

The 14-3-3 proteins alpha and delta (YWHAA and YWHAD) are phosphorylated forms of YWHAB and YWHAZ, respectively.

Presence of large gene families of 14-3-3 proteins in the Viridiplantae kingdom reflects their essential role in plant physiology. A phylogenetic analysis of 27 plant species clustered the 14-3-3 proteins into four groups.

14-3-3 proteins activate the auto-inhibited plasma membrane P-type H + ATPases. They bind the ATPases' C-terminus at a conserved threonine. [11]


Ideal for large-scale protein production

The pcDNA™3.3-TOPO® vector contains a modified, enhanced CMV promoter that enables extremely high protein expression. The vector is ideal for large-scale protein production, especially when used in combination with our FreeStyle™ MAX CHO or 293 Expression Systems. We expressed several proteins and observed 8–30 mg/L of recombinant protein produced using our FreeStyle™ systems (Figure 1).

Figure 1. High yields of recombinant protein. We used pcDNA™3.3-TOPO® vector to clone and express milligram levels of erythropoietin (EPO), Factor IX (FIX), and IgG in FreeStyle™ CHO-S cells. pcDNA™3.3-TOPO® constructs containing the relevant PCR-derived gene sequences were transfected into FreeStyle™ CHO-S cells in 30 mL volumes. Heavy and light chain genes were co-transfected for IgG expression. Four to six days posttransfection, EPO and FIX were quantified using immobilized goat anti–human IgG antibody. Each bar shows the average of two expression runs.

Figure 2. pcDNA™3.3 vector map. The latest version in the pcDNA vector family, pcDNA™3.3 offers you the flexibility to express a native protein or add your favorite purification tag to the gene of interest prior to TOPO® cloning.

Our pcDNA™3.3 vector incorporates TOPO® cloning technology, which removes time-intensive and unreliable steps from your cloning workflow, allowing you to perform bench-top cloning reactions in typically just 5 minutes. With up to 95% recovery of your desired clone, you always have the clone you need for downstream experiments.

Figure 3.We cloned and measured expression of luciferase (panel A) and ß-galactosidase (panel B) in pcDNA™3.3-TOPO® vector, pCI and pCMV-Script® vectors. After transient transfection into adherent GripTite™ 293 MSR cells, ß-galactosidase activity and luciferase activity is 2- and 5-fold higher, respectively, using pcDNA™3.3 vector compared to competitor vectors. Error bars shown represent standard deviation (n = 6).

The pcDNA™3.3 TOPO® TA Cloning Kit includes high-efficiency One Shot® TOP10 Competent Cells for superior cloning performance. Our OptiCHO™ Antibody Express Kit, which includes the pcDNA™3.3 TOPO® TA Cloning Kit, enables stable cell line creation and antibody production in a serum-free dihydrofolate reductase-deficient (DHFR–) Chinese hamster ovary (CHO) DG44 cell line.


Results

Identification and structural characterization of rainbow trout 14-3-3 genes

The first Omy14-3-3 gene was found in the subtracted library prepared from stressed rainbow trout brains and was observed to be similar to the mammalian beta protein. To identify other isoforms, we compared the sequences of 47395 rainbow trout ESTs from the normalized rainbow trout library with human 14-3-3 proteins using stand-alone blastx(Altschul et al., 1997). Blastn distributed the clones encoding putative 14-3-3 genes into 10 clusters, which contained identical sequences of different length. Furthermore, we sequenced 10 clones whose 5′-ends included predicted start codons. We analyzed all salmonid fish ESTs deposited in National Center for Biotechnology Information (NCBI) dbEST(http://www.ncbi.nlm.nih.gov/dbEST/)and The Institute for Genomic Research (TIGR http://www.tigr.org/tdb/tgi/rtgi/)and did not find any new isoforms. Multiple sequence alignment with ClustalW(Thompson et al., 1994 http://www.ebi.ac.uk/clustalw/)divided 10 Omy14-3-3 genes into five pairs, which were designated as A, B (similar to beta), C, E (similar to epsilon) and G (similar to gamma). The protein coding sequences were highly similar in every pair (Fig. 1),whereas the 5′- and 3′-UTRs were divergent in all genes except Omy14-3-3A.

Structure of rainbow trout 14-3-3 proteins. The sequences were aligned with ClustalW and the conserved amino acids were highlighted with Boxshade.

Structure of rainbow trout 14-3-3 proteins. The sequences were aligned with ClustalW and the conserved amino acids were highlighted with Boxshade.

For phylogenetic analyses, we used 14-3-3 proteins of teleost fish(Fundulus and Danio) and mammals whose sequences were retrieved from Swiss-Prot(http://us.expasy.org/sprot/)the outroot was from ascidia (Ciona intestinalis). In addition to five Dr14-3-3 proteins from Swiss-Prot, five sequences were available from Ensembl(http://www.ensembl.org/)and four more were found by comparison of UniGene EST clusters(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene)with human 14-3-3 proteins using blastx (Table S2 in supplementary material). The total number of putative Dr14-3-3 genes was 14 however, two short sequences were not included in the analyses. Phylogenetic study provided additional evidence for duplication of ancestral rainbow trout genes, since 10 Omy14-3-3 proteins were divided into five separate clades(Fig. 2). Ten of 12 Dr14-3-3 genes were duplicates, and three Dr14-3-3G genes could arise as a result of two subsequent duplications. Some cyprinids are,like salmonids, tetraploid, and the possibility of ancient genome duplication in zebrafish is debated. All types of Omy14-3-3 proteins were found in zebrafish. In rainbow trout, there was no ortholog to the Fundulus(F) gene however, this was assigned to the clade including Dr14-3-3F1 and F2. Three of six types of fish 14-3-3 proteins (E, G and B) were clustered with mammalian epsilon, gamma and beta proteins at high bootstrap values. The A, C and F types were specific for fish, whereas zeta, sigma, eta and tau 14-3-3 proteins were found exclusively in mammals. Interestingly, in four of five pairs of Omy14-3-3 proteins (B, C, G and E), one gene was assigned to a clade containing the second gene and either the mammalian or zebrafish ortholog. The 14-3-3 cladogram was markedly different from the organismic tree and this suggested rapid divergence of duplicated Omy14-3-3 genes. To verify this finding, we compared synonymous (Ks) and nonsynonymous (Ka) divergence in rainbow trout and mammalian (human and mouse) 14-3-3 genes(Table 1). The mean Ks in fish genes was 1.65 times greater than in mammalian 14-3-3 genes, whereas Ka was increased 10.5-fold.

Cladogram of vertebrate 14-3-3 proteins. Protein distant matrix was computed and the tree was constructed using the neighbor-joining method. The bootstrap values for 1000 replicates are indicated at the nodes of clades that include rainbow trout proteins. Accession numbers of proteins used in phylogenetic analyses are given in Tables S2, S3 (supplementary material.

Cladogram of vertebrate 14-3-3 proteins. Protein distant matrix was computed and the tree was constructed using the neighbor-joining method. The bootstrap values for 1000 replicates are indicated at the nodes of clades that include rainbow trout proteins. Accession numbers of proteins used in phylogenetic analyses are given in Tables S2, S3 (supplementary material.

Genes . Ks . Ka .
Rainbow trout
14-3-3A0.182 0.0074
14-3-3G0.624 0.0243
14-3-3E1.023 0.0266
14-3-3B0.689 0.0539
14-3-3C1.038 0.0866
Mammals
14-3-3 gamma0.437 0
14-3-3 epsilon0.127 0
14-3-3 eta0.704 0.0035
14-3-3 tau0.322 0.0035
14-3-3 zeta0.391 0.007
14-3-3 beta0.597 0.0087
Genes . Ks . Ka .
Rainbow trout
14-3-3A0.182 0.0074
14-3-3G0.624 0.0243
14-3-3E1.023 0.0266
14-3-3B0.689 0.0539
14-3-3C1.038 0.0866
Mammals
14-3-3 gamma0.437 0
14-3-3 epsilon0.127 0
14-3-3 eta0.704 0.0035
14-3-3 tau0.322 0.0035
14-3-3 zeta0.391 0.007
14-3-3 beta0.597 0.0087

Expression of rainbow trout 14-3-3 genes

Expression of 14-3-3 genes in tissues of rainbow trout was analyzed by RT-PCR. The PCR primers were designed to distinguish between the duplicated genes however, we were unable to separate the Omy14-3-3A1and A2 isoforms due to their high sequence similarity. Distribution of 14-3-3 genes was ubiquitous, and transcripts of six genes(Omy14-3-3B1, B2, C1, C2, G1 and G2) were found in 10 of 11 analyzed tissues (Fig. 3). Brain, ovary and testis harbored a complete set of Omy14-3-3 genes. The number of expressed isoforms in other tissues ranked from eight (gill and kidney) to two (skin). Expression of Omy14-3-3 genes was also studied in embryos. Omy14-3-3B1 and B2 were expressed at all analyzed developmental stages (Fig. 4). Three more genes (Omy14-3-3C1, C2 and A)were detectable in blastulas. This could be due to persistence of maternal transcripts, because expression of these genes was interrupted at the subsequent developmental stages. Six of nine analyzed Omy14-3-3 genes were already expressed in the late gastrulas. Expression of Omy14-3-3E2 and C2 began at early somitogenesis (15 somites), whereas G2 was the latest isoform (34 somites).

(A) Expression of Omy14-3-3 genes was analyzed with RTPCR in rainbow trout spleen (sp), kidney (k), intestine (i), liver (l), gill (g),skin (sk), heart (h), skeletal muscle (sm), ovary (o), testis (t) and brain(b). In each sample, RNA was pooled from three individuals and analyses were repeated twice. (B) The gel picture presents differential expression of duplicated Omy14-3-3 genes.

(A) Expression of Omy14-3-3 genes was analyzed with RTPCR in rainbow trout spleen (sp), kidney (k), intestine (i), liver (l), gill (g),skin (sk), heart (h), skeletal muscle (sm), ovary (o), testis (t) and brain(b). In each sample, RNA was pooled from three individuals and analyses were repeated twice. (B) The gel picture presents differential expression of duplicated Omy14-3-3 genes.

(A) Expression of Omy14-3-3 genes in rainbow trout embryos was determined with RT-PCR at different developmental stages: blastula (b), early gastrula (eg), late gastrula (eg), 15 somites (15 sp) and 34 somites (34 sp). In each sample, RNA was pooled from 10 embryos and analyses were repeated twice. (B) The gel picture presents differential expression of duplicated Omy14-3-3 genes.

(A) Expression of Omy14-3-3 genes in rainbow trout embryos was determined with RT-PCR at different developmental stages: blastula (b), early gastrula (eg), late gastrula (eg), 15 somites (15 sp) and 34 somites (34 sp). In each sample, RNA was pooled from 10 embryos and analyses were repeated twice. (B) The gel picture presents differential expression of duplicated Omy14-3-3 genes.

We analysed differential expression of Omy14-3-3 genes in 31 microarray experiments that dealt with response of rainbow trout to stress,environmental pollutants and bacterial antigens. Isoforms were compared by correlation of expression profiles with other genes presented on the slide and the data were analysed with multi-dimensional scaling. Distance metrics were calculated for every pair of duplicated genes and differences of expression profiles were clearly related to the non-synonymous divergence of duplicates(Fig. 5 Table 1). However, it is possible that difference of expression between the A1 and A2isoforms was underestimated. In contrast to other Omy14-3-3 genes,the 3′-UTR sequences of these mRNAs are highly conserved, and cross-hybridization could affect the results of microarray analyses. We observed tight coordination of expression of 10 isoforms in studies of stress response in the rainbow trout brain. All Omy14-3-3 genes were downregulated after 1 day, which was followed by a subsequent increase of expression levels (Fig. 6). We selected genes with significantly similar profiles (Pearson r,P<0.05) and analyzed over-representation of Gene Ontology functional classes (Ashburner et al.,2000) in this group (Table 2). This result suggested that Omy14-3-3 genes could share regulatory mechanisms with nuclear proteins, chaperones and proteins involved in signal transduction, nucleotide binding and metabolism.

Duplicated Omy14-3-3 genes were compared by similarity of expression profiles in 31 microarray experiments. The distances were determined with multidimensional scaling.

Duplicated Omy14-3-3 genes were compared by similarity of expression profiles in 31 microarray experiments. The distances were determined with multidimensional scaling.

Expression of Omy14-3-3 genes in the brain of stressed rainbow trout. One-year-old fish were stressed by netting (2 min) and confinement (20 min) and this treatment was repeated over 5 days. The brain samples were collected 1, 3 and 5 days after the first stress exposure. In each sample, RNA was pooled from four individuals. A dye-swap design was used for hybridization, and expression of genes was measured in 12 replicates.

Expression of Omy14-3-3 genes in the brain of stressed rainbow trout. One-year-old fish were stressed by netting (2 min) and confinement (20 min) and this treatment was repeated over 5 days. The brain samples were collected 1, 3 and 5 days after the first stress exposure. In each sample, RNA was pooled from four individuals. A dye-swap design was used for hybridization, and expression of genes was measured in 12 replicates.

Significantly over-represented functional classes (exact Fisher's test, P<0.05) in the list of genes whose expression correlated with Omy 14-3-3 genes in the brain of stressed rainbow trout

Functional classes . Number of genes .
Nucleus 47
Cell communication 39
Signal transduction 33
Nucleotide binding 28
Intracellular signaling cascade 17
Chaperone activity 12
Small GTPase mediated signal transduction 7
Receptor signaling protein activity 6
Purine ribonucleotide biosynthesis 4
Chromatin binding 4
Functional classes . Number of genes .
Nucleus 47
Cell communication 39
Signal transduction 33
Nucleotide binding 28
Intracellular signaling cascade 17
Chaperone activity 12
Small GTPase mediated signal transduction 7
Receptor signaling protein activity 6
Purine ribonucleotide biosynthesis 4
Chromatin binding 4

Expression of six Omy14-3-3 genes (A1, A2, E1, E2, G1 and G2) in somitic (40 somites) and postsomitic embryos was analyzed with in situ hybridization. To separate closely related isoforms, we used the PCR-amplified 3′-UTRs as templates for preparation of probes. We were unable to find any marked difference in the expression patterns of Omy14-3-3 genes therefore Omy14-3-3B1 is shown as an example (Fig. 7). In somitic embryos (Fig. 7A,B),transcripts were found in the neural crest, eyes, yolk syncytium, tail bud and caudal somites. Interestingly, expression of 14-3-3 genes in the tail bud and caudal somites was seen in some of the analyzed embryos. In postsomitic embryos (Fig. 7C,D), transcripts were detected in the neural crest, gill covers and gill arches and in pectoral fins however, there was no expression in the tail and eyes.

Expression of Omy14-3-3B1 was analyzed in somitic (A,B) and postsomitic (C,D) rainbow trout embryos with in situ hybridization. 15 embryos were analyzed at each developmental stage, and representative examples are shown. Transcripts were detected in the neural crest (nc), eyes(e), yolk syncytium layer (ysl), caudal somites (s), tail bud (tb), gill covers (gc), gill arches (ga) and pectoral fins (pf). The areas of active expression are shown at higher magnification in B and D.

Expression of Omy14-3-3B1 was analyzed in somitic (A,B) and postsomitic (C,D) rainbow trout embryos with in situ hybridization. 15 embryos were analyzed at each developmental stage, and representative examples are shown. Transcripts were detected in the neural crest (nc), eyes(e), yolk syncytium layer (ysl), caudal somites (s), tail bud (tb), gill covers (gc), gill arches (ga) and pectoral fins (pf). The areas of active expression are shown at higher magnification in B and D.


Background

14-3-3 proteins are known to regulate diverse processes via binding phosphorylated target proteins in all eukaryotes [1–5]. Although hundreds of potential 14-3-3-interacting proteins have been identified [1, 5], there have been limited studies that confirm in vivo interactions and/or elucidate the regulating functions of 14-3-3 proteins [6–10]. The most intensively characterized 14-3-3 target proteins are nitrate reductase and H + -ATPase. 14-3-3 proteins activate H + -ATPase [11] and inhibit nitrate reductase activity [12]. Our previous study suggests that three 14-3-3 isoforms (kappa, chi and psi) also play important roles in nitrogen and sulfur metabolic processes by regulating the activities of phosphoenolpyruvate carboxylase and O-acetylserine lyase [13].

Plant 14-3-3 proteins are mainly thought to be regulators of carbon and nitrogen metabolism [2]. However, this assumption is based on studies of only a few target proteins, such as nitrate reductase and sucrose-phosphate synthase [14]. Nitrate reductase is phosphorylated in the dark by the calcium-dependent protein kinase (CDPK) and the sucrose non-fermenting related kinase 1 (SnRK1) that initiates the interaction of the enzyme with the 14-3-3 proteins and its inactivation. In the light, nitrate reductase is dephosphorylated by a protein phosphatase 2A, leading to the dissociation of the 14-3-3 and the activation of nitrate reductase [15–18]. In carbon metabolism, some carbon metabolic enzymes such as sucrose phosphate synthase [19], and the dual function protein 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase [20], have been identified as interacting targets of 14-3-3 proteins. The functional relevance of 14-3-3 proteins in the regulatory mechanism of their targets, however, is still not clear. Considering the hundreds of possible 14-3-3 target proteins revealed through multiple screening studies, the roles so far described are likely to be only a small part of the functions of 14-3-3 proteins [5, 13, 21].

Metabolite profiling is a powerful tool that has contributed to the understanding of plant physiology, including phenotypic differences, gene annotations, metabolite regulation, and characterization of stress responses [22, 23]. Moreover, the integration of metabolomics with other 'omics,' such as genomics, enzymomics, and interactomics, leads not only to construction of metabolic networks but also to understanding the roles particular proteins play within the metabolic network [24, 25]. In this study, by combining metabolomics and genetical, enzymological, biochemical, and molecular approaches, we were able to draw a comprehensive map of the functional roles 14-3-3 proteins play in essential metabolic processes.

Our study further confirms that 14-3-3 proteins are important regulators of both nitrogen and carbon metabolic processes. Specifically, we show that 14-3-3 proteins play roles to control the tricarboxylic acid (TCA) cycle and the shikimate pathway.


For Students & Teachers

For Teachers Only

ENDURING UNDERSTANDING
ENE-1
The highly complex organization of living systems requires constant input of energy and the exchange of macromolecules.

LEARNING OBJECTIVE
ENE-1.F
Explain how changes to the structure of an enzyme may affect its function.

ENE-1.G
Explain how the cellular environment affects enzyme activity.

ESSENTIAL KNOWLEDGE
ENE-1.F.1
Change to the molecular structure of a component in an enzymatic system may result in a change of the function or efficiency of the system –

  1. Denaturation of an enzyme occurs when the protein structure is disrupted, eliminating the ability to catalyze reactions.
  2. Environmental temperatures and pH outside the optimal range for a given enzyme will cause changes to its structure, altering the efficiency with which it catalyzes reactions.

ENE-1.F.2
In some cases, enzyme denaturation is reversible, allowing the enzyme to regain activity.

ENE-1.G.1
Environmental pH can alter the efficiency of enzyme activity, including through disruption of hydrogen bonds that provide enzyme structure.

ENE-1.G.2
The relative concentrations of substrates and products determine how efficiently and enzymatic reaction proceeds.

ENE-1.G.3
Higher environmental temperatures increase the speed of movement of molecules in a solution, increasing the frequency of collisions between enzymes and substrates and therefore increasing the rate of reaction.

ENE-1.G.4
Competitive inhibitor molecules can bind reversibly or irreversibly to the active site of the enzyme. Noncompetitive inhibitors can bind allosteric sites, changing the activity of the enzyme.


14-3-3 Proteins: Regulators of numerous eukaryotic proteins

14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins. IUBMB Life, 57: 623-629, 2005


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