The human and bovine 14-3-3 eta protein mRNAs are highly conserved
Evolutionary conservation of the 14-3-3 protein
Expression and structural analysis of 14-3-3 proteins
Crystal structure of the zeta isoform of the 14-3-3 protein
Structure of a 14-3-3 protein and multiple signalling pathways
Chromosome assignment of human brain 14-3-3 protein eta chain
Subcellular localisation of 14-3-3 isoforms in rat brain using specific antibodies

Site of interaction of 14-3-3 protein with phosphorylated tryptophan hydroxylase
Association of a 14-3-3 protein with CMP-NeuAc:GM1 alpha 2,3-sialyltransferase
Interaction of 14-3-3 with signaling proteins is mediated by phosphoserine
Activation-modulated association of 14-3-3 proteins with Cbl
14-3-3 binding sequence in cytoplasmic domain of adhesion receptor, platelet glycoprotein Ib alpha

14-3-3 brain protein homologs in plants
The 14-3-3 proteins, BMH1 and BMH2, are essential in Saccharomyces cerevisiae
A single Arabidopsis protein has characteristics of diverse 14-3-3 homologues

Expression and structural analysis of 14-3-3 proteins.

Jones DH; Martin H; Madrazo J; Robinson KA; Nielsen P; Roseboom PH; Patel Y; Howell SA; Aitken A 
Laboratory of Protein Structure, National Institute for Medical Research, London, U.K.
J Mol Biol 245: 375-84 (1995)

The 14-3-3 family of proteins plays a role in a wide variety of cellular functions including regulation of protein kinase C and exocytosis. Using antisera specific for the N termini of 14-3-3 isoforms described previously and an additional antiserum specific for the C terminus of epsilon isoform, protease digestion of intact 14-3-3 showed that the N-terminal half of 14-3-3 (a 16 kDa fragment) was an intact, dimeric domain of the protein. Two isoforms of 14-3-3, tau and epsilon, were expressed in E. coli and their secondary structure was shown by circular dichroism to be identical to wild-type protein, and expression of N-terminally-deleted epsilon 14-3-3 protein showed that the N-terminal 26 amino acids are important for dimerization. Intact 14-3-3 is a potent inhibitor of protein kinase C, but the N-terminal domain does not inhibit PKC activity. Site-specific mutagenesis of several regions in the tau isoform of 14-3-3, including the mutation of a putative pseudosubstrate site to a potential substrate sequence, did not alter its inhibitory activity. Intact 14-3-3 proteins are phosphorylated by protein kinase C with a low stoichiometry, but truncated isoforms are phosphorylated much more efficiently by this kinase. This may imply that the proteins may adopt a different structural conformation, possibly upon binding to the membrane, which could modulate their activity. 14-3-3 proteins are found at high concentration on synaptic plasma membranes and this binding is mediated through the N-terminal 12 kDa of 14-3-3.


Activation-modulated association of 14-3-3 proteins with Cbl in T cells

Liu YC; Elly C; Yoshida H; Bonnefoy-Berard N; Altman A
Division of Immunobiology, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037, USA
J Biol Chem 271: 14591-5 (1996)
14-3-3 proteins have recently been implicated in the regulation of intracellular signaling pathways via their interaction with several oncogene and protooncogene products. We found recently that 14-3-3 associates with several tyrosine-phosphorylated proteins and phosphatidylinositol 3-kinase (PI3-K) in T cells. We report here the identification of the 120-kDa 14-3-3tau-binding phosphoprotein present in activated T cell lysates as Cbl, a protooncogene product of unknown function which was found recently to be a major protein-tyrosine kinase (PTK) substrate, and to interact with several signaling molecules including PI3-K, in T lymphocytes. The association between 14-3-3tau and Cbl was detected both in vitro and in intact T cells and, in contrast to Raf-1, was markedly increased following T cell activation. The use of truncated 14-3-3tau fusion proteins demonstrated that the 15 C-terminal residues are required for the association between 14-3-3 and three of its target proteins, namely, Cbl, Raf-1, and PI3-K. The findings that 14-3-3tau binds both PI3-K and Cbl, together with recent reports of an association between Cbl and PI3-K, suggest that 14-3-3 dimers play a critical role in signal transduction processes by promoting and coordinating protein-protein interactions of signaling proteins.

Association of a 14-3-3 protein with CMP-NeuAc:GM1 alpha 2,3-sialyltransferase

Gao L; Gu XB; Yu DS; Yu RK; Zeng G 
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0614, USA
Biochem Biophys Res Commun 224: 103-7 (1996)
CMP-NeuAc:GM1 alpha 2,3-sialyltransferase (ST-IV) was purified to homogeneity from rat brain. Microsequencing of the tryptic peptides derived from the purified enzyme revealed two amino acid sequences homologous to the 14-3-3 proteins. A polyclonal antibody was raised against purified ST-IV. A 33 kDa protein was co-immunoprecipitated from rat brain extracts with the anti-(ST-IV) antibody as detected by Western blot analysis. This protein was identified as a subtype of 14-3-3 family by an anti-(14-3-3) antibody. Screening of a rat brain lambda gt11 library using the anti-(ST-IV) antibody resulted in the identification of a cDNA clone coding for the subtype of 14-3-3 protein. These results indicate an association of the 14-3-3 protein with the sialyltransferase. Since the 14-3-3 protein has PKC inhibitor activities and the activity of sialyltransferases is, at least in part, regulated by PKC, the association of the 14-3-3 protein with ST-IV may indicate a role for this protein in the post-translational regulation of the sialyltransferase activity through the processes of phosphorylation and dephosphorylation.

Subcellular localisation of 14-3-3 isoforms in rat brain using specific antibodies

Martin H; Rostas J; Patel Y; Aitken A 
Laboratory of Protein Structure, National Institute for Medical Research, London, England
J Neurochem 63: 2259-65 (1994)
The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the epsilon, eta, gamma, beta, and zeta isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the eta, epsilon, and gamma isoforms but not the beta and zeta isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the epsilon and gamma isoforms were present and the beta and zeta isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.

Evolutionary conservation of the 14-3-3 protein

Martens GJ; Piosik PA; Danen EH 
Department of Animal Physiology, University of Nijmegen, Toernooiveld, The Netherlands
Biochem Biophys Res Commun 184: 1456-9 (1992)
The novel family of 14-3-3 proteins may be involved in the regulation of neuronal activity. During our search for proteins coordinately expressed with the prohormone proopiomelanocortin in the melanotrope cells of the Xenopus intermediate pituitary gland, we cloned and sequenced a pituitary cDNA encoding a Xenopus 14-3-3 protein. Alignment of the Xenopus protein with known mammalian, Drosophila and plant 14-3-3 polypeptide and with a mammalian protein kinase C inhibitor protein revealed that the neuron-specific 14-3-3-related proteins are highly conserved (60-88%) throughout eukaryotic evolution.

The human and bovine 14-3-3 eta protein mRNAs are highly conserved in both their translated and untranslated regions

Swanson KD; Dhar MS; Joshi JG 
Department of Biochemistry, University of Tennessee, Knoxville 37996
Biochim Biophys Acta 1216: 145-8 (1993)
14-3-3 proteins form a highly conserved protein family whose members have been shown to activate tyrosine and tryptophan hydroxylases, inhibit protein kinase C and possess phospholipase A2 activity in vitro. We have isolated and analyzed a 14-3-3 protein cDNA clone (H14-3-3) from a human fetal brain cDNA library and found it to possess a high level of sequence identity with the bovine 14-3-3 eta protein cDNA in both the translated and untranslated regions, suggesting the presence of cis-regulatory elements in the untranslated regions of these mRNAs. The proteins encoded by these two cDNAs are 98.4% identical. Two different sized RNA species, approx. 1.9 and 3.5 kb in size that are expressed in a variety of tissues hybridize with this cDNA. However, only the 1.9 kb RNA is detected in the fetal brain. Northern blot analysis of poly(A)+ RNA isolated from eight different human tissues shows that 14-3-3 protein mRNAs are expressed in many tissues in the body. In agreement with previous reports, the highest abundance of RNA hybridizing with this cDNA is seen in the brain.

Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways

Xiao B; Smerdon SJ; Jones DH; Dodson GG; Soneji Y; Aitken A; Gamblin SJ 
Division of Protein Structure, National Institute for Medical Research, Mill Hill, London, UK
Nature 376: 188-91 (1995)
A broad range of organisms and tissues contain 14-3-3 proteins, which have been associated with many diverse functions including critical roles in signal transduction pathways, exocytosis and cell cycle regulation. We report here the crystal structure of the human T-cell 14-3-3 isoform (tau) dimer at 2.6 A resolution. Each monomer (Mr 28K) is composed of an unusual arrangement of nine antiparallel alpha-helices organized as two structural domains. The dimer creates a large, negatively charged channel approximately 35 A broad, 35 A wide and 20 A deep. Overall, invariant residues line the interior of this channel whereas the more variable residues are distributed on the outer surface. At the base of this channel is a 16-residue segment of 14-3-3 which has been implicated in the binding of 14-3-3 to protein kinase C.

A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

Korthout HA; de Boer AH
Department of Plant Physiology and Biochemistry, Vrije Universiteit, Amsterdam, The Netherlands
Plant Cell 6: 1681-92 (1994) The fusicoccin binding protein (FCBP) is a highly conserved plasma membrane protein present in all higher plants tested thus far. It exhibits high- and low-affinity binding for the fungal toxin fusicoccin (FC). We purified the active FCBP from a fraction highly enriched in plasma membrane by selective precipitation and anion exchange chromatography. After SDS-PAGE, the two FCBP subunits of 30 and 31 kD were detected as major bands. Amino acid sequence analysis of the 31-kD polypeptide displayed a high degree of identity with so-called 14-3-3 proteins, a class of mammalian brain proteins initially described as regulators of neurotransmitter synthesis and protein kinase C inhibitors. Thereafter, we affinity purified the 30- and 31-kD FCBP subunits, using biotinylated FC in combination with a monomeric avidin column. Immunodecoration of these 30- and 31-kD FCBP subunits with polyclonal antibodies raised against a 14-3-3 homolog from yeast confirmed the identity of the FCBP as a 14-3-3 homolog. Similar to all 14-3-3 protein homologs, the FCBP seems to exist as a dimer in native form. Thus far, the FCBP is the only 14-3-3 homolog with a receptor-like function. The conserved structure of the 14-3-3 protein family is a further indication that the FCBP plays an important role in the physiology of higher plants.

The 14-3-3 proteins, BMH1 and BMH2, are essential in Saccharomyces cerevisiae

van Heusden GP; Griffiths DJ; Ford JC; Chin-A-Woeng TF; Schrader PA; Carr AM; Steensma HY 
Institute of Molecular Plant Sciences, Leiden University, The Netherlands
Eur J Biochem 229: 45-53 (1995)
The 14-3-3 proteins comprise a family of highly conserved acidic proteins. Several activities have been ascribed to these proteins, including activation of tyrosine and tryptophan hydroxylases in the presence of calcium/calmodulin-dependent protein kinase II, regulation of protein kinase C, phospholipase A2 activity, stimulation of exocytosis and activation of bacterial exoenzyme S (ExoS) during ADP-ribosylation of host proteins. In addition, a plant 14-3-3 protein is present in a G-box DNA/protein-binding complex. Previously, we isolated the BMH1 gene from Saccharomyces cerevisiae encoding a putative 14-3-3 protein. Using the polymerase chain reaction method, we have isolated a second yeast gene encoding a 14-3-3 protein (BMH2). While disruption of either BMH1 or BMH2 alone had little effect, it was impossible to obtain viable cells with both genes disrupted.

Crystal structure of the zeta isoform of the 14-3-3 protein

Liu D; Bienkowska J; Petosa C; Collier RJ; Fu H; Liddington R 
Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA
Nature 376: 191-4 (1995)
The 14-3-3 family of proteins have recently been identified as regulatory elements in intracellular signalling pathways: 14-3-3 proteins bind to oncogene and proto-oncogene products, including c-Raf-1 (refs 2-5), c-Bcr (ref. 6) and polyomavirus middle-T antigen; overexpression of 14-3-3 activates Raf kinase in yeast and induces meiotic maturation in Xenopus oocytes. Here we report the crystal structure of the major isoform of mammalian 14-3-3 proteins at 2.9 A resolution. Each subunit of the dimeric protein consists of a bundle of nine antiparallel helices that form a palisade around an amphipathic groove. The groove is large enough to accommodate a tenth helix, and we propose that binding to an amphipathic helix represents a general mechanism for the interaction of 14-3-3 with diverse cellular proteins. The residues in the dimer interface and the putative ligand-binding surface are invariant among vertebrates, yeast and plants, suggesting a conservation of structure and function throughout the 14-3-3 family.

Chromosome assignment of human brain 14-3-3 protein eta chain

Ichimura-Ohshima Y; Morii K; Ichimura T; Araki K; Takahashi Y; Isobe T; Minoshima S; Fukuyama R; Shimizu N; Kuwano R 
Research Laboratory for Molecular Genetics, Niigata University, Japan
J Neurosci Res 31: 600-5 (1992)
We present the nucleotide sequence of a cDNA clone of mRNA encoding human 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases and an endogenous inhibitor of protein kinase C. The 1,730-nucleotide sequence of the cloned cDNA contains 191 bp of a 5'-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3'-noncoding region containing three canonical polyadenylation signals. The 14-3-3 protein eta chain cDNA encoded a polypeptide of 246 amino acids with a predicted molecular weight 28,196. The predicted amino acid sequence of human 14-3-3 protein eta was highly homologous to that of previously reported bovine and rat 14-3-3 proteins with only two amino acid differences. The sequence carries structural features as putative regions responsible for activation of tyrosine and tryptophan hydroxylases and for inhibition of Ca2+/phospholipid-dependent protein kinase C. Northern blot analysis demonstrated widespread expression of the 14-3-3 protein eta chain in cultured cell lines derived from various human tumors. These findings suggest the conservative functions of the 14-3-3 protein among species. Spot blot hybridization analysis with flow-sorted chromosomes showed that the human 14-3-3 protein eta chain gene is assigned to chromosome 22.

Interaction of 14-3-3 with signaling proteins is mediated by phosphoserine

Muslin AJ; Tanner JW; Allen PM; Shaw AS 
Department of Medicine, Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri, 63110, USA
Cell 84: 889-97 (1996)
The highly conserved and ubiquitously expressed 14-3-3 family of proteins bind to a variety of proteins involved in signal transduction and cell cycle regulation. The nature and specificity of 14-3-3 binding is, however, not known. Here we show that 14-3-3 is a specific phosphoserine-binding protein. Using a panel of phosphorylated peptides based on Raf-1, we have defined the 14-3-3 binding motif and show that most of the known 14-3-3 binding proteins contain the motif. Peptides containing the motif could disrupt 14-3-3 complexes and inhibit maturation of Xenopus laevis oocytes. These results suggest that the interactions of 14-3-3 with signaling proteins are critical for the activation of signaling proteins. Our findings also suggest novel roles for serine/threonine phosphorylation in the assembly of protein-protein complexes.

A single Arabidopsis GF14 isoform possesses biochemical characteristics of diverse 14-3-3 homologues

Lu G; de Vetten NC; Sehnke PC; Isobe T; Ichimura T; Fu H; van Heusden GP; Ferl RJ 
Department of Horticulture, University of Florida, Gainesville 32611
Plant Mol Biol 25: 659-67 (1994)
Arabidopsis cDNA clones of GF14 proteins originally were isolated on the basis of their association with the G-box DNA/protein complex by a monoclonal antibody screening approach. GF14 proteins are homologous to the 14-3-3 family of mammalian proteins. Here we demonstrate that recombinant GF14 omega, one member of the Arabidopsis GF14 protein family, is a dimeric protein that possesses many of the attributes of diverse mammalian 14-3-3 homologues. GF14 omega activates rat brain tryptophan hydroxylase and protein kinase C in a manner similar to the bovine 14-3-3 protein. It also activates exoenzyme S of Pseudomonas aeruginosa as does bovine brain factor activating exoenzyme S (FAS), which is itself a member of 14-3-3 proteins. In addition, GF14 omega binds calcium, as does the human 14-3-3 homologue reported to be a phospholipase A2. These results indicate that a single isoform of this plant protein family can have multiple functions and that individual GF14 isoforms may have multiple roles in mediating signal transductions in plants. However, GF14 omega does not regulate growth in an in vivo test for functional similarity to the yeast 14-3-3 homologue, BMH1. Thus, while a single plant GF14 isoform can exhibit many of the biochemical attributes of diverse mammalian 14-3-3 homologues, open questions remain regarding the physiological functions of GF14/14-3-3 proteins.

A binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib alpha

Du X; Fox JE; Pei S 
Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem 271: 7362-7 (1996)
The zeta-form 14-3-3 protein (14-3-3zeta) regulates protein kinases and interacts with several signaling molecules. We reported previously that a platelet adhesion receptor, glycoprotein (GP) Ib-IX, was associated with a 29-kDa protein with partial sequences identical to 14-3-3zeta. In this study, the interaction between GPIb-IX and recombinant 14-3-3zeta is reconstituted. Further, we show that the 14-3-3zeta binding site in GPIb is within a 15 residue sequence at the C terminus of GPIb-alpha, as indicated by antibody inhibition and direct binding of 14-3-3zeta to synthetic GPIb-alpha cytoplasmic domain peptides. The 14-3-3zeta binds to recombinant wild type GPIb-IX but not to the GPIb-alpha mutants lacking C-terminal 5 or more residues, suggesting that the C-terminal 5 residues of GPIb-alpha are critical. Similarity between the GPIb-alpha C-terminal sequence and the serine-rich regions of Raf and Bcr kinases suggests a possible serine-rich recognition motif for the 14-3-3 protein.

Identification of the site of interaction of the 14-3-3 protein with phosphorylated tryptophan hydroxylase

Ichimura T; Uchiyama J; Kunihiro O; Ito M; Horigome T; Omata S; Shinkai F; Kaji H; Isobe T 
Department of Biochemistry, Faculty of Science, Niigata University, Japan
J Biol Chem 270: 28515-8 (1995)
The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are not known. We have previously shown that the 14-3-3 protein binds with and activates phosphorylated tryptophan hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmitter serotonin) and proposed that this activity might be mediated through the COOH-terminal acidic region of the 14-3-3 molecules. In this report we demonstrate, using a series of truncation mutants of the 14-3-3 eta isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH. This restricted region, which we termed 14-3-3 box I, is one of the structural regions whose sequence is highly conserved beyond species, allowing that the plant 14-3-3 isoform (GF14) could also activate rat brain TPH. The 14-3-3 box I is the first functional region whose activity has directly been defined in the 14-3-3 sequence and may represent a common structural element whereby 14-3-3 interacts with other target proteins such as Raf-1 kinase. The result is consistent with the recently published crystal structure of this protein family, which suggests the importance of the negatively charged groove-like structure in the ligand binding.