A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.     

Post-translational modification of 14-3-3 isoforms and regulation of cellular function

14-3-3 is now well established as a family of dimeric proteins that can modulate interaction between proteins involved in a wide range of functions. In many cases, these proteins show a distinct preference for a particular isoform(s) of 14-3-3 and in many cases a specific repertoire of dimer formation influences the particular proteins that 14-3-3 interact. Well over 200 proteins have been shown to interact with 14-3-3. The purpose of this review is to give an overview of the recently identified post-translational modifications of 14-3-3 isoforms and how this regulates function, interaction, specificity of dimerisation between isoforms and cellular location of target proteins. The association between 14-3-3 and its targets usually involves phosphorylation of the interacting protein which has been the subject of many reviews and discussion of this is included in other reviews in this series. However, it is now realised that in some cases the phosphorylation and a number of other, novel covalent modifications of 14-3-3 isoforms may modulate interaction and dimerisation of 14-3-3. Since this aspect is now emerging to be of major importance in the mechanism of regulation by 14-3-3 isoforms and has not been the focus of previous reviews, this will be detailed here.     

Structure and Sites of Phosphorylation of 14-3-3 Protein: Role in Coordinating Signal Transduction Pathways

The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of adaptor protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX_1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.     

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC₅₀=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.     

The cyclin-dependent kinase 11 interacts with 14-3-3 proteins

Cyclin-dependent kinase 11 isoforms (CDK11) are members of the p34(cdc2) superfamily. They have been shown to play a role in RNA processing and apoptosis. In the present study, we investigate whether CDK11 interacts with 14-3-3 proteins. Our study shows that the putative 14-3-3 binding site (113-RHRSHS-118) within the N-terminal domain of CDK11(p110) is functional. Endogenous CDK11(p110) binds directly to 14-3-3 proteins and phosphorylation of the serine 118 within the RHRSHS motif seems to be required for the binding. Besides, CDK11(p110) is capable of interacting with several different isoforms of 14-3-3 proteins both in vitro and in vivo. The interaction of 14-3-3 gamma with CDK11(p110) occurs throughout the entire cell cycle and reaches maximum at the G2/M phase. Interestingly, 14-3-3 gamma shows strong interaction with N-terminal portion of caspase-cleaved CDK11(p110) (CDK11(p60)) product at 48 h after Fas treatment, which correlates with the maximal cleavage level of CDK11(p110) and the maximum activation level of CDK11 kinase activity during apoptosis. Collectively, these results suggest that CDK11 kinases could be regulated by interaction with 14-3-3 proteins during cell cycle and apoptosis.     

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.     

Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase

The highly conserved family of 14-3-3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 suggest functional isoform specificity of 14-3-3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14-3-3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14-3-3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14-3-3 isoforms. We found that 14-3-3C is unavailable in dark harvested barley leaf extract and 14-3-3A is functionally not capable to efficiently inhibit NR activity, leaving 14-3-3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14-3-3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14-3-3A to the alternative residue, as found in 14-3-3B and 14-3-3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14-3-3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14-3-3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14-3-3 isoforms as well as binding affinity determine isoform-specific regulation of NR activity.     

Subcellular Localisation of 14-3-3 Isoforms in Rat Brain Using Specific Antibodies

Abstract: 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 ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ 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.     

Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo

The 14-3-3 protein family associates with many proteins involved in intracellular signalling. In many cases, there is a distinct preference for a particular isoform(s) of 14-3-3. A specific repertoire of 14-3-3 dimer formation may therefore influence which of the interacting proteins could be brought together. We have analysed the pattern of dimer formation for two of the most abundant isoforms of 14-3-3, epsilon ( epsilon ) and gamma (gamma), following their stable expression. This revealed a distinct preference for particular dimer combinations that is largely independent of cellular conditions. gamma 14-3-3 occurred as homodimers and also formed heterodimers, mainly with epsilon 14-3-3 (In PC12 and Cos cells). The epsilon isoform formed heterodimers with 14-3-3 beta, gamma, zeta, and eta, but no homodimers were detected. The two 14-3-3 homologues, BMH1 and BMH2 from Saccharomyces cerevisiae, were mainly heterodimers.     

Molecular evolution of the 14-3-3 protein family

Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene fromCaenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2–5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins fromEntamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/ HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species. A possible ancestral 14-3-3 sequence is proposed. Correspondence to: D.C. Shakes     

PRAS40与14-3-3蛋白各亚型相互作用的酵母双杂交系统检测

PRAS40是近几年新发现的Akt作用底物,14-3-3结合蛋白。为确定PRAS40与14-3-3蛋白7种亚基间相互作用关系,利用gateway方法构建用于酵母双杂交系统的诱饵质粒pEG-PRAS40及转录激活质粒pJG-PRAS40,将PRAS40和14-3-3各亚型质粒分别作为诱饵蛋白质粒及转录激活质粒共转化酵母细胞EGY48,通过氨基酸营养缺陷生长实验及β-半乳糖苷酶显色反应分析两种蛋白相互作用程度。酶切鉴定证实成功地构建了pEG-PRAS40和pJG-PRAS40质粒,酵母双杂交实验结果显示PRAS40可以和14-3-3亚型tau,beta,zeta及epsilon相结合,epsilon较强,beta和zeta次之,tau较弱。此结果将为深入研究PRAS40与14-3-3蛋白生物学功能及发现药物靶标奠定基础。     

14-3-3蛋白在细胞周期调节中的作用

14-3-3是一个在真核细胞中广泛表达、功能复杂的蛋白家族,主要通过磷酸化依赖的方式与靶蛋白结合,从而发挥其调控作用。细胞周期的调节对维持基因组的稳定性至关重要。近年来的研究发现,14-3—3蛋白可以和越来越多的细胞周期调节蛋白相互作用,调节G2／M期和G1／S期转换,从而对细胞周期起调控作用。简要综述了14—3—3蛋白在细胞周期调节中的作用。     

Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation

Voltage-gated Ca2+ channels (VDCCs) are heteromultimeric proteins that mediate Ca2+ influx into cells upon membrane depolarization. These channels are involved in various cellular events, including gene expression, regulation of hormone secretion and synaptic transmission. Kir/Gem, Rad, Rem, and Rem2 belong to the RGK family of Ras-related small G proteins. RGK proteins interact with the beta-subunits and downregulate VDCC activity. Kir/Gem was proposed to prevent surface expression of functional Ca2+ channels, while for Rem2 the mechanism remains controversial. Here, we have analyzed the mechanism by which Rad and Rem regulate VDCC activity. We show that, similar to Kir/Gem and Rem2, 14-3-3 and CaM binding regulate the subcellular distribution of Rad and Rem, which both inhibit Ca2+ channel activity by preventing its expression on the cell surface. This function is regulated by calmodulin and 14-3-3 binding only for Rad and not for Rem. Interestingly, nuclear targeting of Rad and Rem can relocalize and sequester the beta-subunit to the nucleus, thus providing a novel mechanism for Ca2+ channel downregulation.