植物中14-3-3蛋白的主要功能

14-3-3蛋白家族广泛存在于真核生物中,序列高度保守。主要以同源或异源二聚体形式存在,可以同时与两个靶蛋白或者与一个靶蛋白的两个结构域相互作用,通过与靶蛋白上的一小段共有序列的磷酸化丝氨酸/苏氨酸残基结合来发挥其调控功能。本文综述了植物中的14-3-3蛋白及其主要功能,并重点综述了14-3-3蛋白对植物基本碳、氮代谢的调控。     

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.     

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.     

Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1′ insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal α-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.     

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     

Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

Two-dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of Creutzfeldt-Jakob disease (CJD). One of the two diagnostic protein spots was identified as isoform(s) of the 14-3-3 family of abundant brain proteins. This has led to the development of one-dimensional 14-3-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14-3-3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias. The results clearly show that the 14-3-3 isoforms beta, gamma, epsilon, and eta are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14-3-3eta also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14-3-3eta in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform-specific 14-3-3 antibodies against beta, gamma, and epsilon should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.     

A multimeric membrane protein reveals 14-3-3 isoform specificity in forward transport in yeast

Arginine (Arg)-based endoplasmic reticulum (ER) localization signals are sorting motifs involved in the quality control of multimeric membrane proteins. They are distinct from other ER localization signals like the C-terminal di-lysine [-K(X)KXX] signal. The Pmp2p isoproteolipid, a type I yeast membrane protein, reports faithfully on the activity of sorting signals when fused to a tail containing either an Arg-based motif or a -KKXX signal. This reporter reveals that the Arg-based ER localization signals from mammalian Kir6.2 and GB1 proteins are functional in yeast. Thus, the machinery involved in recognition of Arg-based signals is evolutionarily conserved. Multimeric presentation of the Arg-based signal from Kir6.2 on Pmp2p results in forward transport, which requires 14-3-3 proteins encoded in yeast by BMH1 and BMH2 in two isoforms. Comparison of a strain without any 14-3-3 proteins (Deltabmh2) and the individual Deltabmh1 or Deltabmh2 shows that the role of 14-3-3 in the trafficking of this multimeric Pmp2p reporter is isoform-specific. Efficient forward transport requires the presence of Bmh1p. The specific role of Bmh1p is not due to differences in abundance or affinity between the isoforms. Our results imply that 14-3-3 proteins mediate forward transport by a mechanism distinct from simple masking of the Arg-based signal.     

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蛋白家族的调控机制和生物学功能

14-3-3蛋白家族在真核细胞中广泛表达并高度保守,它们主要以同源／异源二聚体形式存在,可以同时与两个靶蛋白或一个靶蛋白的两个结构域相互作用。14-3-3蛋白通过磷酸化丝氨酸／苏氨酸介导和靶蛋白结合,从而发挥其调控功能。现对14-3-3蛋白的识别序列、与配体相互作用的特点,及其在细胞周期、凋亡、信号转导、线粒体／叶绿体前体蛋白跨膜转运中的调控机制和发挥的生物学功能进行综述。     

Protozoal sequences may reveal additional isoforms of the 14-3-3 protein family

The phylogenetic position of eleven 14-3-3 proteins from five protozoal species was tested relative to other eukaryotic 14-3-3 versions representing many of the previously described isoforms. The protozoal proteins, four from Entodinium caudatum, three from Entameoba histolytica and four from apicomplexan parasites formed clusters closer to the plant and animal epsilon isoforms than to the animal beta, gamma/eta, sigma/theta, and zeta isoforms. This extends the preliminary findings of Wang and Shakes (1996) but data from a wider range of genera are still required to strengthen our hypothesis that the protozoan isoforms may constitute novel isoforms of the 14-3-3 family.     

A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin

The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.     

The association of 14-3-3gamma and actin plays a role in cell division and apoptosis in astrocytes

The 14-3-3 protein family plays critical regulatory roles in signaling pathways in cell division and apoptosis. 14-3-3gamma is mainly expressed in brain. Using primary cultures of cerebral cortical astrocytes, we investigated the relationships between 14-3-3gamma proteins and actin in astrocytes in cell division and under ischemia. Our results showed that endogenous 14-3-3gamma proteins in immature astrocytes appeared filamentous and co-localized with filamentous actin (F-actin). During certain stages of mitosis, 14-3-3gamma proteins first aggregated and then formed a ring-like structure that surrounded the daughter nuclei and enclosed the F-actin. In 4-week-old cultures of astrocytes, 14-3-3gamma proteins appeared as punctate aggregates in the cytoplasm. Under ischemia, 14-3-3gamma proteins formed filamentous structures and were closely associated with F-actin in surviving astrocytes. However, in apoptotic astrocytes, the intensity of immunostaining of 14-3-3gamma proteins in the cytoplasm decreased. The proteins aggregated around the nucleus and dissociated from the actin filaments. Reciprocal co-immunoprecipitations demonstrated that endogenous 14-3-3gamma proteins bound to detergent-soluble actin and the level of binding increased after 4h of ischemia. As actin is a critical structural protein heavily involved in cell division and apoptotic death, our findings suggest that 14-3-3gamma proteins play a role in cytoskeletal function during the process of cell division and apoptosis in astrocytes in association with actin.