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Regulatory 14-3-3 protein–protein interactions in plant cells
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Regulatory 14-3-3 protein–protein interactions in plant cells Michael R Roberts Many biological roles for plant 14-3-3 proteins have been suggested in recent months. The most significant of these include roles in the import of nuclear-encoded chloroplast proteins, in the assembly of transcription factor complexes and in the regulation of enzyme activity in response to intracellular signal transduction cascades. Addresses The Plant Laboratory, Department of Biology, University of York, PO Box 373, York, YO10 5YW, UK; e-mail: [email protected] Current Opinion in Plant Biology 2000, 3:400–405 1369-5266/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations FC fusicoccin NR nitrate reductase SnRK1 SNF1-related protein kinase 1 SPS sucrose phosphate synthase
Introduction The 14-3-3 proteins form a conserved eukaryotic family of regulatory proteins that function via phosphorylationdependent interactions with a wide range of target proteins (Figures 1 and 2). Interactions occur at defined sites within the target proteins and are generally regulated by the activities of protein kinases, which phosphorylate these sites in response to intracellular signals [1,2]. Research on 14-3-3 proteins in animal cells has predominantly focused on their function as mediators of signalling and gene expression. In plants, 14-3-3 proteins appear to play important roles in regulating enzymes of primary metabolism, though functions in signal transduction and gene expression are also evident. A number of recent advances in these fields are discussed in this review. In addition, major roles have recently emerged for 14-3-3 proteins in organellar protein trafficking, including chloroplast protein import in higher plants and nuclear exclusion (examples of which will be presented from yeast and animal systems). Finally, the issues surrounding the existence of 14-3-3 gene families will be addressed, as will possible mechanisms for examining 14-3-3 gene function individually and collectively in the plant.
14-3-3 proteins and the regulation of metabolism One of the earliest defined roles for plant 14-3-3 proteins was the light/dark-transition-regulated inhibition of nitrate reductase (NR), a key enzyme in nitrogen metabolism. The interaction between 14-3-3 proteins and NR has now been well characterised, with 14-3-3s binding to a phosphoserine residue in the hinge-1 region of NR, resulting in a block in electron transport between enzyme cofactors. In addition to the direct and immediate inhibitory effect of 14-3-3 binding, it has recently been
demonstrated that the complexed NR is also specifically targeted for proteolytic degradation, providing an additional longer-term mechanism for the reduction of NR activity in the dark [3•]. It is now known that 14-3-3s also bind several other enzymes of primary metabolism, including sucrose phosphate synthase (SPS), trehalose-6-phosphate synthase, glyceraldehyde-3-phosphate dehydrogenase and glutamine synthase [4,5••,6••]. A number of these proteins were identified using gel overlay assays and affinity purification techniques [5••], which have also been used to monitor the patterns of proteins that bind to 14-3-3 that are present in Arabidopsis cells grown under different nutrient conditions [6••]. The results show that 14-3-3 binding proteins are proteolytically degraded en masse in response to sugar starvation, and that interaction with 14-3-3s inhibits this process [6••]. 14-3-3s may thus be involved in a global nutrient-sensing pathway. How might these effects be mediated? Protein kinases that phosphorylate NR at the 14-3-3 binding site include calcium-dependent protein kinases [5••,7] and calciumindependent SNF1-related protein kinase 1 (SnRK1) kinases [8]. SnRK1 kinases are implicated in the global regulation of metabolism in response to cellular energy levels [8], and it is tempting to speculate that they might function by creating 14-3-3 binding sites on various metabolic enzymes. This is an attractive idea, because the same SnRK1 enzymes phosphorylate both NR and SPS [8]. The SnRK1 phosphorylation site is not, however, that identified as the 14-3-3 binding site in SPS [4,8]. Also in the context of metabolic regulation, it has been demonstrated that pH and AMP concentration, which might be expected to change during metabolic stress, can influence the ability of 14-3-3s to bind target proteins via conformational change [9,10]. Interestingly, AMP has also been found to regulate the degradation of 14-3-3 targets in the Arabidopsis sugar-starvation response [6••] and the activity of SnRK1 kinases [8].
Interactions with the plasma membrane H+-ATPase Probably the most intensively studied property of 14-3-3 proteins in plants is their ability to complex with and activate the plasma-membrane proton-pumping ATPase in the presence of the phytotoxin fusicoccin (FC). Recent advances in determining the molecular nature of this interaction and the demonstration of a role for 14-3-3s in regulating H+-ATPase activity in vivo are a highlight of the period covered by this review. Experiments using in vivo 32P labelling initially identified a carboxy-terminal phosphothreonine residue in the
Regulatory 14-3-3 protein–protein interactions in plant cells Roberts
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Figure 1 14-3-3 proteins are conserved phosphopeptide-binding proteins. 14-3-3 proteins exist as dimers, with each monomer able to bind a target peptide sequence within the groove created on the conserved inner face of the protein. Shown here are (a) side and (b) front views of a molecular model of a human 14-3-3ζ monomer with a bound phosphopeptide shown in black [2]. The phosphopeptide, derived from the polyomavirus middle-T antigen 14-3-3 binding site, conforms to one of the major consensus sequences for 14-3-3 binding, that is R(S)XpSXP (using the single-letter code for amino acids), where pS is phosphoserine. The phosphate group in the peptide is circled.
(a)
(b)
Current Opinion in Plant Biology
H+-ATPase that was protected by FC-induced 14-3-3 binding [11]. Subsequently, it has been conclusively demonstrated that in vivo phosphorylation of the penultimate threonine residue of the Arabidopsis H+-ATPase 2 (AHA2) and Nicotiana plumbaginifolia H+-ATPase 2 (PMA2) isoforms, a residue that is also conserved in other H+-ATPase proteins, creates a binding site for 14-3-3s [12•,13•,14]. Interestingly, FC-induced 14-3-3 binding to the H+-ATPase is phosphorylation-independent, but FC significantly increases the affinity of 14-3-3s for the phosphorylated form of the enzyme. Despite the mass of literature on the activation of the H+-ATPase by 14-3-3 proteins in the presence of FC, clear evidence for a physiological regulatory interaction between these two proteins has been provided only recently. One of the effects of FC on plant tissues is stomatal opening, which is induced as a result of H+ extrusion and consequent K+ influx into guard cells, bringing about the changes in turgor responsible for opening. Elegant experiments presented by Kinoshita and Shimazaki [15••], show that blue light, a physiological signal for stomatal opening, induces both phosphorylation of the H+-ATPase carboxy terminus in Vicia guard cells and subsequent enzyme activation that is correlated with the binding of 14-3-3 proteins. Other research has identified protein kinase and phosphatase activities that are able to influence the interaction between the proton pump and 14-3-3s in the absence of FC [12•,16]. It can be assumed that, in the future, 14-3-3 proteins will be found to play broader physiological roles in regulating H+-ATPase activity in response to a range of environmental and hormonal signals. One possible example of this is the activation of the proton pump by cold stress in sugar beet cells, which is correlated with an increased interaction between 14-3-3s and the H+-ATPase [17].
14-3-3 proteins and transcription One of the first group of 14-3-3 proteins to be identified in plants was discovered during studies of transcription. 14-3-3 proteins that interact with G-box-binding complexes were
purified from maize and Arabidopsis and termed G-box factor 14-3-3 (GF14) proteins [18,19], although their function in these complexes remains unknown. More recently, in both rice and maize, 14-3-3 proteins were found to interact with VIVIPAROUS 1 (VP1) and Em-BINDING PROTEIN 1 (EmBP1), transcription factors that are involved in ABA-regulated gene expression in embryos [20]. As the amino terminus of 14-3-3 proteins was required for these interactions, Schultz et al. [20] suggested that the transcription factors interact with the 14-3-3 amino-terminal dimerisation domain. This would be a unique mechanism because all other characterised 14-3-3-protein–protein interactions involve the carboxy terminus. An alternative explanation for the lack of interaction of VP1 and EmBP1 with the amino-terminally truncated 14-3-3 is that only dimeric 14-3-3 is functional. Nevertheless, the intriguing possibility remains that 14-3-3s mediate the formation of complexes between site-specific DNA-binding proteins and tissue-specific regulatory proteins. A potential role for 14-3-3s in regulating transcription more generally was demonstrated when 14-3-3s were found to interact with the TATA-box binding protein (TBP) from Arabidopsis and humans, with the general transcription factor TFIIB, and with the TBP-associated protein hTAFII32 [21•]. In this context, one possible function of 14-3-3s is to mediate interactions between the transcription pre-initiation complex and promoter-specific factors. However, 14-3-3s also appear able to act as direct transcriptional activators in both plant and yeast experimental systems [21•,22]. Nevertheless, no physiological evidence for a function in either transcriptional-complex formation or direct transcriptional activation has yet been presented.
14-3-3 proteins mediate organellar protein trafficking Perhaps the most unexpected newly recognised function for plant 14-3-3s to be uncovered recently is their involvement in chloroplast protein import. Several independent studies have identified interactions between 14-3-3s and
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Figure 2
Protein X
Protein kinase
Protein X
Protein phosphatase
P
14-3-3
Change in activity Change in stability Change in sub-cellular location Interaction with other proteins
PKUα [28] and the mammalian apoptosis-promoting transcription factor forkhead-related 1 (FKHRL1) [29]. Most research in this area, however, has focused on cell-cycle regulation by 14-3-3s. Data from a number of laboratories have shown that phosphorylation-dependent binding of 14-3-3s to Cdc25C phosphatase in response to DNA damage sequesters Cdc25C in the cytoplasm, contributing to cell-cycle arrest in G2 (e.g. [30•,31,32]). Structural analysis [33•] suggests that potential ligand-dependent interactions take place between a nuclear-export signal identified in 14-3-3s [30•] and the nuclear-export machinery. A second cell-cycle regulatory component, the Cdc2-kinase–cyclin-B1 complex, is also sequestered in the cytoplasm by 14-3-3 binding during cell-cycle arrest [34••]. The experiments that uncovered this response also made a significant discovery regarding the functions of different 14-3-3 isoforms. Human cells carrying a knock-out of the 14-3-3σ isoform failed to sequester Cdc2 in the cytoplasm but successfully sequestered Cdc25C [34••], demonstrating clear isoform specificity between individual 14-3-3 proteins involved in the same response within the same cell.
Current Opinion in Plant Biology
Generic function of 14-3-3 proteins. Proteins phosphorylated on serine residues within specific motifs become targets for 14-3-3 binding. Interaction can result in an altered behaviour of the target protein through one of a range of different mechanisms that are summarised in the figure.
nuclear-encoded chloroplast proteins, including glutamine synthase [5••] and the photosystem I N-subunit [23•], which interacts with 14-3-3 via its amino-terminal leader sequence. May and Soll [24••] explained the significance of these interactions when they recognised that a conserved phosphorylation site in the transit peptide sequence of chloroplast precursor proteins was a target for 14-3-3 binding, and that the interaction stimulated import of these proteins into chloroplasts. 14-3-3s had also previously been shown to act as stimulators of mitochondrial protein import in rats [25]. May and Soll presented the fascinating result that plant mitochondrial precursor proteins do not interact with 14-3-3s when translated in a plant lysate, but do so when translated in reticulocyte lysate [24••]. Conversely, chloroplast precursor proteins were only competent for 14-3-3 binding when produced in a plant system. How this specificity is imparted is unclear because rat liver cytosolic 14-3-3 proteins do bind mitochondrial protein precursors synthesised in wheat germ lysate [25,26]. Intriguingly, 14-3-3 proteins have also been localised within the chloroplast itself [23•], but whether this is related to import or some other function has not yet been addressed. In addition to chloroplast and mitochondrial protein targeting, it has recently become clear that 14-3-3s are also important in regulating the nuclear localisation of a number of proteins. Generally, 14-3-3 binding results in the cytoplasmic sequestration of proteins that would otherwise reside in the nucleus. Examples include the yeast transcription factors MSN2 and MSN4 [27], mammalian
14-3-3 gene families and isoform specificity Structural information and in vitro biochemical studies of 14-3-3–phosphoprotein interactions suggest that most, if not all, 14-3-3 proteins bind to target proteins through a common mechanism and with a similar affinity [1,2]. Many different studies have, however, found that certain isoforms of 14-3-3 proteins appear to bind preferentially to particular target proteins or to fulfil particular roles, suggesting the possibility of isoform-specific function. These studies have produced data using co-purification techniques, yeast two-hybrid analysis and characterisation of 14-3-3 mutants of Drosophila. In many cases, apparent isoform specificity for target-protein interactions could simply reflect restricted 14-3-3 gene expression. In these cases, 14-3-3 isoform specificity would arise not through the different biochemical activities of 14-3-3 proteins, but through specific cell and developmental gene expression patterns. Recent analyses of ten tomato 14-3-3 gene family members showed that they had different expression patterns developmentally and in response to a wide range of challenges ([35•]; G de Bruxelles, MR Roberts, unpublished data). Although the expression of these genes have not been localised at the cellular level, data from barley have shown that whereas three 14-3-3 isoforms show overlapping expression patterns in embryos, one of these isoforms is localised to specific cell types [36]. Furthermore, differential post-translational modification of these 14-3-3 proteins was observed in different subcellular fractions from barley embryos [37]. Although the significance of these differential expression patterns remains to be determined, it is clear that the regulation of Cdc2 and Cdc25C during cell-cycle arrest, at least, relies on isoform-specific 14-3-3 functions. What might form the basis of this specificity? One determinant may lie in the structure of 14-3-3 proteins (Figure 1).
Regulatory 14-3-3 protein–protein interactions in plant cells Roberts
Whereas the inner groove that binds target proteins is well conserved, the outer face of the protein is not. It is not impossible that additional points of contact between the target protein and the outer face of the 14-3-3 may impart specificity on some interactions. One factor that is known to influence 14-3-3 activity, however, is phosphorylation of 14-3-3 proteins themselves. In mammals, several different serine/threonine protein kinases are able to phosphorylate distinct subsets of 14-3-3 isoforms [38–40]. In addition, 14-3-3 phosphorylation inhibits protein–protein interactions [38,40]. In plants, it appears that tyrosine phosphorylation of 14-3-3s inhibits interaction with the plasma-membrane H+-ATPase [41•]. The physiological roles of these types of 14-3-3 phosphorylation event should be a target for future research.
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Conclusions Clearly, plant 14-3-3 proteins should now be considered as multifunctional regulatory proteins (Figure 2) that act through their conserved ability to interact with many different target proteins. They have well understood functions in several major areas of cell biology and biochemistry, including the trafficking of proteins to different subcellular locations, the regulation of metabolic enzymes, ion transport and the assembly of transcription complexes. The range of interacting targets for plant 14-3-3 proteins is, however, expanding rapidly. Previously identified targets and those reviewed here implicate roles for 14-3-3s in the areas of signal transduction, germination and responses against biotic and abiotic stresses, amongst others. The challenge ahead is to discover the physiological importance of these interactions in living plants.
New roles for 14-3-3 proteins in plants? Several recent reports have provoked speculation on potential new roles for 14-3-3s in plant signalling. An earlier report had indicated that 14-3-3s could increase the rate of outward K+ transport in transgenic tobacco cells [42], and recent data from Booij et al. [43] indicate a direct role for 14-3-3s in activating K+-outward-rectifying channels in tomato. Both reports imply that increases in cellular 14-3-3 concentrations, such as might result from inducible gene expression [35•], could alter ion transport at the plasma membrane. 14-3-3s have also recently been found to interact with a specific lipoxygenase isoform from barley embryos, suggesting a possible role in the regulation of germination [44]. Another previously unknown function for 14-3-3s may be in ethylene signalling. Yeast two-hybrid screening has indicated an interaction between Arabidopsis 14-3-3s and CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), a Raf-1 kinase homologue (W Ding, C Chang, personal communication). Finally, the mammalian homologue of the Arabidopsis protein TOUSLED, which is essential for organ primordium development [45], was also recently found to be regulated by a 14-3-3, which mediates shuttling between the nucleus and cytoplasm [28]. It will be of interest to determine whether this mode of regulation is paralleled in plants.
New approaches to investigate 14-3-3 function The observation of differential expression patterns for individual plant 14-3-3 genes suggests that it may be possible to address some of their functions through the use of knockout lines or antisense/co-suppression approaches. In addition, methods are now available with which the functions of the 14-3-3 protein population as a whole can be addressed in vivo. For example, dominant negative forms that suppress 14-3-3 activity in Drosophila, yeast and mammals have recently been reported [46,47,48•]. It has recently become evident that although the vast majority of 14-3-3 targets are phosphorylated, this property is not exclusive, as two high-affinity non-phosphorylated binding peptides have been identified [49,50•,51]. Such peptides could prove useful competitive inhibitors of 14-3-3 function if expressed in a regulated manner in transgenic plants.
Acknowledgements I would like to acknowledge all those researchers who allowed me to cite unpublished data and papers in press. My thanks also to Dawn Worrall for reading a draft of the manuscript and to the Royal Society for supporting me through a University Research Fellowship.
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3. Weiner H, Kaiser WM: 14-3-3 proteins control proteolysis of nitrate • reductase in spinach leaves. FEBS Lett 1999, 455:75-78. The experiments described in this paper show that in addition to its role in reversible enzyme inhibition, 14-3-3 binding to NR has a permanent inhibitory effect on NR activity in the dark by targeting the protein for degradation. 4.
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Cotelle V, Meek SEM, Provan F, Milne FC, Morrice N, MacKintosh C: 14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells. EMBO J 2000, 19:2869-2876. Far-western 14-3-3 overlay assays are used to show wide-scale changes in the patterns of 14-3-3 binding proteins present in extracts from Arabidopsis suspension cells grown under different nutrient conditions. A specific proteolytic activity is identified that is responsible for the loss of 14-3-3 binding proteins in sucrose-starved cells. Proteins bound to 14-3-3 are protected from this proteolytic activity. 7.
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Camoni L, Iori V, Marra M, Aducci P: Phosphorylation-dependent interaction between plant plasma membrane H+-ATPase and 14-3-3 proteins. J Biol Chem 2000, 275:9919-9923.
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23. Sehnke PC, Henry R, Cline K, Ferl RJ: Interaction of a plant 14-3-3 • protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma. Plant Physiol 2000, 122:235-241. A 14-3-3 protein is shown to interact with the leader peptide of an Arabidopsis chloroplast protein, and several 14-3-3 isoforms are immunolocalised to the chloroplast stroma and thylakoid membranes. The role of 14-3-3s in chloroplast protein import has been shown in [24••], but the function of 14-3-3s within the chloroplast itself is still unknown. 24. May T, Soll J: 14-3-3 proteins form a guidance complex with •• chloroplast precursor proteins in plants. Plant Cell 2000, 12:53-63. A ground-breaking publication in which the authors demonstrate that the interaction of 14-3-3s with the transit peptide sequences of nuclearencoded chloroplast proteins stimulates import. This phenomenon parallels, but is distinct from, the involvement of 14-3-3s in mitochondrial protein import in mammals. 25. Alam R, Hachiya N, Sakaguchi M, Kawabata S, Iwanaga S, Kitajima M, Mihara K, Omura T: cDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol. J Biochem 1994, 116:416-425. 26. Hachiya N, Alam R, Sakasegawa Y, Sakaguchi M, Mihara K, Omura T: A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins. EMBO J 1993, 12:1579-1586. 27.
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28. Zhang S, Xing H, Muslin AJ: Nuclear localization of protein α is regulated by 14-3-3. J Biol Chem 1999, kinase U-α 274:24865-24872. 29. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME: Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999, 96:857-868. 30. Lopez-Girona A, Furnari B, Mondesert O, Russell P: Nuclear • localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature 1999, 397:172-175. The first of a series of publications that show that the inhibition of Cdc25 activity by 14-3-3 binding during cell-cycle arrest is mediated by nuclear exclusion of the Cdc25–14-3-3 complex. The nuclear export signal is localised on the 14-3-3 protein. 31. Yang J, Winkler K, Yoshida M, Kornbluth S: Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import. EMBO J 1999, 18:2174-2183. 32. Zeng Y, Piwnica-Worms H: DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding. Mol Cell Biol 1999, 19:7410-7419. 33. Rittinger K, Budman J, Xu JA, Volinia S, Cantley LC, Smerdon SJ, • Gamblin SJ, Yaffe MB: Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol Cell 1999, 4:153-166. The mechanisms of 14-3-3 interaction with two different peptide motifs are identified by X-ray crystallography, and the affect of protein–protein interactions on the nuclear export signal located in 14-3-3s is discussed. 34. Chan TA, Hermeking H, Lengauer C, Kinzler KW, Vogelstein B: 14-3-3 •• is required to prevent mitotic catastrophe after DNA damage. Nature 1999, 401:616-620. This important paper identifies different functions for specific 14-3-3 isoforms in the arrest of the cell cycle. 14-3-3σ, which is upregulated by DNA damage in a p53-dependent manner, is shown to be essential for the maintenance of arrest via interaction with Cdc2/cyclin B1. 14-3-3-dependent initiation of arrest still occurs in 14-3-3σ-knock-out cells as other 14-3-3 isoforms bind Cdc25C. 35. Roberts MR, Bowles DJ: Fusicoccin, 14-3-3 proteins, and • defense responses in tomato plants. Plant Physiol 1999, 119:1243-1250. This work includes a systematic analysis of ten members of the 14-3-3 gene family of tomato. It shows isoform-specific gene expression patterns in response to different challenges, including a pathogen avirulence elicitor. 36. Testerink C, van der Meulen RM, Oppedijk BJ, de Boer AH, Heimovaara-Dijkstra S, Kijne JW, Wang M: Differences in spatial expression between 14-3-3 isoforms in germinating barley embryos. Plant Physiol 1999, 121:81-87.
Regulatory 14-3-3 protein–protein interactions in plant cells Roberts
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van Zeijl MJ, Testerink C, Kijne JW, Wang M: Subcellular differences in post-translational modification of barley 14-3-3 proteins. FEBS Lett 2000, 473:292-296.
38. Dubois T, Rommel C, Howell S, Steinhussen U, Soneji Y, Morrice N, Moelling R, Aitken A: 14-3-3 is phosphorylated by casein kinase I on residue 233 — phosphorylation at this site in vivo regulates Raf/14-3-3 interaction. J Biol Chem 1997, 272:28882-28888. 39. Megidish T, Cooper J, Zhang LX, Fu HA, Hakomori S: A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein. J Biol Chem 1998, 273:21834-21845. 40. van der Hoeven PCJ, van der Wal JCM, Ruurs P, van Dijk MCM, van Blitterswijk WJ: 14-3-3 isotypes facilitate coupling of protein kinase C-ζζ to Raf-1: negative regulation by 14-3-3 phosphorylation. Biochem J 2000, 345:297-306. 41. Olivari C, Albumi C, Pugliarello MC, de Michelis MI: Phenylarsine • oxide inhibits the fusicoccin-induced activation of plasma membrane H+-ATPase. Plant Physiol 2000, 122:463-470. In an attempt to investigate the requirement for phosphorylation of the H+-ATPase for interaction with 14-3-3s, it is discovered that tyrosine phosphorylation of 14-3-3 protein appears to inhibit the interaction. This suggests that 14-3-3 activity is controlled by a mechanism that is independent of target-protein phosphorylation.
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44. Holtman WL, Roberts MR, Oppedijk BJ, Testerink C, van Zeijl MJ, Wang M: 14-3-3 proteins interact with a 13-lipoxygenase, but not with a 9-lipoxygenase. FEBS Lett 2000, 474:48-52. 45. Roe JL, Rivin CJ, Sessions RA, Feldmann KA, Zambryski PC: The Tousled gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development. Cell 1993, 75:939-950. 46. Chang HC, Rubin GM: 14-3-3εε positively regulates Ras-mediated signaling in Drosophila. Genes Dev 1997, 11:1132-1139. 47.
Roth D, Birkenfeld J, Betz H: Dominant-negative alleles of 14-3-3 proteins cause defects in actin organization and vesicle targeting in the yeast Saccharomyces cerevisiae. FEBS Lett 1999, 460:411-416.
48. Xing H, Zhang S, Weinheimer C, Kovacs A, Muslin AJ: 14-3-3 • proteins block apoptosis and differentially regulate MAPK cascades. EMBO J 2000, 19:349-358. Dominant negative 14-3-3 protein variants are used to assess native 14-3-3 function during apoptosis in cultured cells and transgenic mice. The data confirm that 14-3-3s inhibit apoptosis-promoting proteins. 49. Masters SC, Pederson KJ, Zhang LX, Barbieri JT, Fu HA: Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa. Biochemistry 1999, 38:5216-5221.
42. Saalbach G, Schwerdel M, Natura G, Buschmann P, Christov V, Dahse I: Over-expression of plant 14-3-3 proteins in tobacco: enhancement of the plasmalemma K+ conductance of mesophyll cells. FEBS Lett 1997, 413:294-298.
50. Wang BC, Yang HZ, Liu YC, Jelinek T, Zhang LX, Ruoslahti E, Fu H: • Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. Biochemistry 1999, 38:12499-12504. Non-phosphorylated peptide ligands of 14-3-3s are identified by phage-display screening. One peptide in particular shows high affinity for several 14-3-3 isoforms, binds at the same site as physiological target peptides and competes effectively for their interaction with 14-3-3 proteins.
43. Booij PP, Roberts MR, Vogelzang SA, Kraayenhof R, de Boer AH: 14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tomato cells. Plant J 1999, 20:673-683.
51. Petosa C, Masters SC, Bankston LA, Pohl J, Wang B, Fu H, Liddington RC: 14-3-3ζζ binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove. J Biol Chem 1998, 273:16305-16310.
Regulatory 14-3-3 protein–protein interactions in plant cells Michael R Roberts Many biological roles for plant 14-3-3 proteins have been suggested in recent months. The most significant of these include roles in the import of nuclear-encoded chloroplast proteins, in the assembly of transcription factor complexes and in the regulation of enzyme activity in response to intracellular signal transduction cascades. Addresses The Plant Laboratory, Department of Biology, University of York, PO Box 373, York, YO10 5YW, UK; e-mail: [email protected] Current Opinion in Plant Biology 2000, 3:400–405 1369-5266/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations FC fusicoccin NR nitrate reductase SnRK1 SNF1-related protein kinase 1 SPS sucrose phosphate synthase
Introduction The 14-3-3 proteins form a conserved eukaryotic family of regulatory proteins that function via phosphorylationdependent interactions with a wide range of target proteins (Figures 1 and 2). Interactions occur at defined sites within the target proteins and are generally regulated by the activities of protein kinases, which phosphorylate these sites in response to intracellular signals [1,2]. Research on 14-3-3 proteins in animal cells has predominantly focused on their function as mediators of signalling and gene expression. In plants, 14-3-3 proteins appear to play important roles in regulating enzymes of primary metabolism, though functions in signal transduction and gene expression are also evident. A number of recent advances in these fields are discussed in this review. In addition, major roles have recently emerged for 14-3-3 proteins in organellar protein trafficking, including chloroplast protein import in higher plants and nuclear exclusion (examples of which will be presented from yeast and animal systems). Finally, the issues surrounding the existence of 14-3-3 gene families will be addressed, as will possible mechanisms for examining 14-3-3 gene function individually and collectively in the plant.
14-3-3 proteins and the regulation of metabolism One of the earliest defined roles for plant 14-3-3 proteins was the light/dark-transition-regulated inhibition of nitrate reductase (NR), a key enzyme in nitrogen metabolism. The interaction between 14-3-3 proteins and NR has now been well characterised, with 14-3-3s binding to a phosphoserine residue in the hinge-1 region of NR, resulting in a block in electron transport between enzyme cofactors. In addition to the direct and immediate inhibitory effect of 14-3-3 binding, it has recently been
demonstrated that the complexed NR is also specifically targeted for proteolytic degradation, providing an additional longer-term mechanism for the reduction of NR activity in the dark [3•]. It is now known that 14-3-3s also bind several other enzymes of primary metabolism, including sucrose phosphate synthase (SPS), trehalose-6-phosphate synthase, glyceraldehyde-3-phosphate dehydrogenase and glutamine synthase [4,5••,6••]. A number of these proteins were identified using gel overlay assays and affinity purification techniques [5••], which have also been used to monitor the patterns of proteins that bind to 14-3-3 that are present in Arabidopsis cells grown under different nutrient conditions [6••]. The results show that 14-3-3 binding proteins are proteolytically degraded en masse in response to sugar starvation, and that interaction with 14-3-3s inhibits this process [6••]. 14-3-3s may thus be involved in a global nutrient-sensing pathway. How might these effects be mediated? Protein kinases that phosphorylate NR at the 14-3-3 binding site include calcium-dependent protein kinases [5••,7] and calciumindependent SNF1-related protein kinase 1 (SnRK1) kinases [8]. SnRK1 kinases are implicated in the global regulation of metabolism in response to cellular energy levels [8], and it is tempting to speculate that they might function by creating 14-3-3 binding sites on various metabolic enzymes. This is an attractive idea, because the same SnRK1 enzymes phosphorylate both NR and SPS [8]. The SnRK1 phosphorylation site is not, however, that identified as the 14-3-3 binding site in SPS [4,8]. Also in the context of metabolic regulation, it has been demonstrated that pH and AMP concentration, which might be expected to change during metabolic stress, can influence the ability of 14-3-3s to bind target proteins via conformational change [9,10]. Interestingly, AMP has also been found to regulate the degradation of 14-3-3 targets in the Arabidopsis sugar-starvation response [6••] and the activity of SnRK1 kinases [8].
Interactions with the plasma membrane H+-ATPase Probably the most intensively studied property of 14-3-3 proteins in plants is their ability to complex with and activate the plasma-membrane proton-pumping ATPase in the presence of the phytotoxin fusicoccin (FC). Recent advances in determining the molecular nature of this interaction and the demonstration of a role for 14-3-3s in regulating H+-ATPase activity in vivo are a highlight of the period covered by this review. Experiments using in vivo 32P labelling initially identified a carboxy-terminal phosphothreonine residue in the
Regulatory 14-3-3 protein–protein interactions in plant cells Roberts
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Figure 1 14-3-3 proteins are conserved phosphopeptide-binding proteins. 14-3-3 proteins exist as dimers, with each monomer able to bind a target peptide sequence within the groove created on the conserved inner face of the protein. Shown here are (a) side and (b) front views of a molecular model of a human 14-3-3ζ monomer with a bound phosphopeptide shown in black [2]. The phosphopeptide, derived from the polyomavirus middle-T antigen 14-3-3 binding site, conforms to one of the major consensus sequences for 14-3-3 binding, that is R(S)XpSXP (using the single-letter code for amino acids), where pS is phosphoserine. The phosphate group in the peptide is circled.
(a)
(b)
Current Opinion in Plant Biology
H+-ATPase that was protected by FC-induced 14-3-3 binding [11]. Subsequently, it has been conclusively demonstrated that in vivo phosphorylation of the penultimate threonine residue of the Arabidopsis H+-ATPase 2 (AHA2) and Nicotiana plumbaginifolia H+-ATPase 2 (PMA2) isoforms, a residue that is also conserved in other H+-ATPase proteins, creates a binding site for 14-3-3s [12•,13•,14]. Interestingly, FC-induced 14-3-3 binding to the H+-ATPase is phosphorylation-independent, but FC significantly increases the affinity of 14-3-3s for the phosphorylated form of the enzyme. Despite the mass of literature on the activation of the H+-ATPase by 14-3-3 proteins in the presence of FC, clear evidence for a physiological regulatory interaction between these two proteins has been provided only recently. One of the effects of FC on plant tissues is stomatal opening, which is induced as a result of H+ extrusion and consequent K+ influx into guard cells, bringing about the changes in turgor responsible for opening. Elegant experiments presented by Kinoshita and Shimazaki [15••], show that blue light, a physiological signal for stomatal opening, induces both phosphorylation of the H+-ATPase carboxy terminus in Vicia guard cells and subsequent enzyme activation that is correlated with the binding of 14-3-3 proteins. Other research has identified protein kinase and phosphatase activities that are able to influence the interaction between the proton pump and 14-3-3s in the absence of FC [12•,16]. It can be assumed that, in the future, 14-3-3 proteins will be found to play broader physiological roles in regulating H+-ATPase activity in response to a range of environmental and hormonal signals. One possible example of this is the activation of the proton pump by cold stress in sugar beet cells, which is correlated with an increased interaction between 14-3-3s and the H+-ATPase [17].
14-3-3 proteins and transcription One of the first group of 14-3-3 proteins to be identified in plants was discovered during studies of transcription. 14-3-3 proteins that interact with G-box-binding complexes were
purified from maize and Arabidopsis and termed G-box factor 14-3-3 (GF14) proteins [18,19], although their function in these complexes remains unknown. More recently, in both rice and maize, 14-3-3 proteins were found to interact with VIVIPAROUS 1 (VP1) and Em-BINDING PROTEIN 1 (EmBP1), transcription factors that are involved in ABA-regulated gene expression in embryos [20]. As the amino terminus of 14-3-3 proteins was required for these interactions, Schultz et al. [20] suggested that the transcription factors interact with the 14-3-3 amino-terminal dimerisation domain. This would be a unique mechanism because all other characterised 14-3-3-protein–protein interactions involve the carboxy terminus. An alternative explanation for the lack of interaction of VP1 and EmBP1 with the amino-terminally truncated 14-3-3 is that only dimeric 14-3-3 is functional. Nevertheless, the intriguing possibility remains that 14-3-3s mediate the formation of complexes between site-specific DNA-binding proteins and tissue-specific regulatory proteins. A potential role for 14-3-3s in regulating transcription more generally was demonstrated when 14-3-3s were found to interact with the TATA-box binding protein (TBP) from Arabidopsis and humans, with the general transcription factor TFIIB, and with the TBP-associated protein hTAFII32 [21•]. In this context, one possible function of 14-3-3s is to mediate interactions between the transcription pre-initiation complex and promoter-specific factors. However, 14-3-3s also appear able to act as direct transcriptional activators in both plant and yeast experimental systems [21•,22]. Nevertheless, no physiological evidence for a function in either transcriptional-complex formation or direct transcriptional activation has yet been presented.
14-3-3 proteins mediate organellar protein trafficking Perhaps the most unexpected newly recognised function for plant 14-3-3s to be uncovered recently is their involvement in chloroplast protein import. Several independent studies have identified interactions between 14-3-3s and
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Figure 2
Protein X
Protein kinase
Protein X
Protein phosphatase
P
14-3-3
Change in activity Change in stability Change in sub-cellular location Interaction with other proteins
PKUα [28] and the mammalian apoptosis-promoting transcription factor forkhead-related 1 (FKHRL1) [29]. Most research in this area, however, has focused on cell-cycle regulation by 14-3-3s. Data from a number of laboratories have shown that phosphorylation-dependent binding of 14-3-3s to Cdc25C phosphatase in response to DNA damage sequesters Cdc25C in the cytoplasm, contributing to cell-cycle arrest in G2 (e.g. [30•,31,32]). Structural analysis [33•] suggests that potential ligand-dependent interactions take place between a nuclear-export signal identified in 14-3-3s [30•] and the nuclear-export machinery. A second cell-cycle regulatory component, the Cdc2-kinase–cyclin-B1 complex, is also sequestered in the cytoplasm by 14-3-3 binding during cell-cycle arrest [34••]. The experiments that uncovered this response also made a significant discovery regarding the functions of different 14-3-3 isoforms. Human cells carrying a knock-out of the 14-3-3σ isoform failed to sequester Cdc2 in the cytoplasm but successfully sequestered Cdc25C [34••], demonstrating clear isoform specificity between individual 14-3-3 proteins involved in the same response within the same cell.
Current Opinion in Plant Biology
Generic function of 14-3-3 proteins. Proteins phosphorylated on serine residues within specific motifs become targets for 14-3-3 binding. Interaction can result in an altered behaviour of the target protein through one of a range of different mechanisms that are summarised in the figure.
nuclear-encoded chloroplast proteins, including glutamine synthase [5••] and the photosystem I N-subunit [23•], which interacts with 14-3-3 via its amino-terminal leader sequence. May and Soll [24••] explained the significance of these interactions when they recognised that a conserved phosphorylation site in the transit peptide sequence of chloroplast precursor proteins was a target for 14-3-3 binding, and that the interaction stimulated import of these proteins into chloroplasts. 14-3-3s had also previously been shown to act as stimulators of mitochondrial protein import in rats [25]. May and Soll presented the fascinating result that plant mitochondrial precursor proteins do not interact with 14-3-3s when translated in a plant lysate, but do so when translated in reticulocyte lysate [24••]. Conversely, chloroplast precursor proteins were only competent for 14-3-3 binding when produced in a plant system. How this specificity is imparted is unclear because rat liver cytosolic 14-3-3 proteins do bind mitochondrial protein precursors synthesised in wheat germ lysate [25,26]. Intriguingly, 14-3-3 proteins have also been localised within the chloroplast itself [23•], but whether this is related to import or some other function has not yet been addressed. In addition to chloroplast and mitochondrial protein targeting, it has recently become clear that 14-3-3s are also important in regulating the nuclear localisation of a number of proteins. Generally, 14-3-3 binding results in the cytoplasmic sequestration of proteins that would otherwise reside in the nucleus. Examples include the yeast transcription factors MSN2 and MSN4 [27], mammalian
14-3-3 gene families and isoform specificity Structural information and in vitro biochemical studies of 14-3-3–phosphoprotein interactions suggest that most, if not all, 14-3-3 proteins bind to target proteins through a common mechanism and with a similar affinity [1,2]. Many different studies have, however, found that certain isoforms of 14-3-3 proteins appear to bind preferentially to particular target proteins or to fulfil particular roles, suggesting the possibility of isoform-specific function. These studies have produced data using co-purification techniques, yeast two-hybrid analysis and characterisation of 14-3-3 mutants of Drosophila. In many cases, apparent isoform specificity for target-protein interactions could simply reflect restricted 14-3-3 gene expression. In these cases, 14-3-3 isoform specificity would arise not through the different biochemical activities of 14-3-3 proteins, but through specific cell and developmental gene expression patterns. Recent analyses of ten tomato 14-3-3 gene family members showed that they had different expression patterns developmentally and in response to a wide range of challenges ([35•]; G de Bruxelles, MR Roberts, unpublished data). Although the expression of these genes have not been localised at the cellular level, data from barley have shown that whereas three 14-3-3 isoforms show overlapping expression patterns in embryos, one of these isoforms is localised to specific cell types [36]. Furthermore, differential post-translational modification of these 14-3-3 proteins was observed in different subcellular fractions from barley embryos [37]. Although the significance of these differential expression patterns remains to be determined, it is clear that the regulation of Cdc2 and Cdc25C during cell-cycle arrest, at least, relies on isoform-specific 14-3-3 functions. What might form the basis of this specificity? One determinant may lie in the structure of 14-3-3 proteins (Figure 1).
Regulatory 14-3-3 protein–protein interactions in plant cells Roberts
Whereas the inner groove that binds target proteins is well conserved, the outer face of the protein is not. It is not impossible that additional points of contact between the target protein and the outer face of the 14-3-3 may impart specificity on some interactions. One factor that is known to influence 14-3-3 activity, however, is phosphorylation of 14-3-3 proteins themselves. In mammals, several different serine/threonine protein kinases are able to phosphorylate distinct subsets of 14-3-3 isoforms [38–40]. In addition, 14-3-3 phosphorylation inhibits protein–protein interactions [38,40]. In plants, it appears that tyrosine phosphorylation of 14-3-3s inhibits interaction with the plasma-membrane H+-ATPase [41•]. The physiological roles of these types of 14-3-3 phosphorylation event should be a target for future research.
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Conclusions Clearly, plant 14-3-3 proteins should now be considered as multifunctional regulatory proteins (Figure 2) that act through their conserved ability to interact with many different target proteins. They have well understood functions in several major areas of cell biology and biochemistry, including the trafficking of proteins to different subcellular locations, the regulation of metabolic enzymes, ion transport and the assembly of transcription complexes. The range of interacting targets for plant 14-3-3 proteins is, however, expanding rapidly. Previously identified targets and those reviewed here implicate roles for 14-3-3s in the areas of signal transduction, germination and responses against biotic and abiotic stresses, amongst others. The challenge ahead is to discover the physiological importance of these interactions in living plants.
New roles for 14-3-3 proteins in plants? Several recent reports have provoked speculation on potential new roles for 14-3-3s in plant signalling. An earlier report had indicated that 14-3-3s could increase the rate of outward K+ transport in transgenic tobacco cells [42], and recent data from Booij et al. [43] indicate a direct role for 14-3-3s in activating K+-outward-rectifying channels in tomato. Both reports imply that increases in cellular 14-3-3 concentrations, such as might result from inducible gene expression [35•], could alter ion transport at the plasma membrane. 14-3-3s have also recently been found to interact with a specific lipoxygenase isoform from barley embryos, suggesting a possible role in the regulation of germination [44]. Another previously unknown function for 14-3-3s may be in ethylene signalling. Yeast two-hybrid screening has indicated an interaction between Arabidopsis 14-3-3s and CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), a Raf-1 kinase homologue (W Ding, C Chang, personal communication). Finally, the mammalian homologue of the Arabidopsis protein TOUSLED, which is essential for organ primordium development [45], was also recently found to be regulated by a 14-3-3, which mediates shuttling between the nucleus and cytoplasm [28]. It will be of interest to determine whether this mode of regulation is paralleled in plants.
New approaches to investigate 14-3-3 function The observation of differential expression patterns for individual plant 14-3-3 genes suggests that it may be possible to address some of their functions through the use of knockout lines or antisense/co-suppression approaches. In addition, methods are now available with which the functions of the 14-3-3 protein population as a whole can be addressed in vivo. For example, dominant negative forms that suppress 14-3-3 activity in Drosophila, yeast and mammals have recently been reported [46,47,48•]. It has recently become evident that although the vast majority of 14-3-3 targets are phosphorylated, this property is not exclusive, as two high-affinity non-phosphorylated binding peptides have been identified [49,50•,51]. Such peptides could prove useful competitive inhibitors of 14-3-3 function if expressed in a regulated manner in transgenic plants.
Acknowledgements I would like to acknowledge all those researchers who allowed me to cite unpublished data and papers in press. My thanks also to Dawn Worrall for reading a draft of the manuscript and to the Royal Society for supporting me through a University Research Fellowship.
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Cotelle V, Meek SEM, Provan F, Milne FC, Morrice N, MacKintosh C: 14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells. EMBO J 2000, 19:2869-2876. Far-western 14-3-3 overlay assays are used to show wide-scale changes in the patterns of 14-3-3 binding proteins present in extracts from Arabidopsis suspension cells grown under different nutrient conditions. A specific proteolytic activity is identified that is responsible for the loss of 14-3-3 binding proteins in sucrose-starved cells. Proteins bound to 14-3-3 are protected from this proteolytic activity. 7.
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Camoni L, Iori V, Marra M, Aducci P: Phosphorylation-dependent interaction between plant plasma membrane H+-ATPase and 14-3-3 proteins. J Biol Chem 2000, 275:9919-9923.
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23. Sehnke PC, Henry R, Cline K, Ferl RJ: Interaction of a plant 14-3-3 • protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma. Plant Physiol 2000, 122:235-241. A 14-3-3 protein is shown to interact with the leader peptide of an Arabidopsis chloroplast protein, and several 14-3-3 isoforms are immunolocalised to the chloroplast stroma and thylakoid membranes. The role of 14-3-3s in chloroplast protein import has been shown in [24••], but the function of 14-3-3s within the chloroplast itself is still unknown. 24. May T, Soll J: 14-3-3 proteins form a guidance complex with •• chloroplast precursor proteins in plants. Plant Cell 2000, 12:53-63. A ground-breaking publication in which the authors demonstrate that the interaction of 14-3-3s with the transit peptide sequences of nuclearencoded chloroplast proteins stimulates import. This phenomenon parallels, but is distinct from, the involvement of 14-3-3s in mitochondrial protein import in mammals. 25. Alam R, Hachiya N, Sakaguchi M, Kawabata S, Iwanaga S, Kitajima M, Mihara K, Omura T: cDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol. J Biochem 1994, 116:416-425. 26. Hachiya N, Alam R, Sakasegawa Y, Sakaguchi M, Mihara K, Omura T: A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins. EMBO J 1993, 12:1579-1586. 27.
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28. Zhang S, Xing H, Muslin AJ: Nuclear localization of protein α is regulated by 14-3-3. J Biol Chem 1999, kinase U-α 274:24865-24872. 29. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME: Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999, 96:857-868. 30. Lopez-Girona A, Furnari B, Mondesert O, Russell P: Nuclear • localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature 1999, 397:172-175. The first of a series of publications that show that the inhibition of Cdc25 activity by 14-3-3 binding during cell-cycle arrest is mediated by nuclear exclusion of the Cdc25–14-3-3 complex. The nuclear export signal is localised on the 14-3-3 protein. 31. Yang J, Winkler K, Yoshida M, Kornbluth S: Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import. EMBO J 1999, 18:2174-2183. 32. Zeng Y, Piwnica-Worms H: DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding. Mol Cell Biol 1999, 19:7410-7419. 33. Rittinger K, Budman J, Xu JA, Volinia S, Cantley LC, Smerdon SJ, • Gamblin SJ, Yaffe MB: Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol Cell 1999, 4:153-166. The mechanisms of 14-3-3 interaction with two different peptide motifs are identified by X-ray crystallography, and the affect of protein–protein interactions on the nuclear export signal located in 14-3-3s is discussed. 34. Chan TA, Hermeking H, Lengauer C, Kinzler KW, Vogelstein B: 14-3-3 •• is required to prevent mitotic catastrophe after DNA damage. Nature 1999, 401:616-620. This important paper identifies different functions for specific 14-3-3 isoforms in the arrest of the cell cycle. 14-3-3σ, which is upregulated by DNA damage in a p53-dependent manner, is shown to be essential for the maintenance of arrest via interaction with Cdc2/cyclin B1. 14-3-3-dependent initiation of arrest still occurs in 14-3-3σ-knock-out cells as other 14-3-3 isoforms bind Cdc25C. 35. Roberts MR, Bowles DJ: Fusicoccin, 14-3-3 proteins, and • defense responses in tomato plants. Plant Physiol 1999, 119:1243-1250. This work includes a systematic analysis of ten members of the 14-3-3 gene family of tomato. It shows isoform-specific gene expression patterns in response to different challenges, including a pathogen avirulence elicitor. 36. Testerink C, van der Meulen RM, Oppedijk BJ, de Boer AH, Heimovaara-Dijkstra S, Kijne JW, Wang M: Differences in spatial expression between 14-3-3 isoforms in germinating barley embryos. Plant Physiol 1999, 121:81-87.
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44. Holtman WL, Roberts MR, Oppedijk BJ, Testerink C, van Zeijl MJ, Wang M: 14-3-3 proteins interact with a 13-lipoxygenase, but not with a 9-lipoxygenase. FEBS Lett 2000, 474:48-52. 45. Roe JL, Rivin CJ, Sessions RA, Feldmann KA, Zambryski PC: The Tousled gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development. Cell 1993, 75:939-950. 46. Chang HC, Rubin GM: 14-3-3εε positively regulates Ras-mediated signaling in Drosophila. Genes Dev 1997, 11:1132-1139. 47.
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50. Wang BC, Yang HZ, Liu YC, Jelinek T, Zhang LX, Ruoslahti E, Fu H: • Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. Biochemistry 1999, 38:12499-12504. Non-phosphorylated peptide ligands of 14-3-3s are identified by phage-display screening. One peptide in particular shows high affinity for several 14-3-3 isoforms, binds at the same site as physiological target peptides and competes effectively for their interaction with 14-3-3 proteins.
43. Booij PP, Roberts MR, Vogelzang SA, Kraayenhof R, de Boer AH: 14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tomato cells. Plant J 1999, 20:673-683.
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