Download Function and specificity of 14-3-3 proteins in the regulation of

Journal of Experimental Botany, Vol. 54, No. 382,
Regulation of Carbon Metabolism Special Issue, pp. 595±604, January 2003
DOI: 10.1093/jxb/erg057
Function and speci®city of 14-3-3 proteins in the
regulation of carbohydrate and nitrogen metabolism
Sylviane Comparot, Gavin Lingiah and Thomas Martin1
University of Cambridge, Department of Plant Sciences, Downing Site, Cambridge CB2 3EA, UK
Received 13 September 2002; Accepted 26 September 2002
Abstract
Protein phosphorylation is key to the regulation of
many proteins. Altered protein activity often requires
the interaction of the phosphorylated protein with a
class of `adapters' known as 14-3-3 proteins. This
review will cover aspects of 14-3-3 interaction with
key proteins of carbon and nitrogen metabolism
such as nitrate reductase, glutamine synthetase and
sucrose-phosphate synthase. It will also address
14-3-3 involvement in signal transduction pathways
with emphasis on the regulation of plant metabolism.
To date, 14-3-3 proteins have been identi®ed and
studied in many diverse systems, yielding a plethora
of data, requiring careful analysis and interpretation.
Problems such as these are not uncommon when
dealing with multigene families. The number of isoforms makes the question of redundancy versus
speci®city of 14-3-3 proteins a crucial one. This issue
is discussed in relation to structure, function and
expression of 14-3-3 proteins.
Key words: ATPase, C/N metabolism, metabolic signalling,
multigene family, nitrate reductase, 14-3-3 proteins,
regulation, sucrose-phosphate synthase.
Introduction
14-3-3 proteins were ®rst identi®ed as abundant, acidic,
soluble brain proteins with a molecular weight of 25±
32 kDa (Moore and Perez, 1967). Their enigmatic name
was assigned according to fractionation on DEAE cellulose and electrophoretic mobility on starch gel electrophoresis during the puri®cation of a number of bovine
brain proteins of unknown function. Currently, individual
members of 14-3-3 proteins are mostly designated by
Greek letters. Arabidopsis 14-3-3 proteins are also named
General Regulatory Factors (GRFs) thus taking into
account that these proteins regulate a great number of
cellular processes (Rooney and Ferl, 1995, 1996).
Originally 14-3-3 proteins were thought to be found
only in brain tissue. However, it became clear that 14-3-3
proteins belong to a family of proteins present in all the
eukaryotic organisms investigated. A database analysis
performed by Rosenquist et al. (2000) listed 153 isoforms
in 48 different species. Since then the number of 14-3-3
proteins and 14-3-3 harbouring organisms has probably
grown by an order of magnitude. The number of putative
14-3-3 encoding genes ranges from two in yeast to 15 in
Arabidopsis (van Heusden et al., 1992, 1995; Rosenquist
et al., 2001). 14-3-3 proteins exhibit a high degree of
sequence similarity, even between species (Wang and
Shakes, 1996). In Arabidopsis, the amino acid sequences
of 14-3-3 proteins are between 60% and 92% conserved
amongst the various isoforms whilst 14-3-3 encoding
genes are up to 86% identical (Wu et al., 1997). 14-3-3
proteins mainly exist as saddle-shaped homo- or heterodimers in which a broad central groove is able to bind to
target proteins. Thus, 14-3-3 proteins can function as
`adapters', leading speci®c target proteins to each other or
to a speci®c location.
The ®rst description of a possible biological function
was suggested by Ichimura et al. (1987) who correlated the
amino acid sequence of 14-3-3 proteins to tryptophan
hydroxylase activators. Since then, 14-3-3 proteins have
been shown to play diverse roles in many biological
processes, such as the activation of protein kinase C (Toker
et al., 1990), the stimulation of calcium-dependent
1
To whom correspondence should be addressed. Fax: +44 (0)1223 333953. E-mail: [email protected]
Abbreviations: AICAR, 5-aminoimidazole-4-carboxamide riboside; GS, glutamine synthetase; NIP, nitrate reductase inhibitor protein; NR, nitrate reductase;
PM, plasma membrane; SPS, sucrose-phosphate synthase.
Journal of Experimental Botany, Vol. 54, No. 382, ã Society for Experimental Biology 2003; all rights reserved
596 Comparot et al.
exocytosis (Morgan and Burgoyne, 1992), involvement in
mitochondrial and chloroplastic import (Alam et al., 1994;
May and Soll, 2000), the regulation of proton pumping
mechanisms via the plant plasma membrane H+-ATPase
(Oecking et al., 1994), and of key plant metabolic enzymes
(Bachmann et al., 1996a; Moorhead et al., 1996; Toroser
et al., 1998).
Therefore, it appears that 14-3-3 proteins display a
broad spectrum of activities, with a common feature being
the interaction with other proteins as binding partners
(Palmgren et al., 1998; Fu et al., 2000). Binding partners
usually display a phosphorylated motif which is recognized by 14-3-3 proteins and forms part of the interacting
domain. The most common motif identi®ed so far is
RSXpSXP (pS: phosphorylated serine, X: any amino acid)
(Muslin et al., 1996). However, 14-3-3 proteins also bind
to other phosphoserine peptide motifs (Yaffe et al., 1997)
and binding might also occur to unphosphorylated ligands
(Alam et al., 1994; Petosa et al., 1998; Masters et al.,
1999).
14-3-3 proteins in plants
In plants, 14-3-3 proteins have been isolated from many
species, including Arabidopsis (Lu et al., 1992), barley
(Brandt et al., 1992), maize (de Vetten et al., 1992),
spinach (Hirsch et al., 1992), tobacco (Piotrowski and
Oecking, 1998), potato (Wilczynski et al., 1998), and
tomato (Roberts and Bowles, 1999). The recent completion
of the Arabidopsis genome project facilitated the identi®cation of up to 15 putative 14-3-3 genes, of which 12 are
also represented as EST or cDNA clones (Rosenquist et al.,
2001). An exon-based alignment enabled Wu et al. (1997)
to classify Arabidopsis 14-3-3 genes into two main groups;
the epsilon group and the non-epsilon group. This delineation is observed in plants as well as in animals, indicating
that this divergence of 14-3-3 proteins is ancient. The
different isoforms display a very high conservation of
amino acid sequences in the core region, with the N- and Ctermini being the most divergent.
Piotrowski and Oecking (1998) proposed the existence
of at least ®ve different subgroups of 14-3-3 encoding
genes in plants; four of those are possible results of early
gene duplication and divergence, whilst the ®fth group is
speci®c for monocotyledonous plants.
As observed in other eukaryotic organisms, plant 14-3-3
proteins are involved in many crucial processes interacting
with numerous proteins involved in various biological
processes. Examples include proteins of the G-box binding
complex (de Vetten et al., 1992; Lu et al., 1992), the
transcription factors and activators EmBP1 and RSG
(Schultz et al., 1998; Igarashi et al., 2001), the plasma
membrane H+-ATPase (Oecking et al., 1994), enzymes of
carbon and nitrogen metabolism (Bachmann et al., 1996a;
Toroser et al., 1998; Moorhead et al., 1999; Sehnke et al.,
2001), a lipoxygenase from barley (Holtman et al., 2000),
a membrane-bound ascorbate peroxidase (Zhang et al.,
1997), and a tomato outward-rectifying K+ channel (Booij
et al., 1999).
The interaction of 14-3-3 proteins with a number of
metabolic enzymes suggests their involvement in metabolic regulation. The impact of 14-3-3 proteins on
metabolism can be seen in antisense and overexpression
experiments. Overexpression of 14-3-3 proteins in potato
disturbed sugar, catecholamine and lipid content, whilst a
14-3-3 antisense experiment in¯uenced tuber starch content, nitrate reductase activity and amino acid composition
(Prescha et al., 2001; Swiedrych et al, 2002).
14-3-3 proteins regulate plant H+-ATPase and
ion channel activity
The interaction between the plasma membrane (PM) H+ATPase, the fungal metabolite fusicoccin and 14-3-3
proteins has been extensively studied and is so far the best
understood interaction of 14-3-3s and target proteins. The
PM H+-ATPase is known to play crucial roles in the plant
cell by generating a proton gradient, thereby providing the
driving force for nutrient uptake, phloem loading, water
movements, stomatal closure, and opening. Its activity is
regulated by many environmental stimuli and hormones. It
is now clearly established that activity is modulated by the
displacement of an auto-inhibitory C-terminal domain (Sze
et al., 1999). Fusicoccin, a phytotoxin, affects PM H+ATPase-mediated processes such as stomatal opening and
cell elongation, by irreversibly activating the PM H+ATPase via interaction with the auto-inhibitory domain
(de Boer, 1997). The involvement of 14-3-3 proteins in this
process has been investigated in great detail. 14-3-3
protein dimers bind to the phosphorylated PM H+-ATPase
and form a complex that activates the pump. In addition,
binding of 14-3-3 proteins to the PM H+-ATPase generates
a fusicoccin receptor. Binding of fusicoccin stabilizes the
labile ATPase:14-3-3 complex and induces a strong and
persistent activation of the ATPase in vivo (Korthout and
de Boer, 1994; Oecking et al., 1997). Binding of 14-3-3
proteins to AHA2, the Arabidopsis plasma membrane H+ATPase, occurs at the last 98 C-terminal amino acids (Jahn
et al., 1997). In spinach, phosphothreonine-948 in the
C-terminal end of the pump forms an integral part of the
14-3-3 binding motif (Olsson et al., 1998). Oecking and
Hagemann (1999) also showed that the binding site for
14-3-3 dimers is located in the C-terminal autoinhibitory
domain. Moreover, these authors demonstrated the absolute requirement of the C-terminus for the formation of a
functional fusicoccin complex.
To summarize, the binding of 14-3-3 proteins to the
autoinhibitory domain of the H+-ATPase is suf®cient to
lead to the formation of unstable complexes with high
activity. However, it is the binding of fusicoccin that
14-3-3 proteins and plant C/N metabolism 597
stabilizes the interaction between 14-3-3 proteins and the
enzyme, thereby giving rise to the formation of stable
complexes with high proton pumping activity.
The PM H+-ATPase is not the only membrane protein
regulated by 14-3-3 proteins. Addition of 14-3-3 proteins
during patch clamp activity measurements of a tomato
potassium outward-rectifying channel increased the activity of the channel (Booij et al., 1999). The authors
suggested that 14-3-3 proteins may recruit `sleepy' channels and bring them to a voltage-activated state. In the
same way, 14-3-3 proteins from Vicia faba are able to
enhance K+ conductance in tobacco (Saalbach et al.,
1997). These authors propose that the ion regulation is
performed by modulating kinase activities or by direct
binding of 14-3-3 proteins to the channels.
14-3-3 proteins regulate key enzymes in the
nitrogen assimilation pathway
The electrochemical gradient generated by the H+-ATPase
drives many metabolic processes, including nitrate uptake
across the plasma membrane of root cells. Nitrogen
assimilation is an important step in the metabolism of the
whole plant and involves the reduction of nitrate to nitrite
by nitrate reductase (NR) (Crawford, 1995). Coarse control
of NR expression is exerted by light and metabolites such
as nitrate and sucrose, demonstrating cross-talk of primary
carbohydrate and nitrogen metabolism. In addition, nitrate
reductase is subject to ®ne control via inactivation by
phosphorylation. Phosphorylation occurs in the hinge 1
region of nitrate reductase, at Ser-534 in Arabidopsis and
Ser-543 in spinach (Douglas et al., 1995; Su et al., 1996).
However, phosphorylation of the enzyme alone does not
induce the inactivation. The process is completed after
reversible binding of a nitrate reductase inhibitor protein
(NIP; Bachmann et al., 1995). Puri®cation of NIP in the
late 90s showed that it is composed of one or more 14-3-3
isoforms (Bachmann et al., 1996b). The amino acid
sequence surrounding the phosphorylated serine residues
resembles the mammalian 14-3-3:Raf-1 interaction site
and experiments using a raf-derived peptide, including the
14-3-3 binding site, blocked NIP activity on NR.
(Moorhead et al., 1996; Huber et al., 1996) Further
evidence came from the analysis of mutated NR demonstrating the conditional requirement of Ser-534 in the
hinge 1 region of Arabidopsis NR for binding of 14-3-3
proteins (Kanamaru et al., 1999). Lillo et al. (1997)
compared nitrate reductases from various plant species and
all showed a down-regulation by phosphorylation and
binding of 14-3-3 upon light-to-dark transition.
As mentioned previously, 14-3-3 binding to target
proteins is sequence speci®c. In spinach NR, the motif
RTApSTP appears to be the sequence recognized and closely
resembles the consensus 14-3-3 binding motif RSXpSXP
(Bachmann et al., 1996a; Moorhead et al., 1996; Kaiser et al.,
2002). In spinach, nitrate reductase kinase has been identi®ed as a calmodulin-domain protein kinase able to
phosphorylate the spinach enzyme at Ser-543 (Douglas
et al., 1998). Interestingly, 14-3-3 binding to nitrate
reductase not only inhibits NR activity but can also mark
the enzyme for degradation (Weiner and Kaiser, 1999).
Therefore it appears that NR is highly regulated, ®rstly
by the steady-state level of the enzyme protein (Lillo,
1994; Crawford, 1995; Campbell, 1996) and secondly by
reversible protein phosphorylation (Huber et al., 1992,
1994; MacKintosh, 1992).
Glutamine synthetase (GS) also binds 14-3-3 proteins
(Moorhead et al., 1999). Finnemann and Schjoerring
(2000) studied Brassica napus GS1, a cytosolic enzyme
active in senescing leaves. This phosphoprotein is regulated post-translationally by reversible phosphorylation
catalysed by a protein kinase and a microcystin-sensitive
serine/threonine protein phosphatase. The phosphorylation
status of the enzyme changes during light-to-dark transition, is dependent on the ATP/AMP ratio in vitro (Kaiser
and Spill, 1991) and thus affects degradation susceptibility. In young photosynthetically active leaves, the
requirement for ATP decreases the ATP/AMP ratio,
thereby reducing the phosphorylation of the GS1 pool. In
darkness, respiration increases ATP production, thereby
inducing an increase of the ATP/AMP ratio. This leads to
an increase of GS1 phosphorylation. During senescence,
the rate of photosynthesis is decreased while the respiration is maintained or increased (KroÈmer, 1995). The
amount of phosphorylated GS1 consequently increases,
thus possibly allowing the remobilization of nitrogen.
Phosphorylated GS1 interacts with 14-3-3 proteins, and the
highest activity is measured if GS1 is both phosphorylated
and bound to 14-3-3 (Finnemann and Schjoerring, 2000).
Riedel et al. (2001) also showed interaction of the tobacco
chloroplastic GS isoform (GS2) with 14-3-3 proteins. Only
GS2 strongly associated with 14-3-3 proteins was found to
be catalytically active.
Interaction of 14-3-3 proteins with enzymes of
carbohydrate metabolism
Not only do 14-3-3 proteins regulate enzymes of the
nitrogen assimilation pathway and proteins involved in
generating proton gradients which serve as the driving
force for nutrient uptake, they also regulate the activity of
enzymes involved in primary carbon metabolism. Sucrosephosphate synthase (SPS) is a good example of this. SPS
catalyses the conversion of UDP-glucose and fructose-6phosphate to sucrose-6-phosphate, the second last step in
sucrose biosynthesis. SPS is regulated by allosteric
effectors and protein turnover (Huber and Huber, 1996).
The enzyme has several putative phosphorylation sites
which regulate its activity by 14-3-3 dependent and
independent mechanisms. Non-14-3-3 events include
598 Comparot et al.
phosphorylation of SPS on Ser-424 and Ser-158 which are
thought to be responsible for light/dark modulation and
osmotic stress activation of the enzyme (McMichael et al.,
1993; Toroser and Huber, 1997). However, there is a sitespeci®c regulatory interaction between 14-3-3 proteins and
Ser-229 of spinach SPS which inhibits SPS activity
(Toroser et al., 1998).
The interaction of 14-3-3s and target proteins can be
reduced in vitro by 5¢-AMP which interacts with 14-3-3s
and directly reduces their binding to target ligands (Kaiser
and Spill, 1991; Athwal et al., 1998b). It is noteworthy that
there is evidence for an AMP binding site in a spinach
14-3-3 isoform. Thus 14-3-3 protein activity might also be
under metabolic control (Athwal et al., 1998b). The effect
of 5¢-AMP can be mimicked in vivo using 5-aminoimidazole-4-carboxamide riboside (AICAR), a cell permeable
precursor of the 5¢-AMP analogue ZMP (5¢-aminoimidazole-4-carboxamide ribonucleoside monophosphate).
Addition of AICAR to leaf tissue in the dark, for example,
led to activation of nitrate reductase and accumulation of
ZMP in the tissue without changing the general phosphate
metabolism (Huber and Kaiser, 1996). Using AICAR
feeding prior to analysing SPS activity, Toroser et al.
(1998) showed a reduced 14-3-3 binding to SPS leading to
higher SPS activity in the dark. The effect of AICAR may
suggest a metabolic control of SPS activity in vivo.
Moorhead et al. (1999) describe the effect of 14-3-3s on
sucrose-phosphate synthase (SPS) activities plus trehalose6-phosphate synthase (TPS) in cauli¯ower extracts. Using
a phosphopeptide which competes with target proteins for
14-3-3 binding, the authors showed a slight activation of
sucrose-phosphate synthase activity. Although the authors
state that the assay mostly measures SPS activity, it has to
be kept in mind that TPS might contribute to the increase in
sucrose-phosphate synthase activity. Furthermore, the use
of different plant material or analytical techniques by
Toroser et al. (1998) and Moorhead et al. (1999) might
explain, at least partially, the different effects of 14-3-3
proteins on SPS activity.
Further evidence for the regulation of carbohydrate
metabolism by 14-3-3 proteins came from Sehnke et al.
(2001) who showed that reduction of the e-subgroup of
Arabidopsis 14-3-3 proteins by antisense techniques,
resulted in a 2±4-fold increase in leaf starch accumulation.
Breakdown of starch in the dark was not affected and
14-3-3 proteins were found as internal intrinsic granule
proteins. Interestingly, all members of the starch synthase
III family contain the conserved 14-3-3 phosphoserine/
threonine-binding motif (RYGSIP) making them putative
targets for 14-3-3 action.
Far Western overlay experiments also indicated binding
of 14-3-3 proteins to trehalose-6-phosphate synthase,
glyceraldehyde-3-phosphate dehydrogenase and ATP
synthase suggesting regulation of these enzymes by
14-3-3-mediated processes (Moorhead et al., 1999;
Cotelle et al., 2000). Additionally, evidence for the
interaction and regulation of mitochondrial and chloroplastic ATP synthases by 14-3-3 proteins was shown by
Bunney et al. (2001).
The targeting and regulation of key enzymes of carbon
and nitrogen metabolism by 14-3-3 proteins suggest a
common 14-3-3 mediated mechanism in regulating and
possibly co-ordinating the use and generation of metabolites in plants. It is worth noting that primary carbon and
nitrogen metabolites can also be sensed, leading to
physiological and developmental changes. For example,
nitrogen appears to regulate root system morphology,
partitioning of photosynthetic carbon, and ¯owering.
Nitrate, in addition to its metabolic role, has been identi®ed
as a signalling molecule. Nitrate can induce the expression
of genes encoding for proteins of nitrogen metabolism,
such as NR, nitrite reductase, GS and nitrate transporters.
On the other hand, nitrate is a crucial signal in the
regulation of carbon metabolic genes as shown by nitrate
repression of AGPS, encoding for the regulatory subunit of
ADP-glucose pyrophosphorylase, a key enzyme of starch
synthesis (Scheible et al., 1997).
At the same time, sugars act in a complementary manner
to nitrogen. For example, they induce genes encoding for
nitrate transporters, NR, GS, and they also stimulate NR
activity (Koch, 1996; Jang and Sheen, 1997; Stitt, 1999).
Thus, a cross-talk between the two pathways, which allows
plants to optimize the use of available resources, can be
speculated (Coruzzi and Zhou, 2001). Other forms of
regulation involve the sensing of the carbohydrate to
nitrogen (C:N) ratio. Indeed, it has been shown that the
C:N ratio, rather than the carbohydrate status or the nitrate
availability alone, signi®cantly affects the growth and
development of Arabidopsis seedlings (Martin et al.,
2002). Finally, as noted above, plasma membrane H+ATPase and K+ channel activities are necessary to provide
the electrochemical gradient needed for the transport of
many metabolites, including sugars and amino acids.
14-3-3 proteins interact with proteins of
signalling pathways
Evidence for the involvement of 14-3-3 proteins in
metabolic sensing/signalling pathways comes from
nutrient starvation experiments (Cotelle et al., 2000).
Binding of 14-3-3 proteins to target enzymes such as NR or
SPS was lost upon sugar starvation or feeding with nonmetabolizable glucose analogues in Arabidopsis cell
cultures. Loss of 14-3-3 binding resulted in proteolytic
degradation of 14-3-3 binding enzymes, suggesting that
14-3-3 proteins may participate in a nutrient sensing
pathway controlling the stability of many target proteins.
From the overall discussion above it is tempting to
attribute a central role in the control of plant primary
metabolism to 14-3-3 proteins.
14-3-3 proteins and plant C/N metabolism 599
In addition to the direct regulation of metabolism by
14-3-3 proteins via enzyme activation/inactivation, 14-3-3
proteins have been shown to interact with components of
signalling pathways. For instance, 14-3-3 proteins modulate the activity of a number of protein kinases such as
protein kinase C (Robinson et al., 1995) and the calciumdependent protein kinase CPK-1 in Arabidopsis (Camoni
et al., 1998). Ikeda et al. (2000) demonstrated that 14-3-3
proteins are able to bind to the autophosphorylated
C-terminal region of WPK4, a wheat protein kinase
related to the yeast metabolite signalling kinase SNF1.
WPK4 expression itself is regulated by light, general
nutrient deprivation and the plant hormone cytokinin
(Sano and Yousse®an, 1994). Additionally, WPK4 phosphorylates NR in the hinge 1 region prior to 14-3-3 binding
(Ikeda et al., 2000).
Apart from interaction with protein kinases, 14-3-3
proteins interact with other components of signalling
pathways, for example with RGS3, a negative regulator of
the G-alpha subunits of heterotrimeric G proteins (Niu
et al., 2002). Thus, 14-3-3 proteins may be involved in
controlling regulation of G-protein signalling pathways.
14-3-3 proteins bind to transcription factor
complexes
Nuclear localization of 14-3-3 proteins has been shown
using immunological analysis and laser scanning confocal
microscopy (Bihn et al., 1997). This localization is
somehow puzzling as 14-3-3 proteins lack recognizable
nuclear localization signals. However, 14-3-3 association
with transcription factor complexes such as the G-box
binding complex may account for their presence in nuclei
(de Vetten et al., 1992). G-box elements are moderately
conserved elements in many gene promoters including the
anaerobic response element of ADH1 promoter in
Arabidopsis and maize (DeLisle and Ferl, 1990;
McKendree and Ferl, 1992), the promoter of a chalcone
synthase gene (Faktor et al., 1996), and promoters of the
CabE gene and of the RbcS gene family (Schindler and
Cashmore, 1990; Giuliano et al., 1988). It is tempting to
suggest that 14-3-3 proteins exert a second level of
metabolic control via interaction with protein complexes
regulating promoter activities via G-box elements, found
in several metabolic genes. Interestingly, G-box elements
have also been found in maize 14-3-3 genes (de Vetten and
Ferl, 1994). This may indicate possible autoregulatory
mechanisms of 14-3-3 gene expression.
14-3-3 proteins in plants also interact with components
of other transcription factor components. For instance,
14-3-3 proteins interact with the site-speci®c DNA binding
proteins EmBP1 and the tissue-speci®c regulatory factor
VP1 (Schultz et al., 1998).
RSG (Repression of Shoot Growth) is a bZIP transcriptional activator regulating endogenous GA amounts
through the control of GA biosynthetic enzyme expression.
It has been described as a 14-3-3 binding partner (Igarashi
et al., 2001). A mutated form of RSG, that is unable to
interact with 14-3-3s, has a higher transcriptional activity
than the wild-type protein. This might be explained by the
predominant localization of the mutant form in the
nucleus, whilst the wild-type protein is found throughout
the cell. Thus 14-3-3 proteins seem to control the nuclear
localization of a transcription factor, possibly restricting its
activity to a limited number of cells during development
and in a restricted time frame.
Structure, function, modulation
Animal 14-3-3 monomeric proteins consist of nine
antiparallel helices organized into an N-terminal domain
(four helices) and a C-terminal domain (®ve helices) (Liu
et al., 1995). Dimerization in animal 14-3-3 proteins
requires the ®rst three helices in the N-terminal domain
(Luo et al., 1995). Because of the high degree of
conservation between plant and animal 14-3-3 proteins
one could predict the presence of nine helices within plant
14-3-3s. However, plant 14-3-3 proteins show distinct
differences to animal 14-3-3s. Firstly, in plants, dimerization relies only on the fourth helix, a signi®cant difference
between animal and plant forms (Abarca et al., 1999).
Secondly, some plant 14-3-3 proteins possess an EF-handlike motif in the C-terminal region indicating possible
interactions with calcium.
The structure of 14-3-3 proteins has a key role in
interactions with target proteins. A common binding site
for target proteins is located in the box-1 region (helices 7
and 8) in the C-terminus. This box is common to all 14-3-3
isoforms. In animal systems binding speci®city can be
affected by only a few amino acid variations in box 1 or in
regions closely adjacent to box 1 (Ichimura et al., 1997).
In both plants and yeast 14-3-3 proteins bind to their
target proteins in a cation-stimulated manner. Calcium
possibly interacts with helices 8 and 9 causing conformation changes of the protein (Athwal et al., 1998a).
Activation of 14-3-3s by cations may be due to
neutralization of negative charges associated with acidic
residues in the loop involving helices 8 and 9 (Athwal and
Huber, 2002). Furthermore, calcium decreases the homodimerization capacity of RCI14A (Abarca et al., 1999).
This effect involves the C-terminal end of RCI14A and
might also modulate the interaction of this 14-3-3 protein
with its binding partners. The cationic effect of calcium
seems to be speci®c as Mg2+ has no effect on dimerization.
A requirement for millimolar concentrations of divalent
cations like calcium, magnesium and manganese has also
been demonstrated for the formation of inactive 14-3-3:NR
complexes at pH 7.5 (Athwal et al., 1998a). However, this
requirement is strictly pH-dependent and is far less
pronounced at pH 6.5. Binding of cations and lowering
600 Comparot et al.
the pH cause conformational changes of 14-3-3 proteins,
leading to an increase in surface hydrophobicity. These
conformational changes might be necessary to `prime' 143-3 proteins prior to binding of target proteins as suggested
by Athwal et al. (1998a). Physiologically, magnesium
seems to be the only divalent cation present in suf®cient
concentrations to cause such an effect. However, a recent
publication introduced the possibility that polyamines may
act as in vivo effectors of 14-3-3 activity (Athwal and
Huber, 2002). The authors showed that the effect observed
with millimolar concentrations of divalent cations at pH
7.5 could be achieved with micromolar concentrations of
polyamines. Thus a product of metabolism itself might be
required to switch 14-3-3 proteins into a state in which
binding to target proteins becomes possible.
14-3-3 structure may also affect its own phosphorylation. As shown by Megidish et al. (1998), a sphingosineor N,N¢-dimethylsphingosine-activated protein kinase
(SDKs) speci®cally phosphorylates certain endogenous
mouse 14-3-3 isoforms. The phosphorylation site is
located on the dimer interface of 14-3-3 proteins, and it
is likely that phosphorylation alters dimerization or
induces conformational changes that alter the association
of 14-3-3 proteins with other kinases.
Phosphorylation of 14-3-3 proteins may also prevent
association with certain target proteins. In mammals, a
casein kinase I a was able to phosphorylate 14-3-3 proteins
(Dubois et al., 1997). The mammalian 14-3-3 isoforms z
and t possess a phosphorylatable serine residue and it has
been shown previously that only the unphosphorylated z
associates with the regulatory domain of c-Raf (Rommel
et al., 1996).
Regulation and localization of 14-3-3 expression
It is known that 14-3-3 proteins can modulate responses to
environmental changes via interactions with key enzymes,
but the expression of 14-3-3s can also be affected by
different environmental challenges. Expression of a rice
14-3-3 cDNA is regulated by external stresses such as salt
and low temperatures (Kidou et al., 1993). Similarly, the
expression of two Arabidopsis 14-3-3 cDNAs is induced
by low temperatures (Jarillo et al., 1994).
Numerous investigations of 14-3-3 expression patterns
in animal systems performed in the early 90s showed
organ- and tissue-speci®c expression of diverse 14-3-3
genes, and developmental expression was seen in rat brain
(Watanabe et al., 1991, 1993, 1994). Later, Daugherty et al.
(1996) showed that the Arabidopsis gene coding for
14-3-3c also exhibits cell- and tissue-speci®c expression in
roots of seedlings and mature plants.
Recently, Rosenquist et al. (2001) identi®ed two new
14-3-3 genes showing tissue-speci®c expression in
Arabidopsis. Cell- or tissue-speci®c expression of 14-3-3
genes can also be found in tomato. Expression analysis of ten
tomato 14-3-3 isoforms revealed that speci®c subsets of the
genes were induced in leaves during a gene-for-gene
resistance response. Different developmental patterns of 143-3 expression in response to fusicoccin or elicitor treatments
were also shown (Roberts and Bowles, 1999). In potato, the
expression of ®ve 14-3-3s in leaves clearly depends on the
developmental stage (Wilczynski et al., 1998).
However, changes in gene expression do not necessarily
re¯ect the quantity of 14-3-3s. Despite the clear induction
of three barley 14-3-3 mRNAs upon imbibition, protein
levels remain constant during germination (Testerink et al.,
1999). More signi®cance may lie in the ®nding of
subcellular, germination-dependent differences in posttranslational protein modi®cations between the three
barley 14-3-3 isoforms (van Zeijl et al., 2000).
In addition to regulatory factors, subcellular localization
of 14-3-3 isoforms may contribute to their speci®city.
Although 14-3-3 proteins were thought to be solely soluble
cytosolic proteins, Martin et al. (1994) demonstrated that
some mammalian isoforms were tightly associated with
membranes. In plants, 14-3-3 isoforms have been identi®ed in the cytosol, the nucleus (Bihn et al., 1997), in the
mitochondrial matrix (Bunney et al., 2001) and in the
chloroplast stroma and the stromal side of thylakoid
membranes (Sehnke et al., 2000). Sehnke et al. (2000) also
showed that localization of 14-3-3 proteins can be isoformexclusive, as only the Arabidopsis 14-3-3 isoforms n, e, m,
and u are present in the chloroplast stromal extract whereas
the cytosolic isoform w is excluded from chloroplasts.
Despite clear spatial and temporal regulation of 14-3-3
gene expression and organelle speci®c localization of
14-3-3 proteins, the question whether many 14-3-3
isoforms are necessary or redundant remains unclear. As
outlined above, 14-3-3 proteins present a high degree of
structural conservation in the binding groove. This may
allow all isoforms to bind to the same target proteins.
However, the less conserved C-terminal region may confer
some degree of isoform speci®city during binding (Finnie
et al., 1999). It has also been suggested that the presence of
different 14-3-3 isoforms allows the formation of speci®c
heterodimers with unique recognition of ligands (Yaffe,
2002). Another hypothesis is that a high number of
isoforms ensures that 14-3-3 proteins are present and
active in all cell compartments (DeLille et al., 2001), and,
of course, it can not be ruled out that each isoform displays
a speci®c activity in vivo.
Although the data published on 14-3-3 speci®city is
growing steadily, many results still appear contradictory.
For example, there is evidence that an Arabidopsis
thaliana isoform can substitute for the functions of yeast
and mammalian isoforms (Lu et al., 1994; van Heusden
et al., 1995). In mammals, the 14-3-3 binding peptide R18
which occupies the general binding groove of 14-3-3z can
bind different mammalian 14-3-3 isoforms with similar
af®nities (Wang et al., 1999). In plants, Holtman et al.
14-3-3 proteins and plant C/N metabolism 601
(2000) have shown that three barley 14-3-3 proteins
interact with similar properties with a 13-lipoxygenase in
surface plasmon resonance experiments, suggesting that
different isoforms do not display binding speci®city.
However, there is an increasing body of evidence for 143-3 binding speci®city. Firstly, both animals and plants
have two groups of 14-3-3 proteins, the e group and the
non-e group. Ferl et al. (1994) suggested that these two
may have different functions in mammals. Evidence for
14-3-3 isoform speci®city has been reported in animal
cells. For example, Vincenz and Dixit (1996) showed that
both the zinc ®nger protein A20 and c-Raf bind preferentially the h-isoform, followed by the e-isoform whilst the
b-, z- and g-isoforms bind less well. In plants, variation in
binding speci®city of 14-3-3 proteins to NR has been
shown by Bachmann et al. (1996a) and Kanamaru et al.
(1999). The binding of only ®ve of the Arabidopsis 14-3-3
isoforms to GST-hTFIIB beads also shows af®nity differences (Pan et al., 1999). In this case, no dimer formation
was required and no known motif containing phosphoserine was identi®ed. Even the plasma membrane H+-ATPase
of Vicia faba has been shown to have a higher af®nity for
vf14-3-3a than for vf14-3-3b (Emi et al., 2001). Surface
plasmon resonance experiments demonstrated strong
variations in relative binding af®nity between nine of the
Arabidopsis 14-3-3 isoforms and a peptide representing the
last 15 amino acids of the plasma membrane H+-ATPase
AHA2 (Rosenquist et al., 2000).
Conclusion and perspectives
It is believed that the evidence summarized in this review
clearly demonstrates the importance of 14-3-3 proteins in
the regulation of primary plant metabolism. The possible
co-ordination of primary carbon and nitrogen metabolism
via 14-3-3 proteins presents an interesting perspective.
Depending on the developmental and environmental
requirements, 14-3-3 activity could direct carbon either
into sucrose and storage carbohydrate synthesis or, via
inactivation of carbon metabolism and activation of
nitrogen assimilation, divert carbon skeletons into the
synthesis of amino acids.
Despite many investigations into the role of individual
14-3-3 proteins and their interaction with target proteins,
the means of 14-3-3 protein speci®city remains largely
unknown. First insights into this question have been gained
by analysing gene-speci®c expression of 14-3-3 genes.
However, these analyses require clari®cation by determining the fate of 14-3-3 proteins. It is believed that the great
number of 14-3-3 proteins, rather than being a nuisance to
researchers due to their apparent redundancy, offer the
chance of studying ®ne regulatory mechanisms of metabolic pathways. Plants could use the various possible
combinations of 14-3-3 homo- and heterodimers for ®ne-
tuning of metabolism in response to changes in the
environment or internal demands.
As exempli®ed in this review, many details are known
about 14-3-3 proteins. Unfortunately, these facts are spread
across different phyta and are made using different
experimental systems. What is needed is a systematic
approach to investigate the roles of each isoform in a single
system. Completion of the Arabidopsis genome sequencing programme and the opportunities offered by functional genomics, proteomics and metabolomics facilities
make Arabidopsis an excellent plant model for such an
approach. A reverse genetic approach is currently being
adopted to identify mutants in 14-3-3 genes. These mutants
will be systematically compared to wild-type plants with
the ultimate aim being the resolution of 14-3-3 function
and speci®city in the regulation of plant metabolism.
Acknowledgements
We gratefully acknowledge ®nancial support for our work by the
BBSRC and the Royal Society.
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