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Seminars in Cell & Developmental Biology 22 (2011) 663– 672

Contents lists available at SciVerse ScienceDirect

Seminars in Cell & Developmental Biology

jo u rn al hom epa ge: www.elsev ier .com/ locate /semcdb

eview

tructural basis of 14-3-3 protein functions

omas Obsil a,b,∗, Veronika Obsilovab

Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, 12843 Prague, Czech RepublicInstitute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic

r t i c l e i n f o

rticle history:vailable online 6 September 2011

eywords:4-3-3

a b s t r a c t

The 14-3-3 proteins, a family of conserved regulatory molecules, participate in a wide range of cellularprocesses through binding interactions with hundreds of structurally and functionally diverse proteins.Several distinct mechanisms of the 14-3-3 protein function were described, including conformationalmodulation of the bound protein, masking of its sequence-specific or structural features, and scaffold-

tructurerotein–protein interactionshosphorylation

ing that facilitates interaction between two simultaneously bound proteins. Details of these functionalmodes, especially from the structural point of view, still remain mostly elusive. This review gives anoverview of the current knowledge concerning the structure of 14-3-3 proteins and their complexes aswell as the insights it provides into the mechanisms of their functions. We discuss structural basis of targetrecognition by 14-3-3 proteins, common structural features of their complexes and known mechanismsof 14-3-3 protein-dependent regulations.

© 2011 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6642. Structure of 14-3-3 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6643. Target recognition by 14-3-3 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664

3.1. Recognition of consensus phosphorylated binding motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6643.2. Binding of unphosphorylated peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 666

4. Structural basis of 14-3-3 protein functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6674.1. Common aspects of 14-3-3 protein complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667

4.1.1. The rigidity of the 14-3-3 protein dimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6674.1.2. Simultaneous use of multiple 14-3-3 binding motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6674.1.3. Presence of 14-3-3 binding motifs inside disordered regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667

4.2. Mode of action: conformational change of the target protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6674.3. Mode of action: physical occlusion of sequence-specific or structural features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669

4.3.1. Masking of protein–protein interaction sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6694.3.2. Masking of protein–DNA interaction sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6694.3.3. Protection against dephosphorylation and proteolytic degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669

4.4. Mode of action: scaffolding that anchors proteins within close proximity of one another . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 670

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: AANAT, serotonin N-acetyltransferase; AcCoA, acetyl-coenzyme A; FRExport sequence; NLS, nuclear localization sequence; pSer, phosphoserine; pThr, phospho∗ Corresponding author at: Faculty of Science, Charles University, Hlavova 8, 12843 Pra

E-mail address: obsil@natur.cuni.cz (T. Obsil).

084-9521/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2011.09.001

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 670. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 670

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 670

T, Förster resonance energy transfer; GAP, GTPase-activating protein; NES, nuclearthreonine; RGS, regulator of G protein signaling; TH, tyrosine hydroxylase.

gue, Czech Republic. Tel.: +420 221951303; fax: +420 224919752.

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64 T. Obsil, V. Obsilova / Seminars in Cell &

. Introduction

Reversible phosphorylation, one of the most important andell-studied post-translational modifications, plays many crucial

oles in the regulation of cellular processes [1]. Phosphoryla-ion on serine/threonine residues alters protein functions eitherirectly or by inducing the assembly of protein complexes throughodules that recognize specific phosphorylated motifs in another

rotein. The 14-3-3 proteins were the first molecules identifiedo specifically bind phosphoserine/phosphothreonine (pSer/pThr)ontaining motifs. Subsequent studies led to the identification ofther pSer/pThr-binding domains including WW domains, FHAomains as well as WD40 and LRR domains of F-box proteinsreviewed by Yaffe and Elia [2]).

14-3-3 proteins are a family of highly conserved, acidic pro-eins expressed in all eukaryotic cells. The unusual name “14-3-3”eflects their particular migration pattern on two-dimensionalEAE-cellulose chromatography and starch gel electrophoresis [3].he 14-3-3 proteins are highly conserved and many organismsxpress multiple isoforms. While lower eukaryotes, e.g. yeast, con-ain only two 14-3-3 genes, higher eukaryotes possess up to fifteen4-3-3 genes [4,5]. In mammals seven isoforms (�, �, �, �, �, � and) have been identified to date. With exception of sigma isoform,ll 14-3-3 proteins can form both homo- and heterodimers [6–8].he discovery that 14-3-3 proteins bind to specific pSer/pThr con-aining motifs in protein targets not only suggested their utmostmportance in signal transduction, but also pointed out a roleor Ser/Thr phosphorylation in the assembly of protein–proteinomplexes [9]. The subsequent studies revealed that 14-3-3 pro-eins can also recognize unphosphorylated motifs [10–12], furtherxpanding the repertoire of their binding interactions. Due to largeumber and diversity of interacting partners the 14-3-3 proteinsre implicated in the regulation and coordination of many cellularrocesses including cell cycle progression, apoptosis, metabolism,ranscriptional regulation of gene expression, the DNA damageesponse, and more [13].

The aim of this review is to give an overview of the currentnowledge concerning the structure of 14-3-3 protein complexesnd the structural basis of their functions. The reader is referredo several excellent reviews on 14-3-3 proteins and their functionsor further information [8,14–19].

. Structure of 14-3-3 proteins

First crystal structures of human 14-3-3 [20] and 14-3-3�21] revealed that 14-3-3 proteins are dimeric and highly helical.ach monomer consists of a bundle of nine antiparallel -helicesH1–H9) and its concave surface contains an amphipathic ligand-inding groove formed by -helices H3, H5, H7 and H9 (Fig. 1A). Theimeric molecule has a characteristic cup-like shape with a largeentral channel approximately 35 A broad, 35 A wide and 20 A deep.he invariant residues form the dimer interface and line the inneralls of the central channel, while the more variable residues areistributed on the outer convex surface (Fig. 1B). Maximal sequenceariability occurs within the C-terminal loop that is disordered inll available structures.

Crystal structures of all seven mammalian 14-3-3 isoforms arevailable today, especially due to the effort of structural genomics22]. Although all these structures are very similar in both apo-nd ligand-bound forms, the comparative analysis has revealedeveral structural alterations [8,22]. Individual structures show dif-

erences in relative position of monomers as a result of variationsn the angle between the two subunits (Fig. 2A) [22]. This dimerexibility could facilitate the 14-3-3 binding to ligands of differentizes and shapes. Another structural differences observed among

lopmental Biology 22 (2011) 663– 672

the 14-3-3 isoforms are the various lengths and conformations ofthe loop regions (especially loops between -helices H3 and H4 aswell as H8 and H9) and the length of -helices H3 and H4 (Fig. 2B)[7,8].

The individual 14-3-3 isoforms, although highly conserved, sig-nificantly differ in their propensity to form homo- or heterodimers.These differences result from small, but structurally important,sequence variations. The dimer interface in 14-3-3 homodimeris made up of helices H1 and H2 of one monomer that interactwith helices H3 and H4 of the other monomer through three saltbridges (Arg-18–Glu-89, Glu-5–Lys-74, and Asp-21–Lys-85 in � iso-form numbering) and several other both hydrophobic and polarcontacts [20]. Only one of the three salt bridges and all of the keyhydrophobic/polar interactions are conserved, while the other twosalt bridges are not present in all isoforms [6–8,21–23]. The � iso-form shows higher affinity for other subunits than for itself, andhence preferentially heterodimerizes. The reason is the presenceof only one salt bridge at the homodimer interface, while the het-erodimers are stabilized by up to three salt bridges [22]. On theother hand, the crystal structure of 14-3-3� isoform revealed sev-eral unique interactions, including alternative salt bridge betweenLys-9 and Glu-83, that likely account for its strong propensity toform homodimers [6,7].

Maximal isoform sequence variability occurs within the C-terminal stretch, a flexible region approximately 15–40 aminoacid residues long. The C-terminally truncated 14-3-3� exhibitsincreased binding affinity to several tested ligands [24]. It has beentherefore speculated that the C-terminal stretch functions as asuppressor of unspecific interactions between 14-3-3 and inappro-priate ligands. The structure of this segment is unknown becauseit cannot be seen in any of the available 14-3-3 structures, pre-sumably due to disorder; however, its possible location within theligand binding groove has been suggested [20,24]. Indeed, Försterresonance energy transfer (FRET) measurements and moleculardynamics simulation revealed that in the absence of the ligandthe C-terminal stretch occupies the ligand binding groove, but isdisplaced from it upon the phosphopeptide binding [25,26]. Inter-estingly, yeast 14-3-3 protein isoforms BMH1 and BMH2 possessa distinctly variant C-terminal tail which differentiates them fromthe isoforms of higher eukaryotes. Their C-termini are longer andcontain a polyglutamine stretch of unknown function. Recently,we have shown using various biophysical techniques that the C-terminal stretch of BMH proteins adopts a widely opened andextended conformation that likely hinders its folding into the lig-and binding groove [27]. It seems, therefore, that the C-terminalsegment of yeast 14-3-3 protein isoforms does not function as anautoinhibitor.

3. Target recognition by 14-3-3 proteins

3.1. Recognition of consensus phosphorylated binding motifs

The initial discovery that 14-3-3 proteins specifically recognize anovel phosphoserine-containing motif [9] was further refined usinga oriented peptide library screening that identified two consen-sus binding motifs, R[S/�][+]pSXP (mode I) and RX[�/S][+]pSXP(mode II), where pS is phosphoserine, � is an aromatic residue,+ is a basic residue, and X is any type of residue (typically Leu,Glu, Ala, and Met) [28,29]. These motifs, although optimal, are notabsolute as a number of the 14-3-3 protein binding partners iden-tified to date contain either phosphorylated or unphosphorylated

sequences that significantly differ from these optimal motifs [30].Although most of known 14-3-3 binding partners possess motifscontaining phosphoserine, the phosphorylated residue may also bea threonine. For example, serotonin N-acetyltransferase (AANAT)

T. Obsil, V. Obsilova / Seminars in Cell & Developmental Biology 22 (2011) 663– 672 665

F B [29]t

[[

sophaapataag

Fgb

ig. 1. Crystal structure of the 14-3-3 protein (human � isoform, PDB ID code 1QJotally conserved among all seven human isoforms are shaded in dark red.

31,32] or FOXO forkhead transcription factors contain such motifs33,34].

The first co-crystal structures of 14-3-3 protein complexes withynthetic phosphopeptides revealed little change in the structuref the 14-3-3 protein compared with the ligand-free form. Thehosphate group of the phosphopeptide interacts via ionic andydrogen bonds with conserved residues Lys-49, Arg-56, Arg-127nd Tyr-128, which form positively charged pocket within the lig-nd binding groove of 14-3-3 (Fig. 3A and B) [28,29,35]. The boundhosphopeptides adopt an extended main chain conformation with

fixed orientation within the ligand binding groove. This is mainly

he result of interactions between the phosphopeptide backbonend side chains of conserved residues Asn-173 and Asn-224, as wells the presence of conserved hydrophobic patch within the bindingroove that interacts with hydrophobic residues on the C-terminal

ig. 2. Structural comparison of all seven human 14-3-3 protein isoforms. (A) Superimamma (2B05) in red, epsilon (2BR9) in orange, zeta (1QJB) in green, eta (2C74) in cyan,

etter clarity. (B) Comparison of monomers. Only one monomer from each crystal structu

). (A) The ribbon representation. (B) The surface representation. Residues that are

side of the phosphoserine [22,28,29]. Structural data also explainedthe selectivity for sequences of both optimal binding motifs. Forexample, while the preference for basic residue (Arg or Lys) atpSer −3 position arises from interactions with acidic residues inits vicinity, the pSer −4 Arg presumably stabilizes the phospho-peptide conformation by forming an unusual intramolecular saltbridge with the pSer phosphate. The serine at pSer −2 position(mode I consensus) forms strong hydrogen bonds with the 14-3-3 side chain carboxyl of Glu-180 and indole nitrogen of Trp-228(in � isoform numbering). The presence of proline residue at pSer+2 position allows a sharp change in peptide main chain direction

and its exit from the ligand binding groove [28,29]. The same func-tion can also be carried out by a glycine residue but the orientedpeptide library screening showed that peptides containing Gly atpSer +2 position have a 4-fold lower binding affinity compared to

position of 14-3-3 dimers. The beta isoform (PDB ID 2C23) is shown in magenta,sigma (1YWT) in blue and tau (2BTP) in yellow. Bound peptides were removed forre was used for this supermposition.

666 T. Obsil, V. Obsilova / Seminars in Cell & Developmental Biology 22 (2011) 663– 672

Fig. 3. Detailed view of interactions within the 14-3-3 ligand binding groove. (A) The “mode 1” phosphopeptide, sequence ARSHpSYPA, bound to 14-3-3� [29]. (B) The “mode2” phosphopeptide, sequence RLYHpSLPA, bound to 14-3-3� [29]. (C) The “mode 3” phosphopeptide derived from the C-terminus of plant plasma membrane H+-ATPase,sequence QSYpTV bound to plant 14-3-3C [37]. Fusicoccin (FC) is shown in magenta. (D) Phosphorylation-independent interaction between 14-3-3� and peptide fromExoenzyme S (ExoS), sequence GHGQGLLDALDLAS [46]. pS and pT denote phosphoserine and phosphothreonine, respectively. Bound peptides and 14-3-3 residues involvedi respea

p1Hic

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n key contacts are rendered as sticks with carbon atoms colored yellow and gray,

re shown in ribbon representation.

roline-containing peptides [28]. The co-crystal structure of the4-3-3� protein with bound N-terminal phosphorylated histone3 peptide provided an explanation for this difference by show-

ng that a glycine residue at pSer +2 position makes a less favorableontribution to binding than a proline residue [36].

More recently, a third consensus binding motif (pS/pT-X1–2-OOH) was identified and it has been shown that ligands containinguch sequence bind to 14-3-3 with weaker affinity comparedo mode I and II motifs [37–40]. In addition, this C-terminal

otif enables consequent binding of additional small moleculehat closes a gap remaining in the 14-3-3 ligand binding groove,nd hence significantly stabilizes the resulting ternary complexFig. 3C). A typical example of such compound is the fungal phy-otoxin fusicoccin, a diterpene glycoside, which is an activator ofhe H+-ATPase. Fusicoccin has been shown to bind to the 14-3-:H+-ATPase complex and it even enables 14-3-3 to bind to thenphosphorylated C terminus of the H+-ATPase [37,41,42]. Twoew compounds structurally unrelated to fusicoccin, pyrrolidone

and epibestatin, were recently identified and showed to selec-

ively activate the H+-ATPase by stabilizing its complex with the4-3-3 protein [43]. Such compounds might represent very usefulnd potent tools for specific modulation of interactions between4-3-3 and their targets.

ctively. The secondary structural elements forming the 14-3-3 ligand binding cleft

3.2. Binding of unphosphorylated peptides

The 14-3-3 proteins can also bind their ligands in aphosphorylation-independent manner. The Exoenzyme S (ExoS)cytotoxin [10,12,44] and the R18 peptide derived from a phage dis-play library [45] are two well-known examples of ligands with suchmode of binding. The crystallographic analysis revealed that R18binds in a similar manner as the phosphorylated peptides via theamphipathic sequence, WLDLE, whose two acidic groups interactwith a cluster of basic residues within the ligand binding groove[35]. On the other hand, the structure of the 14-3-3�:ExoS complexshowed ExoS peptide bound in a reverse orientation that is pri-marily dependent on hydrophobic contacts between four leucineresidues of ExoS and the “roof” of the ligand binding grove of14-3-3� (Fig. 3D) [46]. It seems that in this case the electrostaticinteractions only marginally contribute to overall binding. Thesequence of the ExoS peptide contains two acidic residues (Asp-424and Asp-427) and only the residue Asp-424 makes a contact withthe 14-3-3 residue Lys-49 from the basic cluster within the 14-3-

3 ligand binding groove [46]. In addition, the substitution of bothaspartate residues in the binding motif of ExoS had no significanteffect on its interaction with the 14-3-3 protein or the enzymaticactivation of ExoS [47].

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. Structural basis of 14-3-3 protein functions

Systematic research of the 14-3-3 proteins during the past 20ears has revealed their participation in a wide range of biologicalrocesses through a variety of mechanisms. Available structuralnd biochemical data enabled classification of functional rolesf the 14-3-3 proteins. The generally accepted classification isased on the following modes of action: (i) direct conformationalhange of the target protein; (ii) physical occlusion of sequence-pecific or structural features; and (iii) scaffolding that anchorsroteins within close proximity of one another (Fig. 4) (reviewed by14–16,48]). However, the exact mechanisms behind these “modesf action” are mostly elusive. The majority of structural data on4-3-3 proteins available to date is restricted to complexes withhort synthetic phosphopeptides. Despite the large number of 14--3 binding partners, only two high-resolution structures of 14-3-3omplexes with the protein ligand that exceeds the consensusecognition motifs were reported so far: the 14-3-3�:AANAT com-lex [32] and the 14-3-3:C-terminal region of the plant plasmaembrane H+-ATPase complex [49]. Additional, although limited,

tructural information was obtained from biophysical studies on4-3-3 protein complexes with various ligands, including fork-ead transcription factor FOXO4 [50,51], the tumor suppressor p5352,53], the regulatory domain of tyrosine hydroxylase (TH1R) [54]r the regulator of G protein signaling 3 (RGS3) [55]. Structuralases of known mechanisms of 14-3-3 functions are discussed inetail below.

.1. Common aspects of 14-3-3 protein complexes

Structural, biochemical and bioinformatics studies of complexesormed by 14-3-3 proteins revealed several common features thateem to be important for 14-3-3‘s ability to bind and regulate theirartners: (i) the rigid structure of the 14-3-3 protein dimer; (ii) therequent presence of multiple 14-3-3 binding motifs in one target

olecule; and (iii) the frequent presence of 14-3-3 binding motifsnside disordered regions.

.1.1. The rigidity of the 14-3-3 protein dimerThe co-crystal structures with peptides or proteins revealed

ittle change in the structure of the 14-3-3 dimer compared tohe ligand-free form [6,7,20–22,28,29,32,36,46,49]. This structuraligidity, likely a result of an extensive network of interactionsetween the -helices, suggests that 14-3-3 protein can behaves so-called ‘molecular anvil’ by forming a rigid platform on whichhe bound target protein can be reshaped, while itself undergoesnly a minimal structural change [56].

.1.2. Simultaneous use of multiple 14-3-3 binding motifsIt is now well established that the dimeric nature of 14-3-

proteins with its two ligand binding grooves arranged in anntiparallel fashion is very important for 14-3-3 functions. Many4-3-3 binding partners contain two or more 14-3-3 binding motifs,hich could be used simultaneously to engage both ligand bind-

ng grooves within a 14-3-3 dimer [30,57]. Doubly phosphorylatedeptides have been shown to bind with significantly higher affinityhan the same peptides containing only one motif [28,57]. Thesendings led to the hypothesis that one of the binding motifs may

unction as a dominant site (a ‘gatekeeper’) which presence isbsolutely required for binding to 14-3-3 [56]. The secondary low-ffinity site, although insufficient to promote a stable associationith 14-3-3 in the absence of the gatekeeper motif, is then required

or a full biological activity. The V3 region of PKC� is an example of aeptide that contains two adjacent phosphorylated 14-3-3 bindingotifs that significantly differ in their binding affinities. Structural

nd calorimetric study confirmed that its divergent 14-3-3 binding

lopmental Biology 22 (2011) 663– 672 667

motif has a barely detectable interaction on its own, but is requiredfor high-affinity binding [57]. Thus the identification of such weakdivergent motifs, although difficult as their modification can havevery small effect on the overall binding affinity, would be an essen-tial task to fully understand the mechanism of 14-3-3 functions. Thestudy of Kostelecky et al. [57] also revealed that a minimal linkersequence of approximately ten residues is required between thetwo motifs to generate a tandem 14-3-3 binding motif, as well asthat the affinity of the tandem binding motif can be inhibited by athird phosphorylation allowing further regulatory inputs.

The 14-3-3 protein dimer can also bind simultaneously twomotifs that are remote from each other, for example, when theyborder the functional domain as have been shown for FOXO fork-head transcription factors [33,34] or AANAT [31,32]. It appearsthat such interaction is able to provide not only the high-affinitybinding but also represents an efficient way for structural modula-tion and/or masking of bound partner. In addition to interactionswith partners containing multiple 14-3-3 binding motifs, the 14-3-3 dimerization plays also a key role in systems where the 14-3-3dimer anchors two proteins within close proximity of one another.This topic is discussed in Section 4.4.

4.1.3. Presence of 14-3-3 binding motifs inside disordered regionsThe bioinformatics analysis revealed that 14-3-3 binding sites

are very frequently located within disordered regions either inthe N- and C-terminal tails or bordering the functional domains[30,58,59]. The flexibility and plasticity of inherently unstructuredregions can provide several functional advantages for signalingproteins including the ability to bind to multiple binding part-ners and/or the fine control over the binding affinities (reviewedby [60,61]). Another reason for the participation of unstructuredregions in binding interactions was proposed by Shoemaker et al.[62], who suggested that the unstructured protein (or segment)would have a greater ‘capture radius’ that facilitates the diffusivesearch for a binding target than a compact, folded protein withrestricted conformational flexibility. All these aspects might be rel-evant for binding interactions between the 14-3-3 proteins andtheir targets.

The frequent presence of 14-3-3 binding motifs inside disor-dered regions also suggests that the disorder-to-order transitionof the ligand molecule’s structure might be a common aspectof the binding to 14-3-3 [58]. Formation of the complex, whichinvolves structuring of disordered regions, would be highly disfa-vored entropically, and hence has to be driven by a large decrease inenthalpy. This is consistent with the fact that the primary interac-tion between the 14-3-3 protein and its ligand is, in vast majorityof cases, mediated by formation of numerous polar contacts (e.g.hydrogen bonds and salt bridges) between the phosphorylatedmotif and the ligand binding groove (Fig. 3) [28,29].

4.2. Mode of action: conformational change of the target protein

The 14-3-3 protein-dependent activation of AANAT is an exam-ple of the mechanism based on a direct structural change of thebound target protein. AANAT catalyzes acetyl transfer from acetyl-coenzyme A (AcCoA) to serotonin, yielding N-acetylserotonin, theprecursor of melatonin. The uncomplexed AANAT is catalyticallyinefficient due to low affinity for its substrates. Serotonin Km offree AANAT is ∼170 �M while the cytoplasmic concentration ofserotonin in the nighttime ovine pineal gland is ∼1 �M [31]. How-ever, upon the phosphorylation the 14-3-3 protein binds to andenhances the enzymatic activity of AANAT by significantly increas-

ing substrate affinity [31]. Structural studies have demonstratedthat the catalytic cycle of AANAT is accompanied by a confor-mational change of an 18-residues long loop region [63,64]. Thisreorganization allows AcCoA to bind and structurally complete

668 T. Obsil, V. Obsilova / Seminars in Cell & Developmental Biology 22 (2011) 663– 672

F indinr teins.

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ig. 4. Modes of 14-3-3 protein action (adapted from [15,16]). The 14-3-3 protein begion in the target protein; or (iii) facilitate the interaction between two other pro

he binding site for serotonin. The crystal structure of the 14-3-�:AANAT complex revealed that AANAT is bound in the centralhannel of the 14-3-3� dimer and is held in place by extensive inter-ctions involving both the ligand binding groove and other parts ofhe central channel of 14-3-3� (Fig. 5A) [32]. These contacts includehe loop region of AANAT which undergoes conformational changepon substrate binding. The structure of the complex suggestshat 14-3-3� forces AANAT to adopt a conformation that allowsptimal substrate binding, thus explaining why 14-3-3� bindingncreases substrate affinity. In addition, AANAT is an examplef a protein that possesses two 14-3-3 binding motifs border-ng the functional catalytic domain. The first motif is located athe N-terminus (sequence RRHpTLP) and the second one at theery C-terminus (sequence RRNpSDR-COOH) [31,38]. The struc-ural and biochemical data suggest that AANAT binds to a 14-3-3imer using both motifs simultaneously with the N-terminal motifunctioning as a ‘gatekeeper’ that binds first, followed by bind-

ng of the C-terminal motif [32,38]. It seems that this providesot only the high-affinity interaction but also a more efficientay for structural and functional modulation of the bound targetrotein.

ig. 5. Complexes of 14-3-3 proteins. (A) The crystal structure of the 14-3-3�:AANAT conalog (BI) is shown as sticks. (B) The crystal structure of the 14-3-3:H+-ATPase complexlasma membrane H+-ATPase. (C) Structural model of the 14-3-3�:FOXO4-DBD complex

g can: (i) induce a conformational change of the target protein; (ii) mask a specific

The catalytic activity of several other enzymes, including tryp-tophan and tyrosine hydroxylases [65–67], Raf kinases [68,69],ASK1 kinase [70,71], plant plasma membrane H+-ATPase [49,72],plant nitrate reductase [73,74], plant mitochondrial and chloro-plast adenosine 5′-triphosphate (ATP) synthases [75,76] and more,have been shown to be regulated in the 14-3-3 protein-dependentmanner. Oecking’s group provided, using X-ray crystallography andelectron cryomicroscopy, a structural explanation for the 14-3-3-protein dependent activation of H+-ATPase [49]. The activity of thisenzyme, which is responsible for building up an electrochemicalproton gradient across the plasma membrane, is autoinhibited byits C-terminal domain. The autoinhibitory activity is relieved byphosphorylation of the penultimate threonine residue and subse-quent association with 14-3-3 proteins [41,77]. The structure of thecomplex between the 14-3-3 protein and the C-terminal fragmentof H+-ATPase revealed an interesting mode of interaction wherethe 14-3-3 dimer simultaneously binds two H+-ATPase C-terminal

fragments in a manner that allows exit of both polypeptides fromthe central channel of 14-3-3 in the same direction (Fig. 5B) [49].The model proposed for the activation of H+-ATPase assumes thatfirst the 14-3-3 protein binds to the phosphorylated C-terminus of

mplex [32]. The dimer of 14-3-3� binds two molecules of AANAT. The bisubstrate [49]. The dimer of 14-3-3 binds two molecules of the C-terminal fragment of plantderived from FRET measurements [51].

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Ser-40 (reviewed in [102]) but only the phosphorylation of Ser-19 is requited for high-affinity binding to 14-3-3. The exact roleof 14-3-3 protein in the regulation of TH activity is still unclear

T. Obsil, V. Obsilova / Seminars in Cell &

ne subunit of inactive dimeric ATPase. This induces a conforma-ional change which abolishes interaction between the inhibitory-terminal regions of adjacent subunits of ATPase dimer. Conse-uently, the binding of a second C-terminal region from anotherTPase dimer to the remaining unoccupied 14-3-3 ligand bindingroove leads to conjunction of two H+-ATPase dimers. The last stepould be the closure of the active hexameric complex by associ-

tion of a third 14-3-3 dimer [49]. It seems, therefore, that in thisase the 14-3-3 protein dimer not only changes the conformationf its binding partner but also anchors two proteins within closeroximity of one another, thus facilitating the formation of a largerotein complex.

.3. Mode of action: physical occlusion of sequence-specific ortructural features

.3.1. Masking of protein–protein interaction sitesThe 14-3-3 proteins have been shown to change in many cases

he subcellular localization of their binding partners. For example,hey are involved in the cytoplasmic localization of FOXO fork-ead transcription factors [33,34,78], the cell cycle dual-specificityhosphatase Cdc25 [79,80] or the class IIa histone deacetylases81,82], as well as the nuclear localization of the catalytic sub-nit of telomerase (TERT) [83,84]. If the target protein possesses

nuclear localization sequence (NLS) or a nuclear export sequenceNES) adjacent to the 14-3-3 binding motif then the 14-3-3 pro-ein can interfere with the function of these signaling sequences.heir masking or obscuring would alter the kinetics of dynamicuclear–cytoplasmic transport, and hence shift the equilibrium ofarget protein localization toward the cytoplasm or nucleus [85].nalogous mechanism seems also to be involved in the regulationf cytoplasmic–endoplasmic reticulum (ER) localization of severalroteins whose dibasic ER retention motif overlaps with the 14--3 recognition motif, and hence can be obscured upon the 14-3-3rotein binding (see review by Bridges and Moorhead [16] for moreetails).

Among the best-characterized subjects of such regulationre FOXO proteins, a subgroup of forkhead transcription fac-ors that play a central role in cell-cycle control, differentiation,

etabolism control, stress response and apoptosis (reviewedy [86,87]). Their transcriptional activity is regulated through

nsulin–phosphatidylinositol 3-kinase–protein kinase B (PI3K-KB) signaling pathway. PKB (also known as Akt kinase)hosphorylates FOXO factors at three specific sites and createswo 14-3-3 binding motifs; one is located at the N-terminus whilehe second one is at the C-terminal end of the forkhead DNA-inding domain embedded within NLS [33,34]. Several studiesemonstrated that upon the binding to 14-3-3 protein, the result-

ng FOXO:14-3-3 complex is translocated to the cytosol where theound 14-3-3 protein prevents reentry of FOXO into the nucleus

ikely by masking its NLS [33,78,88–90]. This hypothesis is alsoupported by results of biophysical study that confirmed directhysical contact between 14-3-3 and NLS of transcription factorOXO4 [50]. In addition, Brunet et al. [78] has shown that the 14-3-3rotein binds to phosphorylated FOXO within the nucleus and facil-

tates the nuclear export of the resulting complex through FOXO’sES. Thus, it appears that 14-3-3 proteins control the subcellular

ocalization of FOXO factors by affecting simultaneously the func-ion of both their NLS and NES.

Regulator of G protein signaling (RGS) proteins are anotherxample of proteins regulated through masking of theirrotein–protein interaction surface. RGS proteins function as

TPase-activating proteins (GAPs) for the -subunit of het-rotrimeric G proteins (reviewed by Hepler [91]). It has been foundhat the GAP function of certain RGS proteins, including RGS3,GS4, RGS5, RGS7 and RGS16, is inhibited by 14-3-3 proteins that

lopmental Biology 22 (2011) 663– 672 669

presumably block the interaction between RGS and G subunit[92–95]. Biophysical study based on dynamic tryptophan fluo-rescence spectroscopy revealed that the 14-3-3 protein interactswith not only the N-terminal part of RGS3 containing the phos-phorylated 14-3-3 binding motif, but also with the region withinthe G-interacting portion of the remote C-terminal RGS domain[55]. This is consistent with the hypothesis that 14-3-3 masksthe G-interaction surface of RGS domain, and hence inhibitsits GAP activity. In addition, the isolated RGS domain of RGS3was found to interact very weakly with the 14-3-3 protein in aphosphorylation-independent manner. Thus, it seems that this isanother example of a complex where the 14-3-3 protein bindsto one high-affinity site in a phosphorylation-dependent manner(the N-terminal ‘gatekeeper’ site) and simultaneously interactswith a region which is remote from the phosphorylated motif,insufficient to promote a stable association with 14-3-3 by itselfbut required for a full biological activity (the C-terminal RGSdomain).

4.3.2. Masking of protein–DNA interaction sitesAs mentioned above, the FOXO transcription factors contain two

14-3-3 binding motifs that border the DNA-binding domain andare both necessary for optimal FOXO binding to the 14-3-3 pro-tein [33,34,90]. This arrangement raises the possibility that the14-3-3 binding affects the DNA-binding properties of FOXO fac-tors. Indeed, such 14-3-3 protein-dependent inhibition of FOXObinding to the DNA has been observed for DAF-16 (Caenorhabditiselegans FOXO homologue) [96] and FOXO4 [34]. Biophysical stud-ies based on time-resolved fluorescence spectroscopy and Försterresonance energy transfer (FRET) measurements revealed directphysical contacts between 14-3-3 and the DNA-binding inter-face of FOXO4 [51]. This interaction, on the other hand, does notcause any dramatic conformational change of FOXO4. The modelof the 14-3-3:FOXO4 complex, which was build using distancesobtained from FRET measurements, suggests that the DNA-bindingdomain of FOXO4 is docked within the central channel of the14-3-3 protein dimer (Fig. 5C), consistent with the hypothesisthat 14-3-3 sterically occludes the DNA binding interface of FOXOproteins [51,97].

4.3.3. Protection against dephosphorylation and proteolyticdegradation

Yet another masking-related function of the 14-3-3 protein isto protect the target protein against dephosphorylation and/orproteolytic degradation. Recently, Dobson et al. [98] showed thatthe 14-3-3 protein binding, in addition to the established nega-tive roles in FOXO regulation, stabilizes FOXO3 by inhibiting itsdephosphorylation and degradation rates. Thus, the 14-3-3 pro-tein availability could dictate the fate of phosphorylated FOXOtoward degradation or recycling. Similar protective role for 14-3-3 was also suggested in the regulation of tyrosine hydroxylase(TH) activity. This enzyme catalyzes the first and rate-limiting stepin the biosynthesis of catecholamines and its activity is signifi-cantly enhanced by phosphorylation-dependent binding to 14-3-3[65,67,99–101]. The N-terminal regulatory domain of TH can bephosphorylated at four serine residues, Ser-8, Ser-19, Ser-31, and

but it has been suggested that 14-3-3 protein might protect pro-teolytically very sensitive phosphorylated regulatory domain ofTH, and/or slowdown dephosphorylation of phosphorylated Ser-19and Ser-40 [54,100,101].

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70 T. Obsil, V. Obsilova / Seminars in Cell &

.4. Mode of action: scaffolding that anchors proteins withinlose proximity of one another

The dimeric nature of 14-3-3 proteins implies the possibility ofheir acting as scaffold molecules that anchor two proteins withinlose proximity of one another. The crystal structures of 14-3-3omplexes with AANAT [32] and H+-ATPase [49] confirmed thathe 14-3-3 dimer is able to accommodate two molecules of ligandrotein within its central channel (Fig. 5A and B). The above dis-ussed activation of H+-ATPase is a nice example of the mechanismhere the 14-3-3 protein induces, due to its dimeric nature, the

ormation of a large multi protein complex [49]. Another exampleas emerged from studies on tumor suppressor p53, a transcription

actor involved in different cellular functions, such as cell cycle con-rol, apoptosis, and differentiation. The p53 protein is active when its tetrameric, and in this conformation it binds with high affinity toNA or interacts more efficiently with various other proteins [103].he 14-3-3 proteins both activate the DNA binding affinity andncrease the stability of p53 by binding to a site in its intrinsicallyisordered C-terminal domain [53,104,105]. It has been shown that4-3-3 proteins enhance the binding of sequence-specific DNA to53 by causing p53 dimers to form tetramers at lower concentra-ions [52]. Thus it seems that p53 activity may be a subject of controly 14-3-3’s that lower its tetramer–dimer dissociation constant.ther examples of 14-3-3 scaffolding involve formation of ternaryomplexes where the 14-3-3 dimer promotes interaction betweenwo different ligand proteins. Such a possibility has been, for exam-le, suggested for �-catenin and Cby. Cby is a conserved antagonistf �-catenin, a central protein of the canonical Wnt signaling path-ay [106]. Cby interacts with the C-terminal activation domain

f �-catenin and blocks its transcriptional activation potential. Itas been suggested that 14-3-3 forms ternary complex with phos-horylated �-catenin and Cby, stabilizes their complex, and henceromotes �-catenin nuclear export and termination of its signaling107].

. Conclusions

The 14-3-3 proteins fulfill panoply of regulatory functionsnd constitute key players in many signaling pathways. Avail-ble biochemical, structural and bioinformatics data enabled us tonderstand the structural basis of target recognition by 14-3-3 pro-eins, revealed common structural features of their complexes androvided insight into some of their functions. Many aspects relatedo the mechanisms of 14-3-3 functions still remain unresolved, forxample structural basis of the 14-3-3 isoform binding specificity,etails concerning structural modulation of bound targets or mech-nisms of their own regulation. One of the reasons of this situations that the majority of structural data on 14-3-3 proteins avail-ble to date is restricted to their complexes with short synthetichosphopeptides. More structures of 14-3-3 complexes with therotein ligand that exceeds the consensus recognition motifs wille needed to fully understand all nuances of their functions. Inddition, the use of alternative biophysical techniques that allowharacterization of unstructured proteins might be very useful con-idering the high disorder propensity of 14-3-3 ligands.

cknowledgements

The authors are supported by grants from the Grant Agencyf the Academy of Sciences of the Czech Science Foundation

Projects P305/11/0708 and P207/11/0455); Czech Republic (GrantAA501110801); Grant Agency of the Charles University (Grant8510); Ministry of Education, Youth, and Sports of the Czechepublic Research (Projects MSM0021620857 and Center of Neu-

lopmental Biology 22 (2011) 663– 672

rosciences LC554); and Academy of Sciences of the Czech Republic(Research Projects AV0Z50110509).

References

[1] Cohen P. The origins of protein phosphorylation. Nat Cell Biol2002;4:E127–30.

[2] Yaffe MB, Elia AE. Phosphoserine/threonine-binding domains. Curr Opin CellBiol 2001;13:131–8.

[3] Moore BW, Perez VJ. Specific acidic proteins of the nervous system. In: Carl-son FD, editor. Physiological and biochemical aspects of nervous integration.Woods Hole, MA: Prentice-Hall, Inc., The Marine Biological Laboratory; 1967.p. 343–59.

[4] Wang W, Shakes DC. Molecular evolution of the 14-3-3 protein family. J MolEvol 1996;43:384–98.

[5] Rosenquist M, Alsterfjord M, Larsson C, Sommarin M. Data mining the Ara-bidopsis genome reveals fifteen 14-3-3 genes. Expression is demonstrated fortwo out of five novel genes. Plant Physiol 2001;127:142–9.

[6] Wilker EW, Grant RA, Artim SC, Yaffe MB. A structural basis for 14-3-3sigmafunctional specificity. J Biol Chem 2005;280:18891–8.

[7] Benzinger A, Popowicz GM, Joy JK, Majumdar S, Holak TA, Hermeking H.The crystal structure of the non-liganded 14-3-3sigma protein: insights intodeterminants of isoform specific ligand binding and dimerization. Cell Res2005;15:219–27.

[8] Gardino AK, Smerdon SJ, Yaffe MB. Structural determinants of 14-3-3 bind-ing specificities and regulation of subcellular localization of 14-3-3-ligandcomplexes: a comparison of the X-ray crystal structures of all human 14-3-3isoforms. Semin Cancer Biol 2006;16:173–82.

[9] Muslin AJ, Tanner JW, Allen PM, Shaw AS. Interaction of 14-3-3 withsignaling proteins is mediated by the recognition of phosphoserine. Cell1996;84:889–97.

[10] Fu H, Coburn J, Collier RJ. The eukaryotic host factor that activates exoenzymeS of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. ProcNatl Acad Sci USA 1993;90:2320–4.

[11] Campbell JK, Gurung R, Romero S, Speed CJ, Andrews RK, Berndt MC, et al.Activation of the 43 kDa inositol polyphosphate 5-phosphatase by 14-3-3zeta.Biochemistry 1997;36:15363–70.

[12] Masters SC, Pederson KJ, Zhang L, Barbieri JT, Fu H. Interaction of 14-3-3 with anonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa.Biochemistry 1999;38:5216–21.

[13] Pozuelo Rubio M, Geraghty KM, Wong BH, Wood NT, Campbell DG, Morrice N,et al. 14-3-3-Affinity purification of over 200 human phosphoproteins revealsnew links to regulation of cellular metabolism, proliferation and trafficking.Biochem J 2004;379:395–408.

[14] Fu H, Subramanian RR, Masters SC. 14-3-3 proteins: structure, function, andregulation. Annu Rev Pharmacol Toxicol 2000;40:617–47.

[15] Tzivion G, Avruch J. 14-3-3 proteins: active cofactors in cellular regulation byserine/threonine phosphorylation. J Biol Chem 2002;277:3061–4.

[16] Bridges D, Moorhead GB. 14-3-3 proteins: a number of functions for a num-bered protein. Sci STKE 2004;2004:re10.

[17] Mackintosh C. Dynamic interactions between 14-3-3 proteins and phospho-proteins regulate diverse cellular processes. Biochem J 2004;381:329–42.

[18] Aitken A. 14-3-3 proteins: a historic overview. Semin Cancer Biol2006;16:162–72.

[19] Morrison DK. The 14-3-3 proteins: integrators of diverse signaling cues thatimpact cell fate and cancer development. Trends Cell Biol 2009;19:16–23.

[20] Liu D, Bienkowska J, Petosa C, Collier RJ, Fu H, Liddington R. Crystal structureof the zeta isoform of the 14-3-3 protein. Nature 1995;376:191–4.

[21] Xiao B, Smerdon SJ, Jones DH, Dodson GG, Soneji Y, Aitken A, et al. Structureof a 14-3-3 protein and implications for coordination of multiple signallingpathways. Nature 1995;376:188–91.

[22] Yang X, Lee WH, Sobott F, Papagrigoriou E, Robinson CV, Grossmann JG, et al.Structural basis for protein–protein interactions in the 14-3-3 protein family.Proc Natl Acad Sci USA 2006;103:17237–42.

[23] Verdoodt B, Benzinger A, Popowicz GM, Holak TA, Hermeking H. Character-ization of 14-3-3sigma dimerization determinants: requirement of homod-imerization for inhibition of cell proliferation. Cell Cycle 2006;5:2920–6.

[24] Truong AB, Masters SC, Yang H, Fu H. Role of the 14-3-3 C-terminal loop inligand interaction. Proteins 2002;49:321–5.

[25] Obsilova V, Herman P, Vecer J, Sulc M, Teisinger J, Obsil T. 14-3-3zetaC-terminal stretch changes its conformation upon ligand binding and phos-phorylation at Thr232. J Biol Chem 2004;279:4531–40.

[26] Silhan J, Obsilova V, Vecer J, Herman P, Sulc M, Teisinger J, et al. 14-3-3 pro-tein C-terminal stretch occupies ligand binding groove and is displaced byphosphopeptide binding. J Biol Chem 2004;279:49113–9.

[27] Veisova D, Rezabkova L, Stepanek M, Novotna P, Herman P, Vecer J, et al.The C-terminal segment of yeast BMH proteins exhibits different structurecompared to other 14-3-3 protein isoforms. Biochemistry 2010;49:3853–61.

[28] Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H, et al.

The structural basis for 14-3-3: phosphopeptide binding specificity. Cell1997;91:961–71.

[29] Rittinger K, Budman J, Xu J, Volinia S, Cantley LC, Smerdon SJ, et al. Structuralanalysis of 14-3-3 phosphopeptide complexes identifies a dual role for thenuclear export signal of 14-3-3 in ligand binding. Mol Cell 1999;4:153–66.

Deve

T. Obsil, V. Obsilova / Seminars in Cell &

[30] Johnson C, Crowther S, Stafford MJ, Campbell DG, Toth R, MacKintosh C.Bioinformatic and experimental survey of 14-3-3-binding sites. Biochem J2010;427:69–78.

[31] Ganguly S, Gastel JA, Weller JL, Schwartz C, Jaffe H, Namboodiri MA,et al. Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis. Proc Natl Acad Sci USA2001;98:8083–8.

[32] Obsil T, Ghirlando R, Klein DC, Ganguly S, Dyda F. Crystal structure of the14-3-3zeta:serotonin N-acetyltransferase complex: a role for scaffolding inenzyme regulation. Cell 2001;105:257–67.

[33] Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. Akt promotes cellsurvival by phosphorylating and inhibiting a forkhead transcription factor.Cell 1999;96:857–68.

[34] Obsil T, Ghirlando R, Anderson DE, Hickman AB, Dyda F. Two 14-3-3 bind-ing motifs are required for stable association of forkhead transcription factorFOXO4 with 14-3-3 proteins and inhibition of DNA binding. Biochemistry2003;42:15264–72.

[35] Petosa C, Masters SC, Bankston LA, Pohl J, Wang B, Fu H, et al. 14-3-3zetabinds a phosphorylated Raf peptide and an unphosphorylated peptide via itsconserved amphipathic groove. J Biol Chem 1998;273:16305–10.

[36] Macdonald N, Welburn JP, Noble ME, Nguyen A, Yaffe MB, Clynes D, et al.Molecular basis for the recognition of phosphorylated and phosphoacetylatedhistone h3 by 14-3-3. Mol Cell 2005;20:199–211.

[37] Wurtele M, Jelich-Ottmann C, Wittinghofer A, Oecking C. Structural view of afungal toxin acting on a 14-3-3 regulatory complex. Embo J 2003;22:987–94.

[38] Ganguly S, Weller JL, Ho A, Chemineau P, Malpaux B, Klein DC. Melatoninsynthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205. Proc Natl Acad Sci USA2005;102:1222–7.

[39] Coblitz B, Shikano S, Wu M, Gabelli SB, Cockrell LM, Spieker M, et al. C-terminalrecognition by 14-3-3 proteins for surface expression of membrane receptors.J Biol Chem 2005;280:36263–72.

[40] Coblitz B, Wu M, Shikano S, Li M. C-terminal binding: an expanded repertoireand function of 14-3-3 proteins. FEBS Lett 2006;580:1531–5.

[41] Svennelid F, Olsson A, Piotrowski M, Rosenquist M, Ottman C, Larsson C, et al.Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell1999;11:2379–91.

[42] Ottmann C, Weyand M, Sassa T, Inoue T, Kato N, Wittinghofer A, et al. A struc-tural rationale for selective stabilization of anti-tumor interactions of 14-3-3proteins by cotylenin A. J Mol Biol 2009;386:913–9.

[43] Rose R, Erdmann S, Bovens S, Wolf A, Rose M, Hennig S, et al. Identifica-tion and structure of small-molecule stabilizers of 14-3-3 protein–proteininteractions. Angew Chem Int Ed 2010;49:4129–32.

[44] Henriksson ML, Troller U, Hallberg B. 14-3-3 proteins are requiredfor the inhibition of Ras by exoenzyme S. Biochem J 2000;349(Pt 3):697–701.

[45] Wang B, Yang H, Liu YC, Jelinek T, Zhang L, Ruoslahti E, et al. Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. Biochemistry1999;38:12499–504.

[46] Ottmann C, Yasmin L, Weyand M, Veesenmeyer JL, Diaz MH, Palmer RH, et al.Phosphorylation-independent interaction between 14-3-3 and exoenzyme S:from structure to pathogenesis. Embo J 2007;26:902–13.

[47] Yasmin L, Jansson AL, Panahandeh T, Palmer RH, Francis MS, Hallberg B. Delin-eation of exoenzyme S residues that mediate the interaction with 14-3-3 andits biological activity. FEBS J 2006;273:638–46.

[48] Aitken A, Baxter H, Dubois T, Clokie S, Mackie S, Mitchell K, et al. Specificity of14-3-3 isoform dimer interactions and phosphorylation. Biochem Soc Trans2002;30:351–60.

[49] Ottmann C, Marco S, Jaspert N, Marcon C, Schauer N, Weyand M, et al.Structure of a 14-3-3 coordinated hexamer of the plant plasma membraneH+-ATPase by combining X-ray crystallography and electron cryomicroscopy.Mol Cell 2007;25:427–40.

[50] Obsilova V, Vecer J, Herman P, Pabianova A, Sulc M, Teisinger J, et al. 14-3-3Protein interacts with nuclear localization sequence of forkhead transcriptionfactor FOXO4. Biochemistry 2005;44:11608–17.

[51] Silhan J, Vacha P, Strnadova P, Vecer J, Herman P, Sulc M, et al. 14-3-3 proteinmasks the DNA binding interface of forkhead transcription factor FOXO4. JBiol Chem 2009;284:19349–60.

[52] Rajagopalan S, Jaulent AM, Wells M, Veprintsev DB, Fersht AR. 14-3-3 activa-tion of DNA binding of p53 by enhancing its association into tetramers. NuclAcids Res 2008;36:5983–91.

[53] Schumacher B, Mondry J, Thiel P, Weyand M, Ottmann C. Structure of thep53 C-terminus bound to 14-3-3: implications for stabilization of the p53tetramer. FEBS Lett 2010;584:1443–8.

[54] Obsilova V, Nedbalkova E, Silhan J, Boura E, Herman P, Vecer J, et al. The 14-3-3protein affects the conformation of the regulatory domain of human tyrosinehydroxylase. Biochemistry 2008;47:1768–77.

[55] Rezabkova L, Boura E, Herman P, Vecer J, Bourova L, Sulc M, et al. 14-3-3protein interacts with and affects the structure of RGS domain of regulator ofG protein signaling 3 (RGS3). J Struct Biol 2010;170:451–61.

[56] Yaffe MB. How do 14-3-3 proteins work? Gatekeeper phosphorylation andthe molecular anvil hypothesis. FEBS Lett 2002;513:53–7.

[57] Kostelecky B, Saurin AT, Purkiss A, Parker PJ, McDonald NQ. Recognition ofan intra-chain tandem 14-3-3 binding site within PKCepsilon. EMBO Rep2009;10:983–9.

lopmental Biology 22 (2011) 663– 672 671

[58] Bustos DM, Iglesias AA. Intrinsic disorder is a key characteristic in partnersthat bind 14-3-3 proteins. Proteins 2006;63:35–42.

[59] Oldfield CJ, Meng J, Yang JY, Yang MQ, Uversky VN, Dunker AK. Flexible nets:disorder and induced fit in the associations of p53 and 14-3-3 with theirpartners. BMC Genomics 2008;9(Suppl. 1):S1.

[60] Dyson HJ, Wright PE. Coupling of folding and binding for unstructured pro-teins. Curr Opin Struct Biol 2002;12:54–60.

[61] Wright PE, Dyson HJ. Linking folding and binding. Curr Opin Struct Biol2009;19:31–8.

[62] Shoemaker BA, Portman JJ, Wolynes PG. Speeding molecular recognition byusing the folding funnel: the fly-casting mechanism. Proc Natl Acad Sci USA2000;97:8868–73.

[63] Hickman AB, Klein DC, Dyda F. Melatonin biosynthesis: the structure of sero-tonin N-acetyltransferase at 2.5 A resolution suggests a catalytic mechanism.Mol Cell 1999;3:23–32.

[64] Hickman AB, Namboodiri MA, Klein DC, Dyda F. The structural basisof ordered substrate binding by serotonin N-acetyltransferase: enzymecomplex at 1.8 A resolution with a bisubstrate analog. Cell 1999;97:361–9.

[65] Ichimura T, Isobe T, Okuyama T, Yamauchi T, Fujisawa H. Brain 14-3-3 pro-tein is an activator protein that activates tryptophan 5-monooxygenase andtyrosine 3-monooxygenase in the presence of Ca2+, calmodulin-dependentprotein kinase II. FEBS Lett 1987;219:79–82.

[66] Ichimura T, Isobe T, Okuyama T, Takahashi N, Araki K, Kuwano R, et al.Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a proteinkinase-dependent activator of tyrosine and tryptophan hydroxylases. ProcNatl Acad Sci USA 1988;85:7084–8.

[67] Itagaki C, Isobe T, Taoka M, Natsume T, Nomura N, Horigome T, et al.Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins.Biochemistry 1999;38:15673–80.

[68] Fantl WJ, Muslin AJ, Kikuchi A, Martin JA, MacNicol AM, Gross RW, et al.Activation of Raf-1 by 14-3-3 proteins. Nature 1994;371:612–4.

[69] Fu H, Xia K, Pallas DC, Cui C, Conroy K, Narsimhan RP, et al. Interaction of theprotein kinase Raf-1 with 14-3-3 proteins. Science 1994;266:126–9.

[70] Zhang L, Chen J, Fu H. Suppression of apoptosis signal-regulating kinase1-induced cell death by 14-3-3 proteins. Proc Natl Acad Sci USA1999;96:8511–5.

[71] Goldman EH, Chen L, Fu H. Activation of apoptosis signal-regulating kinase1 by reactive oxygen species through dephosphorylation at serine 967 and14-3-3 dissociation. J Biol Chem 2004;279:10442–9.

[72] Oecking C, Piotrowski M, Hagemeier J, Hagemann K. Topology and targetinteraction of the fusicoccin-binding 14-3-3 homologs of Commelina com-munis. Plant J 1997;12:441–53.

[73] Athwal GS, Huber JL, Huber SC. Phosphorylated nitrate reductase and 14-3-3proteins. Site of interaction, effects of ions, and evidence for an amp-bindingsite on 14-3-3 proteins. Plant Physiol 1998;118:1041–8.

[74] Moorhead G, Douglas P, Morrice N, Scarabel M, Aitken A, MacKintosh C.Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3proteins and activated by fusicoccin. Curr Biol 1996;6:1104–13.

[75] Moorhead G, Douglas P, Cotelle V, Harthill J, Morrice N, Meek S,et al. Phosphorylation-dependent interactions between enzymes of plantmetabolism and 14-3-3 proteins. Plant J 1999;18:1–12.

[76] Bunney TD, van Walraven HS, de Boer AH. 14-3-3 protein is a regulatorof the mitochondrial and chloroplast ATP synthase. Proc Natl Acad Sci USA2001;98:4249–54.

[77] Fuglsang AT, Visconti S, Drumm K, Jahn T, Stensballe A, Mattei B, et al. Bindingof 14-3-3 protein to the plasma membrane H(+)-ATPase AHA2 involves thethree C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation ofThr(947). J Biol Chem 1999;274:36774–80.

[78] Brunet A, Kanai F, Stehn J, Xu J, Sarbassova D, Frangioni JV, et al. 14-3-3 transitsto the nucleus and participates in dynamic nucleocytoplasmic transport. J CellBiol 2002;156:817–28.

[79] Davezac N, Baldin V, Gabrielli B, Forrest A, Theis-Febvre N, Yashida M,et al. Regulation of CDC25B phosphatases subcellular localization. Oncogene2000;19:2179–85.

[80] Margolis SS, Walsh S, Weiser DC, Yoshida M, Shenolikar S, Kornbluth S. PP1control of M phase entry exerted through 14-3-3-regulated Cdc25 dephos-phorylation. Embo J 2003;22:5734–45.

[81] Grozinger CM, Schreiber SL. Regulation of histone deacetylase 4 and 5 andtranscriptional activity by 14-3-3-dependent cellular localization. Proc NatlAcad Sci USA 2000;97:7835–40.

[82] McKinsey TA, Zhang CL, Olson EN. Identification of a signal-responsivenuclear export sequence in class II histone deacetylases. Mol Cell Biol2001;21:6312–21.

[83] Seimiya H, Sawada H, Muramatsu Y, Shimizu M, Ohko K, Yamane K, et al.Involvement of 14-3-3 proteins in nuclear localization of telomerase. Embo J2000;19:2652–61.

[84] Kimura A, Ohmichi M, Kawagoe J, Kyo S, Mabuchi S, Takahashi T, et al. Induc-tion of hTERT expression and phosphorylation by estrogen via Akt cascade inhuman ovarian cancer cell lines. Oncogene 2004;23:4505–15.

[85] Muslin AJ, Xing H. 14-3-3 proteins: regulation of subcellular localization by

molecular interference. Cell Signal 2000;12:703–9.

[86] van der Horst A, Burgering BMT. Stressing the role of FOXO proteins in lifespanand disease. Nat Rev Mol Cell Biol 2007;8:440–50.

[87] Dansen TB, Burgering BM. Unravelling the tumor-suppressive functions ofFOXO proteins. Trends Cell Biol 2008;18:421–9.

6 Deve

[106] Takemaru K, Fischer V, Li FQ. Fine-tuning of nuclear-catenin by Chibby and

72 T. Obsil, V. Obsilova / Seminars in Cell &

[88] Rena G, Prescott AR, Guo S, Cohen P, Unterman TG. Roles of the forkhead inrhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3 bind-ing, transactivation and nuclear targetting. Biochem J 2001;354:605–12.

[89] Brownawell AM, Kops GJ, Macara IG, Burgering BM. Inhibition of nuclearimport by protein kinase B (Akt) regulates the subcellular distribu-tion and activity of the forkhead transcription factor AFX. Mol Cell Biol2001;21:3534–46.

[90] Zhao X, Gan L, Pan H, Kan D, Majeski M, Adam SA, et al. Multiple ele-ments regulate nuclear/cytoplasmic shuttling of FOXO1: characterizationof phosphorylation- and 14-3-3-dependent and -independent mechanisms.Biochem J 2004;378:839–49.

[91] Hepler JR. Emerging roles for RGS proteins in cell signalling. Trends PharmacolSci 1999;20:376–82.

[92] Benzing T, Yaffe MB, Arnould T, Sellin L, Schermer B, Schilling B, et al. 14-3-3interacts with regulator of G protein signaling proteins and modulates theiractivity. J Biol Chem 2000;275:28167–72.

[93] Benzing T, Kottgen M, Johnson M, Schermer B, Zentgraf H, Walz G, et al. Inter-action of 14-3-3 protein with regulator of G protein signaling 7 is dynamicallyregulated by tumor necrosis factor-alpha. J Biol Chem 2002;277:32954–62.

[94] Niu J, Scheschonka A, Druey KM, Davis A, Reed E, Kolenko V, et al. RGS3 inter-acts with 14-3-3 via the N-terminal region distinct from the RGS (regulatorof G-protein signalling) domain. Biochem J 2002;365:677–84.

[95] Abramow-Newerly M, Ming H, Chidiac P. Modulation of subfamily B/R4 RGSprotein function by 14-3-3 proteins. Cell Signal 2006;18:2209–22.

[96] Cahill CM, Tzivion G, Nasrin N, Ogg S, Dore J, Ruvkun G, et al. Phos-

phatidylinositol 3-kinase signaling inhibits DAF-16 DNA binding and functionvia 14-3-3-dependent and 14-3-3-independent pathways. J Biol Chem2001;276:13402–10.

[97] Obsil T, Obsilova V. Structural basis for DNA recognition by FOXO proteins.Biochim Biophys Acta. doi:10.1016/j.bbamcr.2010.11.025.

lopmental Biology 22 (2011) 663– 672

[98] Dobson M, Ramakrishnan G, Ma S, Kaplun L, Balan V, Fridman R, et al.Bimodal regulation of FOXO3 by AKT and 14-3-3. Biochim Biophys Acta.doi:10.1016/j.bbamcr.2011.05.001.

[99] Yamauchi T, Nakata H, Fujisawa H. A new activator protein that activates tryp-tophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence ofCa2+-, calmodulin-dependent protein kinase. Purification and characteriza-tion. J Biol Chem 1981;256:5404–9.

[100] Kleppe R, Toska K, Haavik J. Interaction of phosphorylated tyrosine hydrox-ylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependentassociation. J Neurochem 2001;77:1097–107.

[101] Toska K, Kleppe R, Armstrong CG, Morrice NA, Cohen P, Haavik J. Regula-tion of tyrosine hydroxylase by stress-activated protein kinases. J Neurochem2002;83:775–83.

[102] Dunkley PR, Bobrovskaya L, Graham ME, von Nagy-Felsobuki EI, Dickson PW.Tyrosine hydroxylase phosphorylation: regulation and consequences. J Neu-rochem 2004;91:1025–43.

[103] Chene P. The role of tetramerization in p53 function. Oncogene2001;20:2611–7.

[104] Waterman MJ, Stavridi ES, Waterman JL, Halazonetis TD. ATM-dependentactivation of p53 involves dephosphorylation and association with 14-3-3proteins. Nat Genet 1998;19:175–8.

[105] Yang HY, Wen YY, Chen CH, Lozano G, Lee MH. 14-3-3 sigma posi-tively regulates p53 and suppresses tumor growth. Mol Cell Biol 2003;23:7096–107.

14-3-3. Cell Cycle 2009;8:210–3.[107] Li FQ, Mofunanya A, Harris K, Takemaru K. Chibby cooperates with 14-3-3

to regulate beta-catenin subcellular distribution and signaling activity. J CellBiol 2008;181:1141–54.