Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

Discovered in 1967 [1], 14-3-3 proteins became one of the first known protein modules recognizing protein phosphorylation [2,3]. Although being practically unable to bind unphosphorylated forms of protein clients (rare exceptions exist [4,5]), 14-3-3 proteins are well-known for their ability to interact with a variety of cellular proteins containing phosphorylated Ser/Thr residues within certain sequence contexts. Biochemical studies revealed several optimal 14-3-3-binding sequences flanking the central phospho-Ser/Thr, that can be roughly subdivided into three main classes — internal motif I (R/K)-[S/F/Y/W]-[H/+]-p(S/T)-[L/E/A/M]-(P/G), motif II R-X-[S/Y/F/W]-(+)-p(S/T)-[L/E/A/M]-[P/G], and the C-terminally located motif III p(S/T)X1-2-COOH (where X denotes any amino acid, ‘+’ denotes positively charged amino acid, and p(S/T) denotes phosphorylated Ser or Thr) [6–8]. The C-terminal motif III 14-3-3-binding sites may overlap with the C-terminal PDZ domain-binding motifs (PBMs) and the recent data suggest that phosphorylation of certain PBMs can play a role of a 14-3-3/PDZ-binding switch that links the two protein–protein interaction (PPI) networks [9–11].

14-3-3-binding phosphomotifs tend to locate in the intrinsically disordered protein regions (IDPRs) [12], which brings sufficient plasticity for their accommodation within the binding grooves of 14-3-3 dimers that have a rather rigid structure [13]. 14-3-3 binding to phosphorylated sites of client proteins may have different outcomes including (i) the regulation of their enzymatic activity; (ii) masking of recognition motifs for binding to other proteins and, therefore, modulation of PPI; (iii) the regulation of intracellular localization of client proteins, and, more speculatively, (iv) mediation of protein interactions via bringing together two interacting entities on a ditopic 14-3-3 scaffold [14] (see also below).

14-3-3-binding motifs appear upon phosphorylation by different kinases, e.g. of the AGC and CAMK families phosphorylating (R/K)XX(S/T) sequences [15], and are found in hundreds of different proteins [16]. This explains the involvement of 14-3-3 proteins in sophisticated PPI networks [17,18], in the regulation of many different processes [19,20], and in the development of neurodegenerative diseases and cancer [21–23]. The high biomedical relevance of 14-3-3 complexes has gained much attention during the recent years, and those complexes are considered promising targets for modulation by small molecules in drug discovery and therapy [24,25].

Phosphorylation of client proteins allows members of the 14-3-3 family to ‘read’ this modification. Forming rather stable α-helical homo- and heterodimers from several 14-3-3 isotypes, that are usually named by Greek letters and co-expressed in eukaryotic organisms, 14-3-3 proteins harbor two identical phosphopeptide-binding amphipathic grooves [26], and, therefore, by definition are ditopic. Each subunit of the antiparallel 14-3-3 dimer contains one such phosphopeptide-binding site, where the conserved Lys49, Arg56, Arg127, and Tyr128 residues (human 14-3-3ζ numbering) coordinate the phosphate moiety [8], presenting some filter that selectively accepts phosphorylation. Noteworthy, the same unphosphorylated motifs are usually ignored by 14-3-3, despite the presence of the same positional determinants of binding surrounding the central phosphorylatable Ser/Thr. The optimal consensus 14-3-3-binding motifs derived from peptide libraries emphasize the favorable effect of a positively charged Arg (or Lys) residue upstream of pSer/pThr and a Pro/Gly residue in position +2 relative to pSer/pThr [8]. However, knowledge accumulated in more than 20 years of research says that phosphopeptide sequences with the experimentally confirmed 14-3-3 binding may substantially differ from the consensus motifs I–III, making a sequence-based prediction of binding affinities and even likelihood of binding problematic. This questions the applicability of existing prediction algorithms (e.g. [27,28]) and underlines the remaining importance of an individual experimental approach to 14-3-3/client interactions.

This problem lies deeper because many clients of 14-3-3 proteins bear more than one phosphorylatable 14-3-3-binding site. It was shown that doubly phosphorylated clients bind to 14-3-3 dimers with an increased affinity, exceeding a simple additive effect [8,29] — the occupation of both phosphopeptide-binding grooves of 14-3-3 leads to client stabilization and solidifies the complex. In line with this, the ‘molecular anvil’ and ‘gatekeeper phosphorylation’ hypotheses were put forward [30]. According to the latter, the presence of two binding centers allows stepwise phosphorylation and binding to 14-3-3, whereas the former hypothesis suggests that such a bidentate binding can deform the doubly phosphorylated client on the rather rigid 14-3-3 dimer (an ‘anvil’), thereby regulating client's structure and function [30]. Cdc25B binding to 14-3-3 using one high- and two low-affinity sites limits the access to the catalytic site of this phosphatase and seems to support such a scenario [31]. Further development of the abovementioned ideas led to the insightful suggestion that ditopic 14-3-3 proteins may operate as ‘logic gates’ and ‘coincidence detectors’ that integrate different signaling inputs, i.e. site-specific phosphorylation by different protein kinases, into a timely and weighted output [16]. Indeed, the concerted action of two kinase cascades may be required to trigger the downstream effects mediated by 14-3-3 binding, such as in the case of proapoptotic BAD protein phosphorylation by ERK1/2, PKB/Akt, and PKA [32] or glucose uptake-regulating Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) phosphorylation by PKB/Akt and AMPK protein kinases [33,34]. In such a context, 14-3-3 proteins by right may be considered as readers and translators of the phosphorylation code that is installed by a range of protein kinases (writers) in response to various intra- or extracellular stimuli.

Another aspect of ditopic 14-3-3 protein binding is their so-called scaffolding (or adaptor) function that is fulfilled upon tentative simultaneous binding to two different partners, each containing a 14-3-3-binding phosphosite, whereby 14-3-3 could mediate transient interactions and cross-talk between proteins that do not appreciably interact in the 14-3-3 absence. Although appealing, this hypothesis lacks direct structural confirmations, as ternary complexes formed via a 14-3-3 dimer have not been sufficiently well evidenced and described yet.

Most likely dependent on a particular phosphoprotein, the problem of the stoichiometry of 14-3-3 complexes cannot be neglected. Variations include structurally characterized scenarios when (i) 14-3-3 dimer is bound to some dimer composed of singly phosphorylated polypeptide chains (stoichiometry 2 : 2, such as seen in crystal structures of 14-3-3 with phosphorylated HSPB6 dimer [35] or B-RAF dimer [36]); (ii) 14-3-3 dimer bound to two non-interacting monomers of a client (stoichiometry 1 : 2 : 1, such as seen in the crystal structure of 14-3-3 with serotonin N-acetyltransferase (AANAT) [37]); (iii) 14-3-3 dimer bound to a doubly phosphorylated client (stoichiometry 2 : 1, such as seen in the crystal structure of yeast 14-3-3 with neutral trehalase Nth1 [38], Figure 1). Remarkably, most of these structures have been published in the last 3 years, i.e. the long time after the first structure of the 14-3-3:client complex was published in 2001 [37]. This reflects a great challenge that the community has been facing in studies of 14-3-3 complexes that are enriched with IDPRs and, therefore, difficult for structural biology approaches [12,39,40].

Stoichiometry variations in 14-3-3 complexes.

Figure 1.
Stoichiometry variations in 14-3-3 complexes.

(A) Examples of 2 : 2, 1 : 2 : 1, and 2 : 1 stoichiometries observed in the co-crystal structures of 14-3-3 dimers with client proteins. Protein names are indicated above, PDB codes are presented below. The structures are in scale but shown at different angles for clarity. 14-3-3 dimer is shown in light pink and wheat, client polypeptides are shown by a gradient from blue (N terminus) to red (C terminus), phosphorylated residues are shown by red spheres, binding sites are numbered 1 and 2. Note that due to the C-terminally trimmed construct of AANAT used, which lacks the C-terminal 14-3-3-binding site around Thr205 [37], the observed stoichiometry 1 : 2 : 1 may not be representative of the authentic complex (the expected stoichiometry is 2 : 1). (B) Exemplary binding of the 14-3-3 dimer to a polyvalent protein containing six phosphorylated sites. (C) Schematic showing combinatorics giving different outputs as the result of ditopic 14-3-3 binding (o12, o23, etc. indicate outputs in response to binding at 1 and 2 or 2 and 3 sites, etc.).

Figure 1.
Stoichiometry variations in 14-3-3 complexes.

(A) Examples of 2 : 2, 1 : 2 : 1, and 2 : 1 stoichiometries observed in the co-crystal structures of 14-3-3 dimers with client proteins. Protein names are indicated above, PDB codes are presented below. The structures are in scale but shown at different angles for clarity. 14-3-3 dimer is shown in light pink and wheat, client polypeptides are shown by a gradient from blue (N terminus) to red (C terminus), phosphorylated residues are shown by red spheres, binding sites are numbered 1 and 2. Note that due to the C-terminally trimmed construct of AANAT used, which lacks the C-terminal 14-3-3-binding site around Thr205 [37], the observed stoichiometry 1 : 2 : 1 may not be representative of the authentic complex (the expected stoichiometry is 2 : 1). (B) Exemplary binding of the 14-3-3 dimer to a polyvalent protein containing six phosphorylated sites. (C) Schematic showing combinatorics giving different outputs as the result of ditopic 14-3-3 binding (o12, o23, etc. indicate outputs in response to binding at 1 and 2 or 2 and 3 sites, etc.).

A completely different challenge is presented by binding of principally ditopic 14-3-3 protein to client proteins with multisite phosphorylation, where stoichiometry and binding mechanism is impossible to predict without a careful systematic analysis. These examples include 14-3-3 binding to (i) Tau protein involved in the development of Alzheimer's disease and other tauopathies (at least three to seven 14-3-3-binding sites [41,42]), (ii) cystic fibrosis transmembrane conductance regulator (CFTR) involved in the development of the associated genetic disease (nine 14-3-3-binding sites [43]), and (iii) Parkinson's disease-related LRRK2 (six 14-3-3-binding sites [44]).

Some trivial considerations should be taken into account when studying the interaction of multivalent phosphoproteins with 14-3-3. If the phosphosites are located too close to each other in sequence, they may interfere upon 14-3-3 binding. To contribute to 14-3-3 binding, they have to be separated by at least 15 residues not forming rigid structural elements, otherwise, the antiparallel 14-3-3 dimer can accommodate only one of them. The sites that are too far away from each other, both in sequence and in space (for example, in large multi-domain proteins like ∼2500 amino acid residues-long leucine-rich repeat kinase 2 (LRRK2) [44]), can serve as docking points for different 14-3-3 molecules unless the protein architecture permits sufficient 14-3-3 dimer-mediated deformation. Synergistic binding of three 14-3-3 dimers to the C-terminal tails of the plant plasma membrane H+-ATPase dimers was shown to convert the enzyme into the active hexamer [45,46]. Although somewhat speculative in the absence of direct structural information about 14-3-3 complexes with full-length multivalent proteins, the presence of IDPRs should be especially beneficial for complex formation with large multivalent client proteins composed of several rigid domains. Multivalency may lead to various 14-3-3 binding configurations involving different combinations of binding sites and each combination may provide distinct output. This emphasizes the importance of studies aimed to delineate the binding process that basically decodes the complex multisite phosphorylation signal via 14-3-3.

Multisite phosphorylation indeed creates some complexity that can be interpreted by reader proteins as the phosphorylation code, and this complexity can be expressed in numbers. In the example depicted in Figure 1B, 14-3-3 dimer binding to a hexavalent phosphoprotein generates the 6!/2!*4! = 15 unique combinations of outputs taking into account ditopic binding, plus 6 possible single-site binding events, 21 variants in total. Likewise, for a nonavalent partner, there are 36 unique possible outputs of the ditopic binding, plus 9 single-site binding events (45 in total). Even if some of these variations are not realized due to a low affinity or steric hindrance, the repertoire appears to be pretty rich. Taking into account differences in binding affinity at individual sites, the multivalency creates a very complex hierarchy of the binding events, which complicates the binding mechanism and makes its deciphering an enthralling challenge.

In their recent work, Stevers et al. [44] have systematically studied the 14-3-3 interaction with human LRRK2. This large enzyme contains seven domains and a range of residues, upon phosphorylation of which recruitment of 14-3-3 is achieved. Among 20 PKA phosphorylation sites [47], the six candidate 14-3-3-binding sites (Figure 2) are found at the end of ANK domain (S860), in the long disordered loop connecting ANK and LRR domains (S910, S935, S955, S973), and in the ROC domain (S1444) [44]. The latter site, 1441-RASpSSP-1446, represents motif I 14-3-3-binding site and includes residue Arg1441 that is subject to mutation in Parkinson's disease, which impairs S1444 phosphorylation and 14-3-3 binding [47]. Although being distant from the other five residues, S1444 was earlier shown to be a high-affinity binding site for 14-3-3 (KD ∼ 0.2 µM [47]). As with other 14-3-3 partners, the sites unlikely contribute to 14-3-3 binding unless they are phosphorylated; however, apart from S1444 and S935, the sequences of the rest four tentative 14-3-3-binding sites in LRRK2 are not optimal (Figure 2). Nevertheless, Stevers et al. [44] clearly demonstrated that at least three combinations, involving S910 + S935 (KD ∼ 11 nM), S935 + S1444 (KD ∼ 3 nM), and S910 + S1444 (KD ∼ 6 nM), are among the highest-affinity 14-3-3 binders known to date (Figure 2). Nanomolar affinities were measured by two independent experimental methods, fluorescence polarization and isothermal titration calorimetry, increasing the robustness of the assessment. The authors selected the protein-peptide binding approach and overall used 20 synthetic peptides representing singly and different combinations of doubly phosphorylated peptides to make an insight into the hierarchy of binding events. In addition, crystal structures trapping the 14-3-3 interaction with the S910 (PDB code 5MYC) and S935 (PDB code 5MY9) high-affinity sites were obtained [44]. Although these cannot be fully representative of the 14-3-3/LRRK2 molecular interface, especially given that the S1444 highest-affinity peptide could not be crystallized [44], these atomic resolution structures revealed several remarkable points. First, although binding at a very similar affinity (∼8 µM KD determined by fluorescence polarization [44]), the S910 and S935 peptides show substantially different occupancy and order in the binding grooves of 14-3-3 — in the S935 case, only 4 out of 13 residues are seen in the electron density maps in spite of the relatively high resolution of 1.33 Å. It is especially surprising given that the S935 site is much more similar than the S910 site to the consensus motifs that are thought to be optimal for 14-3-3 binding (Figure 2). This strongly points to the lack of correlation between the peptide sequence, its binding affinity, and the apparent stability of its interface with 14-3-3. Second, it is intriguing that the long peptide co-crystallized with 14-3-3 (VKKKSNSISVGEFYRDAVLQRCSPNLQRHSNSLGPIFD, phosphosites are underlined; PDB code 5MYC), encompassing the two sites S910 + S935 that give a nanomolar affinity to 14-3-3, led to only one site of 14-3-3 occupied (12 residues in the interpretable electron density). The observed peptide conformation features a short α-helix, which is very atypical of the 14-3-3 bound phosphopeptides [44] and raises an interesting question of whether it folds upon 14-3-3 binding or pre-exists in the LRRK2 structure.

Summary of the protein-peptide binding approach used by Stevers et al. [44] to characterize the 14-3-3/LRRK2 interaction.

Figure 2.
Summary of the protein-peptide binding approach used by Stevers et al. [44] to characterize the 14-3-3/LRRK2 interaction.

The picture shows the location of the six phosphorylatable 14-3-3-binding sites in the primary structure of LRRK2, their sequences, and the affinities for the singly or doubly phosphorylated peptides determined by fluorescence polarization (taken from [44]). n.b., not binding. The last line shows the internal sequence motif optimal for 14-3-3 binding. Of note, the bold underlined font marks residues that are seen in the electron density maps in the crystal structures with 14-3-3 (PDB codes 5MYC and 5MY9). In the S910 case, the longer peptide containing the high-affinity sites around S910 and S935 was used for co-crystallization (PDB code 5MYC), which resulted in one site binding (S910), but with two additional residues, YR, observed in the electron density (a gray bold underlined font).

Figure 2.
Summary of the protein-peptide binding approach used by Stevers et al. [44] to characterize the 14-3-3/LRRK2 interaction.

The picture shows the location of the six phosphorylatable 14-3-3-binding sites in the primary structure of LRRK2, their sequences, and the affinities for the singly or doubly phosphorylated peptides determined by fluorescence polarization (taken from [44]). n.b., not binding. The last line shows the internal sequence motif optimal for 14-3-3 binding. Of note, the bold underlined font marks residues that are seen in the electron density maps in the crystal structures with 14-3-3 (PDB codes 5MYC and 5MY9). In the S910 case, the longer peptide containing the high-affinity sites around S910 and S935 was used for co-crystallization (PDB code 5MYC), which resulted in one site binding (S910), but with two additional residues, YR, observed in the electron density (a gray bold underlined font).

The inability of the 14-3-3 complex with the highest-affinity peptide around S1444 to form crystals is also remarkable and hints to potential problems with the solubility of this peptide, as stated by the authors [44]. The set of the mentioned issues (i.e. partial binding occupancy, low peptide solubility, unstable binding stoichiometry even for the high-affinity peptides) is very often encountered in crystallographic studies of 14-3-3/peptide complexes and can to some extent be alleviated by direct tethering of the client peptides to the C terminus of 14-3-3 and co-expression of such fusion proteins in Escherichia coli in the presence of a protein kinase or by in vitro phosphorylation following fusion protein purification [48]. This fortunate opportunity is provided by a natural orientation of phosphopeptides in the binding grooves of 14-3-3, which brings the C-terminal end of 14-3-3 very close to the N-terminal end of the phosphopeptide, and is directly supported by an exotic 14-3-3 structure with the self-bound C-terminal peptide spontaneously phosphorylated upon production in E. coli (PDB code 3EFZ [49]).

For multivalent clients, first binding events inevitably lead to an increase in the effective local concentration of other, yet unbound phosphosites, which can substantially improve the binding efficiency for even low-affinity sites. The great attempt to model the complexity of multivalent phosphoprotein binding to 14-3-3 was undertaken recently by the same research group [50]. Curiously, depending on the set of affinities of the individual binding sites within the polyvalent client, which could only be obtained using the robust systematic approach demonstrated by Stevers et al. for LRRK2 [44] and CFTR [43], the binding regime may be dramatically different. In case of a too large difference in KD for individual sites, only a limited set of those will likely be realized and removal of one of the highest-affinity sites drastically influences the overall affinity (as predicted for LRRK2). For the multiple binding sites with similar affinity, a remarkable buffering may take place, when the low-affinity sites may collectively contribute to the appreciably tight binding and removal of some sites is compensated by the remaining sites, which maintains the overall affinity (as predicted for CFTR) [50]. Importantly, in both CFTR and LRRK2 cases, the stabilization of specific binding sites much more efficiently affects the binding affinity than site-specific inhibition [50].

The focused attention of the community to the issues highlighted above is expected to help elucidate binding mechanisms involving other polyvalent proteins having wide-ranging roles in cellular signaling.

Competing Interests

The author declares that there are no competing interests associated with this manuscript.

Funding

This work was supported by the Russian Science Foundation (grant no. 19-74-10031).

Acknowledgment

I am grateful to Prof. Alfred A. Antson (YSBL, The University of York, U.K.) for critical reading of the manuscript.

Abbreviations

     
  • LRRK2

    Leucine-Rich Repeat Kinase 2

  •  
  • PBMs

    PDZ-binding motifs

  •  
  • IDPRs

    intrinsically disordered protein regions

  •  
  • PPI

    protein–protein interaction

References

References
1
Moore
,
B.W.,
and
Perez
,
V.J.
(
1967
)
Specific Acid Proteins in the Nervous System
,
Prentice-Hall
,
Englewood Cliffs, New Jersey
2
Muslin
,
A.J.
,
Tanner
,
J.W.
,
Allen
,
P.M.
and
Shaw
,
A.S.
(
1996
)
Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine
.
Cell
84
,
889
897
3
Reinhardt
,
H.C.
and
Yaffe
,
M.B.
(
2013
)
Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response
.
Nat. Rev. Mol. Cell. Biol.
14
,
563
580
4
Karlberg
,
T.
,
Hornyak
,
P.
,
Pinto
,
A.F.
,
Milanova
,
S.
,
Ebrahimi
,
M.
,
Lindberg
,
M.
et al (
2018
)
14-3-3 proteins activate pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface
.
Nat. Commun.
9
,
3785
5
Petosa
,
C.
,
Masters
,
S.C.
,
Bankston
,
L.A.
,
Pohl
,
J.
,
Wang
,
B.
,
Fu
,
H.
et al (
1998
)
14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove
.
J. Biol. Chem
273
,
16305
16310
6
Coblitz
,
B.
,
Wu
,
M.
,
Shikano
,
S.
and
Li
,
M.
(
2006
)
C-terminal binding: an expanded repertoire and function of 14-3-3 proteins
.
FEBS Lett.
580
,
1531
1535
7
Rittinger
,
K.
,
Budman
,
J.
,
Xu
,
J.
,
Volinia
,
S.
,
Cantley
,
L.C.
,
Smerdon
,
S.J.
et al (
1999
)
Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding
.
Mol. Cell
4
,
153
166
8
Yaffe
,
M.B.
,
Rittinger
,
K.
,
Volinia
,
S.
,
Caron
,
P.R.
,
Aitken
,
A.
,
Leffers
,
H.
et al (
1997
)
The structural basis for 14-3-3:phosphopeptide binding specificity
.
Cell
91
,
961
971
9
Boon
,
S.S.
and
Banks
,
L.
(
2013
)
High-risk human papillomavirus E6 oncoproteins interact with 14-3-3ζ in a PDZ binding motif-dependent manner
.
J. Virol.
87
,
1586
1595
10
Espejo
,
A.B.
,
Gao
,
G.
,
Black
,
K.
,
Gayatri
,
S.
,
Veland
,
N.
,
Kim
,
J.
et al (
2017
)
PRMT5 C-terminal phosphorylation modulates a 14-3-3/PDZ interaction switch
.
J. Biol. Chem.
292
,
2255
2265
11
Gogl
,
G.
,
Jane
,
P.
,
Caillet-Saguy
,
C.
,
Kostmann
,
C.
,
Bich
,
G.
,
Cousido-Siah
,
A.
et al (
2020
)
Dual specificity PDZ- and 14-3-3-binding motifs: a structural and interactomics study
.
Structure
in press
12
Bustos
,
D.M.
and
Iglesias
,
A.A.
(
2006
)
Intrinsic disorder is a key characteristic in partners that bind 14-3-3 proteins
.
Proteins
63
,
35
42
13
Oldfield
,
C.J.
,
Meng
,
J.
,
Yang
,
J.Y.
,
Yang
,
M.Q.
,
Uversky
,
V.N.
and
Dunker
,
A.K.
(
2008
)
Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners
.
BMC Genomics
9
,
S1
14
Obsil
,
T.
and
Obsilova
,
V.
(
2011
)
Structural basis of 14-3-3 protein functions
.
Semin. Cell. Dev. Biol.
22
,
663
672
15
Douglass
,
J.
,
Gunaratne
,
R.
,
Bradford
,
D.
,
Saeed
,
F.
,
Hoffert
,
J.D.
,
Steinbach
,
P.J.
et al (
2012
)
Identifying protein kinase target preferences using mass spectrometry
.
Am. J. Physiol. Cell. Physiol.
303
,
C715
C727
16
Johnson
,
C.
,
Crowther
,
S.
,
Stafford
,
M.J.
,
Campbell
,
D.G.
,
Toth
,
R.
and
MacKintosh
,
C.
(
2010
)
Bioinformatic and experimental survey of 14-3-3-binding sites
.
Biochem. J.
427
,
69
78
17
Bustos
,
D.M.
(
2012
)
The role of protein disorder in the 14-3-3 interaction network
.
Mol. Biosyst.
8
,
178
184
18
Uhart
,
M.
and
Bustos
,
D.M.
(
2014
)
Protein intrinsic disorder and network connectivity. The case of 14-3-3 proteins
.
Front. Genet.
5,
10
19
Mackintosh
,
C.
(
2004
)
Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes
.
Biochem. J.
381
,
329
342
20
Aitken
,
A.
(
2006
)
14-3-3 proteins: a historic overview
.
Semin. Canc. Biol.
16
,
162
172
21
Wilker
,
E.
and
Yaffe
,
M.B.
(
2004
)
14-3-3 proteins–a focus on cancer and human disease
.
J. Mol. Cell. Cardiol.
37
,
633
642
22
Steinacker
,
P.
,
Aitken
,
A.
and
Otto
,
M.
(
2011
)
14-3-3 proteins in neurodegeneration
.
Semin. Cell. Dev. Biol.
22
,
696
704
23
Freeman
,
A.K.
and
Morrison
,
D.K.
(
2011
)
14-3-3 proteins: diverse functions in cell proliferation and cancer progression
.
Semin. Cell. Dev. Biol.
22
,
681
687
24
Aghazadeh
,
Y.
and
Papadopoulos
,
V.
(
2016
)
The role of the 14-3-3 protein family in health, disease, and drug development
.
Drug Discov. Today
21
,
278
287
25
Stevers
,
L.M.
,
Sijbesma
,
E.
,
Botta
,
M.
,
MacKintosh
,
C.
,
Obsil
,
T.
,
Landrieu
,
I.
et al (
2018
)
Modulators of 14-3-3 protein–protein interactions
.
J. Med. Chem.
61
,
3755
3778
26
Liu
,
D.
,
Bienkowska
,
J.
,
Petosa
,
C.
,
Collier
,
R.J.
,
Fu
,
H.
and
Liddington
,
R.
(
1995
)
Crystal structure of the ζ isoform of the 14-3-3 protein
.
Nature
376
,
191
194
27
Li
,
Z.
,
Tang
,
J.
and
Guo
,
F.
(
2016
)
Identification of 14-3-3 proteins phosphopeptide-binding specificity using an affinity-based computational approach
.
PLoS One
11
,
e0147467
28
Madeira
,
F.
,
Tinti
,
M.
,
Murugesan
,
G.
,
Berrett
,
E.
,
Stafford
,
M.
,
Toth
,
R.
et al (
2015
)
14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides
.
Bioinformatics
31
,
2276
2283
29
Kostelecky
,
B.
,
Saurin
,
A.
,
Purkiss
,
A.
,
Parker
,
P.
and
McDonald
,
N.
(
2009
)
Recognition of an intra-chain tandem 14-3-3 binding site within PKCepsilon
.
EMBO Rep.
10
,
983
989
30
Yaffe
,
M.
(
2002
)
How do 14-3-3 proteins work?– gatekeeper phosphorylation and the molecular anvil hypothesis
.
FEBS Lett.
513
,
53
57
31
Giles
,
N.
,
Forrest
,
A.
and
Gabrielli
,
B.
(
2003
)
14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity
.
J. Biol. Chem.
278
,
28580
28587
32
Lizcano
,
J.M.
,
Morrice
,
N.
and
Cohen
,
P.
(
2000
)
Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155
.
Biochem. J.
349
,
547
557
33
Chen
,
S.
,
Murphy
,
J.
,
Toth
,
R.
,
Campbell
,
D.G.
,
Morrice
,
N.A.
and
Mackintosh
,
C.
(
2008
)
Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators
.
Biochem. J.
409
,
449
459
34
Geraghty
,
K.M.
,
Chen
,
S.
,
Harthill
,
J.E.
,
Ibrahim
,
A.F.
,
Toth
,
R.
,
Morrice
,
N.A.
et al (
2007
)
Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR
.
Biochem. J.
407
,
231
241
35
Sluchanko
,
N.N.
,
Beelen
,
S.
,
Kulikova
,
A.A.
,
Weeks
,
S.D.
,
Antson
,
A.A.
,
Gusev
,
N.B.
et al (
2017
)
Structural basis for the interaction of a human small heat shock protein with the 14-3-3 universal signaling regulator
.
Structure
25
,
305
316
36
Liau
,
N.P.D.
,
Wendorff
,
T.J.
,
Quinn
,
J.G.
,
Steffek
,
M.
,
Phung
,
W.
,
Liu
,
P.
et al (
2020
)
Negative regulation of RAF kinase activity by ATP is overcome by 14-3-3-induced dimerization
.
Nat. Struct. Mol. Biol.
27
,
134
141
37
Obsil
,
T.
,
Ghirlando
,
R.
,
Klein
,
D.C.
,
Ganguly
,
S.
and
Dyda
,
F.
(
2001
)
Crystal structure of the 14-3-3ζ:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme regulation
.
Cell
105
,
257
267
38
Alblova
,
M.
,
Smidova
,
A.
,
Docekal
,
V.
,
Vesely
,
J.
,
Herman
,
P.
,
Obsilova
,
V.
et al (
2017
)
Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1
.
Proc. Natl. Acad. Sci. U.S.A.
114
,
E9811
E9820
39
Sluchanko
,
N.N.
(
2018
)
Association of multiple phosphorylated proteins with the 14-3-3 regulatory hubs: problems and perspectives
.
J. Mol. Biol.
430
,
20
26
40
Sluchanko
,
N.N.
and
Bustos
,
D.M.
(
2019
)
Intrinsic disorder associated with 14-3-3 proteins and their partners
.
Prog. Mol. Biol. Transl. Sci.
166
,
19
61
41
Sluchanko
,
N.N.
,
Seit-Nebi
,
A.S.
and
Gusev
,
N.B.
(
2009
)
Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3ζ
.
FEBS Lett.
583
,
2739
2742
42
Tugaeva
,
K.V.
,
Tsvetkov
,
P.O.
and
Sluchanko
,
N.N.
(
2017
)
Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction
.
PLoS One
12
,
e0178933
43
Stevers
,
L.M.
,
Lam
,
C.V.
,
Leysen
,
S.F.
,
Meijer
,
F.A.
,
van Scheppingen
,
D.S.
,
de Vries
,
R.M.
et al (
2016
)
Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR
.
Proc. Natl. Acad. Sci. U.S.A.
113
,
E1152
E1161
44
Stevers
,
L.M.
,
de Vries
,
R.M.
,
Doveston
,
R.G.
,
Milroy
,
L.G.
,
Brunsveld
,
L.
and
Ottmann
,
C.
(
2017
)
Structural interface between LRRK2 and 14-3-3 protein
.
Biochem. J.
474
,
1273
1287
45
Ottmann
,
C.
,
Marco
,
S.
,
Jaspert
,
N.
,
Marcon
,
C.
,
Schauer
,
N.
,
Weyand
,
M.
et al (
2007
)
Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+ -ATPase by combining X-ray crystallography and electron cryomicroscopy
.
Mol. Cell
25
,
427
440
46
Kanczewska
,
J.
,
Marco
,
S.
,
Vandermeeren
,
C.
,
Maudoux
,
O.
,
Rigaud
,
J.L.
and
Boutry
,
M.
(
2005
)
Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer
.
Proc. Natl. Acad. Sci. U.S.A.
102
,
11675
11680
47
Muda
,
K.
,
Bertinetti
,
D.
,
Gesellchen
,
F.
,
Hermann
,
J.S.
,
von Zweydorf
,
F.
,
Geerlof
,
A.
et al (
2014
)
Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3
.
Proc. Natl. Acad. Sci. U.S.A.
111
,
E34
E43
48
Sluchanko
,
N.N.
,
Tugaeva
,
K.V.
,
Greive
,
S.J.
and
Antson
,
A.A.
(
2017
)
Chimeric 14-3-3 proteins for unraveling interactions with intrinsically disordered partners
.
Sci. Rep.
7
,
12014
49
Brokx
,
S.J.
,
Wernimont
,
A.K.
,
Dong
,
A.
,
Wasney
,
G.A.
,
Lin
,
Y.H.
,
Lew
,
J.
et al (
2011
)
Characterization of 14-3-3 proteins from Cryptosporidium parvum
.
PLoS One
6
,
e14827
50
Stevers
,
L.M.
,
de Vink
,
P.J.
,
Ottmann
,
C.
,
Huskens
,
J.
and
Brunsveld
,
L.
(
2018
)
A thermodynamic model for multivalency in 14-3-3 protein–protein interactions
.
J. Am. Chem. Soc.
140
,
14498
14510