PROTEINAS 14-3-3 e mecanismos

1. Review
The capture of phosphoproteins by
14-3-3 proteins mediates actions
of insulin
Shuai Chen, Silvia Synowsky, Michele Tinti and Carol MacKintosh
MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
How does signalling via PI3K–PKB (AKT)–mTORC1– The discovery that insulin has more widespread influ-
p70S6K and ERK–p90RSK mediate wide-ranging physio- ences than was previously realised implies that complica-
logical responses to insulin? Quantitative proteomics tions of diabetes such as neuropathies, nephropathy, bone
and biochemical experiments are revealing that these disorders and heart disease might not be solely attribut-
signalling pathways induce the phosphorylation of large able to hyperglycaemia, but could be due in part to deregu-
and overlapping sets of proteins, which are then lated insulin action in the damaged tissues [10]. In any
captured by phosphoprotein-binding proteins named case, an understanding of how insulin exerts its diverse
14-3-3 s. The 14-3-3 s are dimers that dock onto dual- effects on different tissues is of enormous practical signifi-
phosphorylated sites in a configuration with special cance given the growing worldwide burden of insulin re-
signalling and mechanical properties. They interact with sistance and type 2 diabetes.
the Rab GTPase-activating proteins AS160 and TBC1D1 The immediate effects of insulin binding to its cognate
to regulate glucose uptake into target tissues in re- tyrosine kinase receptor are relatively well understood.
sponse to insulin and energy stress. Dynamic patterns Phosphatidyinositol 3-kinase (PI3K)–protein kinase B
in the 14-3-3-binding phosphoproteome are providing (PKB, also known as AKT)– mammalian target of rapa-
new insights into how insulin triggers coherent shifts in mycin complex 1 (mTORC1)–p70 ribosomal S6 kinase
metabolism that are integrated with other cellular re- (p70S6K) signalling is activated, and in some cell types
sponse systems. insulin also activates the Ras–Raf– extracellular signal-
regulated kinase (ERK)–p90 ribosomal S6 kinase
Established and emerging actions of insulin (p90RSK) pathway [11,12]. However, too few downstream
Insulin is best known for suppressing blood glucose levels targets are known to explain how these core signalling
by driving glucose from the bloodstream into muscle and pathways stimulate diverse physiological responses to
liver glycogen and into fat in adipose tissue, and for insulin. Here, we review the roles of a family of phospho-
inhibiting glucose production from the liver. However, protein-binding proteins named 14-3-3 s in mediating in-
these actions are only part of an orchestrated shift in global sulin responses. We outline how 14-3-3 proteins work and
metabolism induced by insulin action. Nutrients and ions focus on contributions of 14-3-3 s to key regulatory steps in
surge into our bloodstream after a meal, and insulin directs insulin-regulated glucose homeostasis. Recent develop-
the assimilation of glucose, metal ions, amino acids and ments in 14-3-3 affinity capture and proteomics technolo-
lipids according to the demands of specialized tissues in gies are also highlighted, which show promise as a way to
ways that ensure whole-body homeostasis, electrochemical identify new intracellular targets and help to explain the
balance of ions across membranes, and stoichiometric wider actions of insulin.
balance in fluxes through metabolic pathways.
Although the main targets for insulin-regulated glucose 14-3-3 dimers dock onto specific pairs of
homeostasis are muscle, fat and liver, insulin also prepares phosphorylated sites
other organs in their response to food. For example, it was The 14-3-3 s dock onto and regulate hundreds of phospho-
recently discovered that insulin induces cytoskeletal remo- proteins inside eukaryotic cells, including proteins that
delling in kidney glomerular podocytes [1] and thus readies are deregulated in diabetes, cancer and neurological dis-
the kidney for increased urine filtration following a meal. orders (Figure 1). Mammals have seven 14-3-3 genes
Insulin influences vascular endothelium in ways that may (gamma, epsilon, beta, zeta, sigma, theta, tau), and their
affect tissue blood flow, and regulates fuel use and cell peculiar name refers to the discovery of the protein iso-
survival in the heart [2–5]. Insulin signalling to the hypo- forms as a cluster of spots in position 14-3-3 on an early
thalamus, pituitary, bone, pancreas and reproductive sys- two-dimensional starch gel separation of bovine brain
tems is also being recognised as critical to the whole-body extract (Box 1) [13]. The numbers 14-3-3 have no function-
distribution of resources [6–9]. al significance; perhaps a more meaningful number for
these proteins is ‘two’, for they are dimers that bind two
phosphorylated residues. Often a 14-3-3 dimer docks onto
Corresponding author: MacKintosh, C. (c.mackintosh@dundee.ac.uk). a pair of tandem phosphorylated sites on a target protein,
1043-2760/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2011.07.005 Trends in Endocrinology and Metabolism, November 2011, Vol. 22, No. 11 429

2. Review Trends in Endocrinology and Metabolism November 2011, Vol. 22, No. 11
ITB2
Metabolic Cardiovascular
MEFV
IGF1R GP1BA IRS1 CDN1B
TY3H
Psychiatric/behaviour Tumorigenesis
P53
MDM2
TPH2 TAU CHK1 CTNB1
LRRK2 ACHA4 PRLR AKP13
COF1 SLIK1 GLI2 BRAF
ABL1
TRENDS in Endocrinology & Metabolism
Figure 1. The current 14-3-3 diseasome encompasses tumourigenesis, cardiovascular, metabolic and neurological disorders. Some 480 human disease gene sets described
in the Genetic Association Database were recently mapped to indicate the degree of gene sharing among diseases [68]. We extracted from this disease map the gold
standard proteins with defined 14-3-3-binding phosphorylation sites [15,69]. The 14-3-3 binders map predominantly in four disease classes: tumourigenesis (11 proteins:
P53, BRAF, MDM2, CDN1B, AKP13, IRS1, PRLR, CTNB1, CHK1, ABL1, GLI2), cardiovascular (6 proteins: GP1BA, TY3H, ITB2, MEFV, IRS1, CDN1B), metabolic (5 proteins:
IRS1, GP1BA, TY3H, IGF1R, MEFV) and psychiatric or behaviour (11 proteins: TAU, IGF1R, GP1BA, P53, LRRK2, TY3H, ACHA4, MEFV, COF1, SLIK1, TPH2). The protein
overlap between these classes is illustrated here by Visant graphing (http://visant.bu.edu). This map is just a starting point, with indications that the concentration of 14-3-3-
binding proteins in the tumourigenesis, cardiovascular, metabolic and neurological disease classes will increase. For example, AS160 and TBC1D1, mutations of which are
found in subtypes of diabetes and obesity, respectively [56,61,62], were not in the Genetic Association Database when the disease map was prepared [68]. Moreover, further
proteins linked to these four disease classes are among those that exhibit affinity for 14-3-3 s in high-throughput proteomics studies; these await biochemical studies to
define the specificity of their interactions with 14-3-3 [23,24,26,27].
which is a configuration that confers interesting signalling promoter (BAD) for which the 14-3-3 has been proposed as
and mechanical properties [14–16]. an OR gate because phosphorylation of either site is suffi-
The act of docking onto two phosphorylated residues cient to recruit 14-3-3, which in turn promotes cell survival
turns a 14-3-3 dimer into a type of biochemical microcon- by preventing BAD binding to the anti-apoptotic BCL-XL
troller called a logic gate (Figure 2a). Logic gates are [17]. The 14-3-3 would be an AND gate if both sites must be
fundamental components of digital electronic circuits, phosphorylated, and by different kinases, for a 14-3-3 to
where they perform OR and AND operations on two or bind and exert its action. Such devices would filter out
more inputs to produce a single output. The concept is signalling noise because only the correct combination of
useful because an increasing number of proteins are being inputs would trigger an output. In biological systems, the
identified in which two tandem phosphorylated 14-3-3- digital logic is likely to be more ‘fuzzy’ than in electronic
binding sites can be phosphorylated by distinct protein circuits, because the phosphorylation and binding kinetics
kinases [15]. One is the BCL-XL/BCL2-associated death often differ for the two 14-3-3 interaction sites. Later in this
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3. Review Trends in Endocrinology and Metabolism November 2011, Vol. 22, No. 11
(a) (b)
Mechanical action
P P P P P P
Lever Mask Adapter
P P
‘AND’ ‘OR’
Hypothetical
Kinase 2 Kinase 1 P P clip function
Insulin & growth factors
(c) Proliferative
stimuli
Akt/PKB
Pims PMA,
PKC platelet activators,
etc
PKA
Various stimuli
AMPK in different cell types
Energy
deprivation
Ca2+
TRENDS in Endocrinology & Metabolism
Figure 2. 14-3-3 dimers are logic gates and mechanical devices that dock onto two CAMK- and AGC-phosphorylated sites. (a) Analogy between a 14-3-3 dimer and digital
logic gates. Digital AND and OR gates, which integrate two inputs into a single output, are depicted on the right. The left-hand side shows how the act of docking onto two
sites, which may be phosphorylated by different kinases, turns the 14-3-3 dimer into a digital logic gate whose output is a mechanical action. (b) The 14-3-3 s are dimeric
regulatory proteins that dock onto two phosphorylated residues, which are usually on the same target protein. Mechanically, this means that 14-3-3 can act as levers to force
a conformational change in a target, can mask a functional domain within the central groove, and can potentially clip back flanking regions to present a functional domain
for external interactions. When the two phosphorylated 14-3-3-binding sites are on different proteins, the 14-3-3 dimer would be an adapter that stabilises the interaction
between the two targets. 14-3-3 s are remarkable for having hundreds of phosphoprotein partners. (c) Phosphorylated 14-3-3-binding sites are created by CAMK and AGC
kinases. The frequency plot at the bottom was generated by aligning the sequences around more than 200 14-3-3-binding sites, centred on the phosphorylated 14-3-3-
binding residue, which can be serine or threonine [15]. Most 14-3-3-binding sites have arginine somewhere in the -3 to -5 positions relative to the phosphorylated site, and
just under half have a proline at +2. Such sites are generally phosphorylated by basophilic kinases in the AGC and CAMK sections of the human kinome, including central
mediators of insulin and nutrient signaling.
review we consider cases for which fuzzy 14-3-3 AND gates conformational change in the target. For example, a 14-3-3
might integrate insulin signalling with other cellular dimer alters the conformations of phosphorylated tyrosine
signals. hydroxylase and the GTPase-activating protein RGS3 to
In electronic circuits, the output of a logic gate is an activate these enzymes [19,20]. In other cases, 14-3-3
electrical impulse. In intracellular circuits, a 14-3-3 dock- dimers mask a subcellular targeting sequence or a func-
ing event triggers a mechanical action [18]. The two phos- tional domain. For example 14-3-3 binds to PKB-phosphor-
phorylated 14-3-3-binding sites on target proteins are ylated FOXO4, which shields the DNA binding interface
generally located within disordered regions and/or strad- of this transcription factor and also interferes with its
dle a functional domain [15]. Their positions can give clues nuclear localization [21]. In this way, 14-3-3 inhibits tran-
about how 14-3-3 dimers modulate the conformations and scriptional activation when FOXO4 is phosphorylated in
interactions of their targets (Figure 2b). 14-3-3 s can act as response to insulin and other stimuli. In principle, a 14-3-3
levers, whereby one site acts as a fulcrum for the 14-3-3 dimer could clip back flanking regions to better present a
dimer to exert a force on the other site, which causes a functional domain for external interactions, but there are
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4. Review Trends in Endocrinology and Metabolism November 2011, Vol. 22, No. 11
Box 1. 14-3-3 proteins nutrients. Their identification is aided by recent develop-
ments in high-resolution mass spectrometry and quantita-
14-3-3 s are phosphoprotein-binding proteins with many regula- tive proteomics; thus, proteins within complex mixtures
tory roles in all eukaryotes.
Dimers of curved L-shaped monomers (30 kDa) form a central
can be identified and quantified, and their post-transla-
groove that docks onto two phosphorylated sites on targets. tional modification status can be analysed. For example,
AGC and CAMK kinases phosphorylate many of the known 14-3-3- 14-3-3 affinity capture can be used to isolate 14-3-3-binding
binding sites. proteins from lysates of two or three sets of cells exposed to
The two phosphorylated docking sites must be spatially compa-
˚
different agonists and/or inhibitors. Proteins that bind
tible with fitting into either side of the central groove (35 A
across).
specifically to the immobilised 14-3-3 s can be released
Different eukaryotic lineages have evolved sets of 14-3-3 isoforms by competitive elution with a synthetic 14-3-3-binding
(e.g. gamma, epsilon, beta, zeta, sigma, theta and tau in phosphopeptide. After digestion of the proteins, the pep-
mammals). tides are reacted with formaldehyde and borohydride to
All 14-3-3 isoforms share a similar mode of action, but may differ
dimethylate every primary amine, with the chemicals for
in their ability to form hetero- and homodimers, in affinities for
different targets, in expression patterns and in their post- each preparation labelled with different stable isotopes to
translational regulation. generate a ‘light’, ‘intermediate’ or ‘heavy’ dimethyl moie-
ty. Mass spectrometry is then used to identify the sequence
of the peptides, and to determine the ratio of the light,
no compelling examples of this. A 14-3-3 dimer may also act intermediate and heavy forms of each peptide. In this way,
as an adapter linking two phosphorylated proteins, and a proteins whose peptides are more abundant in, for exam-
convincing case of this mode of action is the stabilisation ple, the 14-3-3-captured sample from insulin-stimulated
and activation of the hexomeric plasma membrane proton than from unstimulated cells or inhibitor-treated cells can
ATPase in plants by phosphorylation-dependent interac- be identified [24,25]. Because the dimethyl labelling step is
tions with three 14-3-3 dimers [22]. carried out after proteins are proteolytically digested, this
method has potential to identify insulin-regulated proteins
AGC and CAMK protein kinases phosphorylate many isolated from their physiological target tissues. Another
14-3-3-binding sites strategy uses a similar principle, except that the differen-
14-3-3 s interact with many phosphoproteins [23] and pro- tial isotope label is introduced into proteins by metabolic
vide a common mechanism for linking signalling pathways incorporation of ‘light’, ‘intermediate’ or ‘heavy’ stable
to cell metabolism, growth, proliferation and morphology. isotopically labelled amino acids that are fed to cells in
With so many targets and over 500 human protein kinases, culture (SILAC) [26,27]. Other quantitation methods, such
it seems daunting to sort out which kinase phosphorylates as differential isobaric tag for relative and absolute quan-
which 14-3-3-binding sites and when. Fortunately, the task titation (iTRAQ) labelling of protein and peptide samples
is simplified because much of the kinome can be disregarded: and even label-free methods, are also possible, and each
14-3-3 s do not bind to phosphorylated tyrosines, nor gener- method has its advantages and disadvantages [27,28].
ally to phosphorylated serines and threonines that are
followed by proline residues. This means that neither 14-3-3 phosphoproteomics data indicate new regulatory
tyrosine kinases nor proline-directed enzymes, such as mechanisms for insulin
mitogen-activated protein (MAP) kinases and cyclin-depen- The first experiments using 14-3-3-phosphoproteomics
dent protein kinases, create docking sites for 14-3-3 s. Mode technology have identified many proteins that bind to
I RXX(pS/pT)XP and mode II RX(F/Y)X(pS)XP sequence 14-3-3 s in response to signalling pathways that are acti-
motifs showed optimal binding to 14-3-3 s in a screen of a vated by insulin [24,26,27]. The insulin-regulated 14-3-3-
phosphopeptide library [16]. In a recent survey of defined binding sites have been verified for fewer proteins. Some of
14-3-3-binding sites in mammalian proteins, mode I these proteins have poorly defined functions, including
RXX(pS/pT)XP motifs dominate, although the +2 proline coiled-coil domain protein 6 (CCDC6), the E3 ubiquitin
residue occurs in less than half (Figure 2c). Interestingly, ligase zinc-finger, RING-finger 2 (ZNRF2), and the sterile
these 14-3-3-binding sequences overlap with the specifici- alpha motif (SAM) and SRC homology 3 (SH3) domain
ties of the protein kinase A/protein kinase G/protein kinase protein SASH1 [24]. Intriguingly, it was found that a
C (AGC) group and Ca2+/calmodulin protein kinase (CAMK) single-nucleotide polymorphism in the SASH1 gene is
group of protein kinases [15], which exhibit individual pre- associated with diabetic nephropathy genes in African
ferences for phosphorylating motifs with basic residues in Americans in a genome-wide study, so study of this protein
the -3 to -5 positions relative to the phosphorylated residue. may reveal new disease mechanisms [10]. Other insulin-
These basophilic kinases include proviral integration of regulated 14-3-3-binding proteins provide more immediate
MMLV (PIM) kinases, protein kinase C (PKC) isoforms, insights into the known actions of the hormone. These
the energy-sensing AMP-activated protein kinase (AMPK), include proteins that regulate insulin-stimulated glucose
and the insulin-activated PKB, SGK, p70S6K and p90RSKs uptake into tissues, namely the Rab GTPase-activating
(Figure 2c). proteins AS160 (TBC1D4) and TBC1D1; the myosin
MYO1C; the cardiac glycolytic activator PFKFB2; the
14-3-3 capture and release combined with differential transcriptional regulator and E3 SUMO-protein ligase
proteomics PIAS2 (Miz1); transcription factors FOXO1, FOXO3,
There are many proteins whose binding to 14-3-3 s is FOXO4 and capicua; ECD3, which is involved in decapping
dynamically regulated by insulin, growth factors and mRNA as a step in its degradation; cell cycle and apoptotic
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5. Review Trends in Endocrinology and Metabolism November 2011, Vol. 22, No. 11
control proteins including CDKN1B (p27, Kip1), beta-cate- glucose homeostasis in individuals with insulin resistance
nin, BAD and BIM; and components of the insulin signal- [50].
ling PI3K–PKB–mTOR pathway itself, namely IRS2 GLUT4 trafficking requires active GTP-bound Rab pro-
(insulin receptor substrate 2), PRAS40 (AKT1S1) and teins. AS160 and the closely related Rab-GAP, TBC1D1,
rictor [3,17,21,24,26,27,29–40]. Overall, a network of 14- promote GTP hydrolysis by Rabs. It has been proposed that
3-3–phosphoprotein interactions is emerging that is pro- these Rab-GAPs are somehow inactivated by phosphoryla-
viding mechanistic insights into how insulin stimulates tion via insulin and AMPK signalling, which allows the
glucose assimilation, activates cardiac glycolysis, and reg- relevant Rabs to be loaded with GTP to facilitate GLUT4
ulates transcription, protein synthesis, feedback control of trafficking [51–54]. AS160 and TBC1D1 have similar do-
insulin signalling, cytoskeletal dynamics and other intra- main architectures, and both proteins have two 14-3-3-
cellular events. binding sites that are located within clusters of multisite
It should be emphasised that none of the above-men- phosphorylation in a part of the protein that also has so-
tioned proteins are regulated solely by insulin. Insulin called PTB domains (Figure 3). Beyond this superficial
signalling and other pathways converge on several 14-3- similarity, however, some key regulatory details for
3-binding sites, and some of these proteins are also modu- AS160 and TBC1D1 are distinct.
lated by energy and nutrient levels [41]. An intriguing idea AS160 is named for its discovery as an AKT (PKB)
is that 14-3-3 dimers as fuzzy AND logic gates might substrate, and the effects of overexpression of a nonpho-
integrate insulin signals with other inputs. For example, sphorylatable mutant [AS160(4P)] first implicated this
there is an interplay between insulin and amino acids in protein in GLUT4 trafficking [53,55]. Furthermore, a re-
stimulating phosphorylation of the 14-3-3-binding site(s) cent genetic analysis identified patients with severe insu-
on PRAS40 [42,43]. In addition, 14-3-3 interacts with the lin resistance during puberty who carry a premature stop
cardiac PFKFB2 via a high-affinity PKB-phosphorylated mutation in one allele of AS160 [56], which underscores the
site and a second site that can be phosphorylated by both need to define precisely how AS160 regulates glucose
PKB and AMPK, which is activated when the energy or homeostasis. Insulin stimulates phosphorylation of several
ATP status of cells is low, for example during ischaemia sites of AS160 and induces its binding to 14-3-3 s mainly
[3,44]. Insulin enhances the ability of cardiomyocytes to through PKB-phosphorylated Thr642, with support from
survive ischaemia, and a functional role for 14-3-3 is phospho-Ser341, which can be phosphorylated by p90RSK
indicated by the finding that the PKB-mediated increases and PKB [29,37,53]. A form of AS160 that binds 14-3-3
in fructose-2-6-bisphosphate levels and glycolysis are constitutively, generated by genetic mutation, could over-
prevented by a cell-permeable 14-3-3-binding phosphopep- ride the inhibitory effect of AS160(4P) on GLUT4 translo-
tide in cells containing cardiac PFKFB2 [3]. cation in adipocytes [29,37,53]. In addition, insulin-
Another key physiological process in which insulin sig- stimulated fusion of GLUT4 vesicles with the plasma
nalling is integrated with other inputs is the uptake of membrane was prevented by blocking the AS160–14-3-3
glucose into muscles [45]. The goal of improving glucose interaction in cell-free assays, which further indicates a
control in individuals means that the underlying mecha- functional role for 14-3-3 in this process [57]. It was ini-
nisms are under intense research, and the next section tially proposed that Thr642 on AS160 was a convergence
outlines the contributions of two 14-3-3-binding proteins, point for phosphorylation by both PKB and AMPK [58].
the related Rab GTPase activating proteins (Rab-GAPs) However, in vitro and cell culture studies suggest that
AS160 (TBC1D4) and TBC1D1. Both proteins are regulat- whereas AMPK may phosphorylate other residues, AMPK
ed by multiple phosphorylations [46,47] and details of does not seem to be a physiological Thr642 kinase [29,59].
relevant kinases and the roles of some sites are still Therefore, 14-3-3 binds to phospho-Thr642 and phospho-
controversial, a situation that is being resolved with im- Ser341 in response to insulin and growth factors, and not
proved reagents for phosphosite identification and quanti- AMPK activators (Figure 3).
fication. Readers should therefore note that we do not In contrast to AS160, AMPK has a clear role in mediat-
attempt to be comprehensive here, but present a personal ing 14-3-3 binding to the related TBC1D1 [60]. TBC1D1
14-3-3-centred perspective with a testable hypothesis. was originally identified as a candidate gene underlying
severe familial female obesity [61] and its natural deletion
Differential regulation of AS160 and TBC1D1 in mice resulted in leanness and protection against diet-
Insulin promotes glucose uptake into muscles and fat induced obesity [62]. TBC1D1 and AS160 share a similar
through GLUT4 glucose transporters [48]. In unstimulated substrate preference towards Rabs, at least in vitro [63],
cells, GLUT4 is localised to internal storage vesicles that and overexpression of a form of TBC1D1 mutated at three
must fuse with the plasma membrane before glucose can of its phosphorylation sites interfered with GLUT4 trans-
flow into cells. GLUT4 trafficking to the plasma membrane location in 3T3-L1 adipocytes [64].
is stimulated by insulin signalling mainly via the PI3K– In cultured L6 myotubes and intact skeletal muscles,
PKB pathway, and impairment of this process is an early whereas AS160 is phosphorylated on Ser341 and Thr642
sign of insulin resistance and type 2 diabetes [46,48]. In and binds to 14-3-3 s in response to insulin, TBC1D1 is
skeletal and cardiac muscle, GLUT4 trafficking is also phosphorylated on Ser237 and binds to 14-3-3 s in response
activated by contraction, which activates protein kinases, to acute exercise [65] and pharmacological AMPK activa-
including AMPK, which is activated by lowering ATP tors [60,66]. Insulin and growth factors also stimulate the
levels [49]. There is considerable interest in understanding phosphorylation of a second 14-3-3-binding site at Thr596
how exercise, as well as drugs that activate AMPK, restore of TBC1D1, which is similar to Thr642 of AS160; thus,
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AMPK activators Insulin
AMPK PKB PKB/p90RSK PKB
14-3-3 dimer 14-3-3 dimer
S237 T596 S341 T642
S585? S588
S566 S570 S666
S565
S235 S263 S507 S318 T568 S751
CBD
PTB PTB
CBD
Rab GAP PTB PTB
TBC1D1 1 2 1 2 Rab GAP
AS160
1 96 153 301 373 721 800 994 1168 1 121 180 367 439 832 918 1121 1299
757 876
R125W
R363X 55aa insert
GLUT4 trafficking
Glucose homeostasis
TRENDS in Endocrinology Metabolism
Figure 3. Hypothesis of complementary regulation of glucose homeostasis by AS160 and TBC1D1 in skeletal muscles. Domains, motifs and disease-associated mutations
(the latter in red) are shown in bar diagrams of AS160 and TBC1D1 (see the text for sources). PTB1 and PTB2 are phosphotyrosine interaction domains (by sequence,
although their interaction partners have not been reported); CBD is a calcium-regulated region; and Rab GAP is the Rab GTPase-activating domain. In the hypothesis shown
here, AMPK controls GLUT4 trafficking through Ser237 phosphorylation and consequent docking of 14-3-3 onto the TBC1D1 protein, whereas the insulin–PKB (AKT)
pathway regulates surface translocation of GLUT4 via Thr642 phosphorylation and consequent 14-3-3 binding of the AS160 protein. This hypothesis does not take into
account that both TBC1D1 and AS160 also receive other regulatory inputs via the other phosphorylations that are clustered close to the 14-3-3-binding sites on these
proteins. Phosphorylated residues are indicated by numbers above the bar diagrams, with the 14-3-3-binding sites in yellow. R125W is an obesity-linked mutation of
TBC1D1 [61], and R363W is a diabetes-linked mutation of AS160 [56].
TBC1D1 seems to be a dual-input 14-3-3 logic gate homeostasis, including glucose intolerance and reduced
(Figure 3). The functional significance of this molecular insulin sensitivity. Despite elevated total GLUT4 protein
arrangement is not yet clear, however, and, at least in levels in skeletal muscles and fat tissues of the knock-ins,
muscles, phosphorylation of Thr596 alone is insufficient for GLUT4 is not efficiently localised to the cell surface, which
14-3-3 binding [60,65]. results in a reduced glucose uptake rate in muscle in re-
sponse to insulin [67]. Together, these findings confirm that
Complementary roles of AS160 and TBC1D1 for insulin insulin-stimulated Thr649 phosphorylation and 14-3-3
and AMPK activators binding to AS160 in muscle has a key role in glucose
We propose that 14-3-3 binding to AS160 regulates glucose homeostasis.
homeostasis in response to insulin, whereas 14-3-3 binding Further experiments are needed to address the com-
to TBC1D1 regulates this process in response to AMPK- plexity of the glucose uptake process, including genetic
activating stimuli (Figure 3). To test this hypothesis of tests of the roles of the AMPK and insulin-regulated 14-3-
complementary roles for AS160–14-3-3 and TBC1D1–14-3- 3-binding sites on TBC1D1. For wider health implications,
3 complexes in GLUT4 regulation, mice with knock-in we need to know if the knock-in mice display altered
mutations in the 14-3-3-binding phosphorylation sites in physiological responses to high-fat diets and ageing. Will
AS160 and TBC1D1 must be generated. Unlike studies the phenotype of TBC1D1 mice provide an insight into the
using ectopic overexpression, introduction of a point mu- obesity of individuals with mutations or polymorphisms in
tation of a phospho-Ser/Thr residue to a nonphosphoryla- this protein or gene? Would genetic crosses between the
table residue using knock-in genetic technology generally knock-in AS160 and TBC1D1 mice and ‘diabetes loci’
results in expression of mutant proteins at endogenous reveal synergism with respect to insulin resistance?
levels [67] (provided that mutations do not cause unexpect- Answers to such questions would help to determine wheth-
ed outcomes such as destabilisation of the protein), without er it would be useful to devise drugs that mimic the effects
exerting dominant-negative effects or competition from of insulin and exercise on AS160 and TBC1D1 as means to
wild-type proteins. improving blood glucose management.
First, a mouse model with Thr649 on AS160 (equivalent
to Thr642 on human AS160) substituted by alanine The future: 14-3-3-phosphoproteome barcodes
was generated to prevent insulin-stimulated binding to More general lessons may also be learned from this narrow
14-3-3 in vivo [67]. The most striking phenotype of the focus on AS160 and TBC1D1. Earlier in this review, we
AS160-Thr649Ala knock-in mice is their altered glucose alluded to the possibility of using 14-3-3 capture and
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phosphoproteomics to identify the commonalities and dif- 19 Obsilova, V. et al. (2008) The 14-3-3 protein affects the conformation of
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Another exciting issue would be the potential of developing 20 Rezabkova, L. et al. (2010) 14-3-3 protein interacts with and affects the
these technologies to track which signalling pathways are structure of RGS domain of regulator of G protein signaling 3 (RGS3).
activated in cells and tissues under normal conditions and J. Struct. Biol. 170, 451–461
in response to therapeutic drugs. As exemplified by the 21 Silhan, J. et al. (2009) 14-3-3 protein masks the DNA binding interface
of forkhead transcription factor FOXO4. J. Biol. Chem. 284, 19349–
contrast between AS160 and TBC1D1, each protein has its
19360
own signalling signature, meaning its own pattern of 22 Ottmann, C. et al. (2007) Structure of a 14-3-3 coordinated hexamer of
responsiveness to one, two or many signalling pathways. the plant plasma membrane H+-ATPase by combining X-ray
In turn, this means that insulin, growth factors and other crystallography and electron cryomicroscopy. Mol. Cell 25, 427–440
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