Proteinas 14 3-3 e insulina

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PROTEINAS 14-3-3 e mecanismos

PROTEINAS 14-3-3 e mecanismos

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  • 1. ReviewThe capture of phosphoproteins by14-3-3 proteins mediates actionsof insulinShuai Chen, Silvia Synowsky, Michele Tinti and Carol MacKintoshMRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UKHow 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, boneand 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 anycaptured by phosphoprotein-binding proteins named case, an understanding of how insulin exerts its diverse14-3-3 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 cognateto 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 Bin 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 kinasemetabolism that are integrated with other cellular re- (p70S6K) signalling is activated, and in some cell typessponse systems. insulin also activates the Ras–Raf– extracellular signal- regulated kinase (ERK)–p90 ribosomal S6 kinaseEstablished and emerging actions of insulin (p90RSK) pathway [11,12]. However, too few downstreamInsulin is best known for suppressing blood glucose levels targets are known to explain how these core signallingby driving glucose from the bloodstream into muscle and pathways stimulate diverse physiological responses toliver 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 andmetabolism induced by insulin action. Nutrients and ions focus on contributions of 14-3-3 s to key regulatory steps insurge 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 toways that ensure whole-body homeostasis, electrochemical identify new intracellular targets and help to explain thebalance 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 ofhomeostasis are muscle, fat and liver, insulin also prepares phosphorylated sitesother 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 thatdelling 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 genesInsulin influences vascular endothelium in ways that may (gamma, epsilon, beta, zeta, sigma, theta, tau), and theiraffect 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 earlythalamus, pituitary, bone, pancreas and reproductive sys- two-dimensional starch gel separation of bovine braintems 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 & MetabolismFigure 1. The current 14-3-3 diseasome encompasses tumourigenesis, cardiovascular, metabolic and neurological disorders. Some 480 human disease gene sets describedin the Genetic Association Database were recently mapped to indicate the degree of gene sharing among diseases [68]. We extracted from this disease map the goldstandard 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 proteinoverlap 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 arefound 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, furtherproteins 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 todefine 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 asand 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 survivalturns a 14-3-3 dimer into a type of biochemical microcon- by preventing BAD binding to the anti-apoptotic BCL-XLtroller called a logic gate (Figure 2a). Logic gates are [17]. The 14-3-3 would be an AND gate if both sites must befundamental components of digital electronic circuits, phosphorylated, and by different kinases, for a 14-3-3 towhere they perform OR and AND operations on two or bind and exert its action. Such devices would filter outmore inputs to produce a single output. The concept is signalling noise because only the correct combination ofuseful because an increasing number of proteins are being inputs would trigger an output. In biological systems, theidentified in which two tandem phosphorylated 14-3-3- digital logic is likely to be more ‘fuzzy’ than in electronicbinding sites can be phosphorylated by distinct protein circuits, because the phosphorylation and binding kineticskinases [15]. One is the BCL-XL/BCL2-associated death often differ for the two 14-3-3 interaction sites. Later in this430
  • 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 & MetabolismFigure 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 digitallogic 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 twosites, 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 dimericregulatory 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 forcea 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 domainfor 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 interactionbetween 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 AGCkinases. 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, andjust 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 centralmediators 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 14-3-3might integrate insulin signalling with other cellular dimer alters the conformations of phosphorylated tyrosinesignals. 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, 14-3-3electrical 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 interfacegenerally located within disordered regions and/or strad- of this transcription factor and also interferes with itsdle 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 ininteractions of their targets (Figure 2b). 14-3-3 s can act as response to insulin and other stimuli. In principle, a 14-3-3levers, whereby one site acts as a fulcrum for the 14-3-3 dimer could clip back flanking regions to better present adimer to exert a force on the other site, which causes a functional domain for external interactions, but there are 431
  • 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-stimulatedand activation of the hexomeric plasma membrane proton than from unstimulated cells or inhibitor-treated cells canATPase in plants by phosphorylation-dependent interac- be identified [24,25]. Because the dimethyl labelling step istions with three 14-3-3 dimers [22]. carried out after proteins are proteolytically digested, this method has potential to identify insulin-regulated proteinsAGC and CAMK protein kinases phosphorylate many isolated from their physiological target tissues. Another14-3-3-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 metabolicvide a common mechanism for linking signalling pathways incorporation of ‘light’, ‘intermediate’ or ‘heavy’ stableto cell metabolism, growth, proliferation and morphology. isotopically labelled amino acids that are fed to cells inWith so many targets and over 500 human protein kinases, culture (SILAC) [26,27]. Other quantitation methods, suchit 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 samplesis simplified because much of the kinome can be disregarded: and even label-free methods, are also possible, and each14-3-3 s do not bind to phosphorylated tyrosines, nor gener- method has its advantages and disadvantages [27,28].ally to phosphorylated serines and threonines that arefollowed by proline residues. This means that neither 14-3-3 phosphoproteomics data indicate new regulatorytyrosine kinases nor proline-directed enzymes, such as mechanisms for insulinmitogen-activated protein (MAP) kinases and cyclin-depen- The first experiments using 14-3-3-phosphoproteomicsdent protein kinases, create docking sites for 14-3-3 s. Mode technology have identified many proteins that bind toI 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 of14-3-3-binding sites in mammalian proteins, mode I these proteins have poorly defined functions, includingRXX(pS/pT)XP motifs dominate, although the +2 proline coiled-coil domain protein 6 (CCDC6), the E3 ubiquitinresidue occurs in less than half (Figure 2c). Interestingly, ligase zinc-finger, RING-finger 2 (ZNRF2), and the sterilethese 14-3-3-binding sequences overlap with the specifici- alpha motif (SAM) and SRC homology 3 (SH3) domainties of the protein kinase A/protein kinase G/protein kinase protein SASH1 [24]. Intriguingly, it was found that aC (AGC) group and Ca2+/calmodulin protein kinase (CAMK) single-nucleotide polymorphism in the SASH1 gene isgroup of protein kinases [15], which exhibit individual pre- associated with diabetic nephropathy genes in Africanferences for phosphorylating motifs with basic residues in Americans in a genome-wide study, so study of this proteinthe -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 immediateMMLV (PIM) kinases, protein kinase C (PKC) isoforms, insights into the known actions of the hormone. Thesethe energy-sensing AMP-activated protein kinase (AMPK), include proteins that regulate insulin-stimulated glucoseand 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; the14-3-3 capture and release combined with differential transcriptional regulator and E3 SUMO-protein ligaseproteomics 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 decappingdynamically regulated by insulin, growth factors and mRNA as a step in its degradation; cell cycle and apoptotic432
  • 5. Review Trends in Endocrinology and Metabolism November 2011, Vol. 22, No. 11control proteins including CDKN1B (p27, Kip1), beta-cate- glucose homeostasis in individuals with insulin resistancenin, 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 that3-3–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 theglucose assimilation, activates cardiac glycolysis, and reg- relevant Rabs to be loaded with GTP to facilitate GLUT4ulates 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 superficialsignalling and other pathways converge on several 14-3- similarity, however, some key regulatory details for3-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 thisthere 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 stopcardiac PFKFB2 via a high-affinity PKB-phosphorylated mutation in one allele of AS160 [56], which underscores thesite and a second site that can be phosphorylated by both need to define precisely how AS160 regulates glucosePKB and AMPK, which is activated when the energy or homeostasis. Insulin stimulates phosphorylation of severalATP 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 fromsurvive ischaemia, and a functional role for 14-3-3 is phospho-Ser341, which can be phosphorylated by p90RSKindicated by the finding that the PKB-mediated increases and PKB [29,37,53]. A form of AS160 that binds 14-3-3in 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 plasmanalling is integrated with other inputs is the uptake of membrane was prevented by blocking the AS160–14-3-3glucose into muscles [45]. The goal of improving glucose interaction in cell-free assays, which further indicates acontrol 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 convergenceoutlines 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 thatAS160 (TBC1D4) and TBC1D1. Both proteins are regulat- whereas AMPK may phosphorylate other residues, AMPKed 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 notproved 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]. TBC1D114-3-3-centred perspective with a testable hypothesis. was originally identified as a candidate gene underlying severe familial female obesity [61] and its natural deletionDifferential 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 similarthrough 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 threemust 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 Thr642sign of insulin resistance and type 2 diabetes [46,48]. In and binds to 14-3-3 s in response to insulin, TBC1D1 isskeletal and cardiac muscle, GLUT4 trafficking is also phosphorylated on Ser237 and binds to 14-3-3 s in responseactivated 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 thelevels [49]. There is considerable interest in understanding phosphorylation of a second 14-3-3-binding site at Thr596how exercise, as well as drugs that activate AMPK, restore of TBC1D1, which is similar to Thr642 of AS160; thus, 433
  • 6. Review Trends in Endocrinology and Metabolism November 2011, Vol. 22, No. 11 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 & MetabolismFigure 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 shownhere, 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 intoaccount 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 theseproteins. 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 ofTBC1D1 [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 proteinarrangement 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, which14-3-3 binding [60,65]. results in a reduced glucose uptake rate in muscle in re- sponse to insulin [67]. Together, these findings confirm thatComplementary roles of AS160 and TBC1D1 for insulin insulin-stimulated Thr649 phosphorylation and 14-3-3and AMPK activators binding to AS160 in muscle has a key role in glucoseWe 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 geneticactivating 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 alteredmutations in the 14-3-3-binding phosphorylation sites in physiological responses to high-fat diets and ageing. WillAS160 and TBC1D1 must be generated. Unlike studies the phenotype of TBC1D1 mice provide an insight into theusing ectopic overexpression, introduction of a point mu- obesity of individuals with mutations or polymorphisms intation of a phospho-Ser/Thr residue to a nonphosphoryla- this protein or gene? Would genetic crosses between thetable 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 effectsexerting dominant-negative effects or competition from of insulin and exercise on AS160 and TBC1D1 as means towild-type proteins. improving blood glucose management. First, a mouse model with Thr649 on AS160 (equivalentto Thr642 on human AS160) substituted by alanine The future: 14-3-3-phosphoproteome barcodeswas generated to prevent insulin-stimulated binding to More general lessons may also be learned from this narrow14-3-3 in vivo [67]. The most striking phenotype of the focus on AS160 and TBC1D1. Earlier in this review, weAS160-Thr649Ala knock-in mice is their altered glucose alluded to the possibility of using 14-3-3 capture and434
  • 7. Review Trends in Endocrinology and Metabolism November 2011, Vol. 22, No. 11phosphoproteomics to identify the commonalities and dif- 19 Obsilova, V. et al. (2008) The 14-3-3 protein affects the conformation of the regulatory domain of human tyrosine hydroxylase. Biochemistryferences in insulin targets in specialised organs of the body. 47, 1768–1777Another exciting issue would be the potential of developing 20 Rezabkova, L. et al. (2010) 14-3-3 protein interacts with and affects thethese 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, 451–461in 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. 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