14-3-3alpha; 14-3-3theta; 14-3-3zeta; 14-3-3β: 14-3-3beta; 14-3-3ε: 14-3-3epsilon; 14-3-3ζ: 14-3-3delta; 14-3-3η: 14-3-3eta; 14-3-3θ: 14-3-3tau; 14-3-3σ: SFN; 1C5, HS1; GW128; HEL2; HEL4; HEL-S-1; HEL-S-3; HEL-S-93; HS1; KCIP-1; MDCR; MDS; PPP1R170; Stratifin; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; YWHA1; YWHAA; YWHAB 14-3-3γ: 14-3-3gammma; YWHAD; YWHAE; YWHAG; YWHAH; YWHAQ; YWHAS YWHAZ
14-3-3 proteins have been purified from the mammalian brain (Boston and Jackson 1980). In humans, 7 isoforms of 14-3-3 with highly conserved sequences are located on individual chromosome loci: 14-3-3β (chromosome 20), 14-3-3γ (chromosome 7), 14-3-3ε (chromosome 17), 14-3-3ζ (chromosome 8), 14-3-3η (chromosome 22), 14-3-3θ/τ (chromosome 2), and 14-3-3σ (chromosome 1). 14-3-3 acts as a scaffold protein to interact with various ligand proteins via formation of homo- or heterodimers. 14-3-3 proteins regulate cell signaling, the cell cycle, apoptosis, subcellular localization, cytoskeletal structure, and transcriptional regulation by binding to phosphorylated serine/threonine motifs. The binding motif of 14-3-3 has been identified in two sequences, RSXpSXP and RXY/FXpSXP (where pS is phosphoserine). Expression of 14-3-3 is involved in the regulation of various cellular functions, including the modulation of kinase and other enzyme activity, alteration of subcellular translocation, prevention of protein dephosphorylation or degradation, disruption or stabilization of protein complexes, interaction with proteins on the plasma membrane, and regulation of exosome secretion.
Modulation of Kinase and Other Enzyme Activity
Tryptophan hydroxylases encoded by TPH (TPH1 and TPH2) genes are the key enzymes involved in biosynthesis of the neurotransmitters serotonin and melatonin. TPH mainly expresses in the neuron and is activated by PKA phosphorylation at Ser19. 14-3-3 proteins interact with phosphorylated TPH and increase stability and enzyme activity of TPH (Fig. 1a, panel 2 and Fig. 1c) (Obsilova et al. 2008).
DNA (cytosine-5)-methyltransferase 1 (DNMT1) is a crucial regulator for maintaining methylation. 14-3-3 colocalizes with and binds to phosphorylated DNMT1 at Ser143 in an AKT1-dependent manner (Fig. 1a, panel 3). Binding of 14-3-3 with phosphorylated DNMT1 results in inhibition of DNA methylation (Fig. 1d) (Esteve et al. 2016).
Apoptosis signal-regulating kinase 1 (ASK1) contributes to induction of apoptosis by stimulating the production of H2O2 and TNF-α. In the progression of the cell cycle, the G1 to S phase transition 1 (GSPT1) gene interacts with and induces autophosphorylation and activation of ASK1. 14-3-3 competes with the binding domain of GSPT1 on ASK1, thereby suppressing the activation of ASK1 (Fig. 1e). Phosphorylated ASK1 on S967 selectively interacts with the conserved amphipathic groove of 14-3-3 isoforms (Fig. 1a, panel 4). Therefore, one mechanism by which 14-3-3 inhibits apoptosis is mediated by inhibition of ASK1 (Lee et al. 2008).
Alteration of Subcellular Translocation
Forkhead box O (Foxo) is a family of transcriptional regulators involved in cell cycle arrest and cell proliferation, survival, stress defense, and longevity. Akt phosphorylates Foxo3a at Thr32 and Ser253 residues, and phosphorylated Foxo3a interacts with 14-3-3 proteins (Fig. 2b, panel 1). Binding with 14-3-3 masks the nuclear localization sequence (NLS) on Foxo3a and sequesters Foxo3a in the cytosol, thereby preventing nuclear translocation of Foxo3a and its downstream transcriptional activation (Fig. 2f) (Singh et al. 2010).
The c-Abl (c-Abelson) tyrosine kinase is implicated in regulation of tumorigenesis and tumor progression. Activated c-Abl is located in the nucleus, and c-Abl phosphorylated at Thr735 responds to oxidative stress and stress-induced apoptosis. Phosphorylation of c-Abl at Thr735 is also essential for binding to 14-3-3 and for nuclear translocation (Fig. 2b, panel 2). JNK phosphorylates 14-3-3, leading to release and accumulation of c-Abl in the cytosol (Fig. 2f) (Yoshida et al. 2005).
The Hippo-YAP (Yes-associated protein) pathway plays an important role in controlling cell proliferation, differentiation, and apoptosis. YAP phosphorylated by Akt or lats1/2 (large tumor suppressor) at Ser127 induces interaction of Ser127 with 14-3-3 proteins (Fig. 2b, panel 3). Interaction with 14-3-3 inhibits nuclear translocation of YAP and results in attenuation of p73-mediated apoptosis (Fig. 2f) (Basu et al. 2003).
Cdc25 (cell division cycle 25) is a phosphatase that activates cyclin-dependent kinase (CDK) by dephosphorylation of CDK. Phosphorylation of Cdc25 at Ser216 by Chk1 (checkpoint kinase 1) is required for 14-3-3 binding to maintain cytoplasmic localization of Cdc25 during the interphase (Fig. 2b, panel 4). Depletion of 14-3-3 leads to nuclear retention of Cdc25c in the G2/M transition of the cell cycle (Fig. 2g) (Peng et al. 1997).
Histone deacetylases (HDACs) function as transcriptional repressors to remove the acetyl group from the histone. Class II HDAC phosphorylated in the N-terminal region generates a 14-3-3 interacting motif. 14-3-3 proteins bind to HDAC5 and HDAC9 with the motifs comprised of Ser220 and Ser451 residues (Fig. 2b, panel 5). Thus, 14-3-3 may affect transcription mediated by epigenetic regulation (Fig. 2h) (Martin et al. 2007).
In the Wnt signal pathway, β-catenin is an important effector in promoting gene transcription. Nuclear translocation and activation of β-catenin induces expression of c-myc and cyclin D1. Akt phosphorylates β-catenin at Ser552 and enhances 14-3-3 binding to β-catenin (Fig. 2c). Binding with 14-3-3 induces β-catenin stability and accumulation in the nucleus. Moreover, 14-3-3 regulates Wnt signaling by binding with Dvl-2 (dishevelled segment polarity protein 2) and stabilizing β-catenin (Fig. 2i) (Tian et al. 2004).
Cell mobility is regulated by cytoskeleton proteins through Rho-associated protein kinase (ROCK) and related factors. ROCK is a GTP-dependent serine/threonine kinase which regulates cofilin organization through phosphorylating LIM (named from the proteins Lin11, Isl-1, and Mec-3) kinase. The kinase activity of ROCK is regulated by Rnd3 (a small signaling G protein in the Rnd family) which is a typical, constitutively GTP-binding Rho protein. Rnd3 is phosphorylated by ROCK1 at Ser240/218 and protein kinase C (PKC) at Ser210. Phosphorylated Rnd3 interacts with 14-3-3, triggering Rnd3 translocation from the plasma membrane to the cytosol and consequently the loss of the inhibitory effect of Rho (Fig. 2d, panel 1). Thus, 14-3-3 associates with Rnd3 to influence cell motility via regulation of Rho/ROCK signaling (Fig. 2j) (Riou et al. 2013).
It has been documented that 14-3-3 interacts with the cysteine-rich domain of PKC (Fig. 2d, panel 2). In T cells, transient overexpression of 14-3-3τ associates with PKCθ and inhibits PKCθ-induced IL-2 secretion by preventing PKC translocation from the cytosol to the membrane (Meller et al. 1996).
14-3-3 is involved in pre-protein trafficking into the mitochondria. Newly synthesized pre-proteins are imported into the mitochondria mainly depending on whether their mitochondrial-targeting sequence (MTS) is initially recognized by a cytoplasmic chaperone. 14-3-3 binds to the MTS-containing pre-protein and forms a complex with Tom70 (mitochondrial import receptor subunit Tom70). 14-3-3-containing complex promotes pre-protein delivery into the matrix through the mitochondrial outer membrane (Fig. 2e) (Inaba and Schnell 2008).
Prevention of Protein Dephosphorylation and Degradation
Cdt2 (CRL4) is a substrate recognition adaptor of E3 ubiquitin ligase complex which targets to proteasomal degradation of cell regulators Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase inhibitor), and Set8 (histone methyltransferase). Cdt2 is phosphorylated by Cdk at Thr464 and generates a 14-3-3 binding motif (Fig. 3a, panel 2). Association with 14-3-3 prevents Cdt2 from polyubiquitination and enhances its protein stability. Depleting of 14-3-3 results in dissociation of Cdt2 with 14-3-3 and promotes Cdt2 interaction with FbxO11 (F-box protein 11) (Fig. 3c) (Dar et al. 2014). Moreover, it has been reported that binding with 14-3-3 attenuates dephosphorylation of the proapoptotic function of Raf-1 and Bad by protein phosphatases (Chiang et al. 2003).
Disruption or Stabilization of Protein Complexes
The activity of GSK-3β is mainly regulated by its phosphorylation on Ser9. Phosphorylated GSK-3β (at Ser9) selectively binds to 14-3-3σ isoforms in mouse embryonic stem (ES) cells (Fig. 4a, panel 2). Overexpression of 14-3-3σ promotes mouse ES cell proliferation through disruption of the APC/Axin/GSK-3β complex formation. 14-3-3σ synergizes with Wnt and AKT signaling to enhance the stability of β-catenin, thereby inducing nuclear translocation of β-catenin (Fig. 4c) (Chang et al. 2012).
Snail is a transcriptional repressor for E-cadherin expression and contributes to epithelial-mesenchymal transition (EMT), cancer cell migration, and tumor metastasis. The interaction of 14-3-3 and Snail was demonstrated in a study by 2D-DIGE electrophoresis analysis. Moreover, 14-3-3 forms complexes with Snail and with LIM protein Ajuba on the promoter region of E-cadherin (Fig. 4a, panel 3), thereby suppressing E-cadherin expression and consequently promoting EMT and cell migration (Fig. 4d) (Hou et al. 2010).
Interaction with Proteins on the Plasma Membrane
Cells interact with the surrounding extracellular matrix (ECM) through integrins. Integrins are transmembrane proteins that regulate multiple cellular functions including signaling. 14-3-3 recognizes and binds to integrin tails through the Ser/Thr-rich sequence KEATSTFT. Phosphorylation of β2 integrin at Thr758 generates a 14-3-3 binding site. Through interaction with 14-3-3, integrin triggers signals that activate Raf or Cdc42 pathways (Fig. 5a, panel 2) (Takala et al. 2008). In addition, 14-3-3 interacts with the cytoplasmic domain of β1 integrin to regulate cell adhesion (Fig. 5c) (Legate and Fassler 2009).
Phosphorylation of Ser394 on the C-terminus of the potassium channels TASK-1 and TASK-3 is required for binding with 14-3-3. 14-3-3 interacts with the cytosolic region enhances of TASK-1, and TASK-3 localize to the plasma membrane (Rajan et al. 2002).
Regulation of Exosome Secretion
Mutations of LRRK2 (leucine-rich repeat kinase 2) can be found in patients with Parkinson’s disease. 14-3-3 interacts with the motif containing phosphorylation at the Ser910 and Ser935 residues in the N-terminal region of LRRK2. This binding with 14-3-3 prevents dephosphorylation and stabilizes the structure of LRRK2 (Li et al. 2011). Exosomes are small vesicles that are released from a cell and fuse to a target cell plasma membrane through biological fluids. Exosomes influence cell signaling and metabolism by transmitting various molecules such as RNA and proteins. LRRK2 is released in exosomes from cells into the extracellular environment. Both LRRK2 and 14-3-3 are abundant exosome proteins. Disruption of 14-3-3-LRRK2 interaction significantly blocks the release of LRRK2 in exosomes (Nichols et al. 2010).
Involvement of 14-3-3 in Human Diseases
Neurological diseases. 14-3-3 proteins are abundantly expressed in the brain and are involved in chaperone-mediated protein refolding and degradation of misfolded protein. Misfolded proteins aggregate and are captured by a chaperone in neuron cells. 14-3-3 connects the chaperone to the motor protein, dynein, leading to lysosomal degradation. Most pathological examination of neuron diseases indicate that aggregation of misfolded protein results in neurodegeneration. Disruption of 14-3-3 expression significantly associates with neurological disorder, including Alzheimer’s disease, Parkinson’s disease, Miller-Dieker syndrome, and Creutzfeldt-Jakob disease (Steinacker et al. 2011).
Neoplastic disorders. 14-3-3 proteins have been implicated in regulating tumor progression of various types of human malignancies, including glioma; neuroblastoma; hepatocellular carcinoma; and cancers of the lung, breast, stomach, mouth, and pancreas. 14-3-3 proteins regulate cancer cell survival, proliferation, epithelial-mesenchymal transition, migration, invasion, and resistance to anticancer drugs. Thus, 14-3-3 proteins promote tumor progression by modulating multiple signaling processes and by transcriptional regulation (Tzivion et al. 2006).
Seven 14-3-3 isoforms have been identified in mammals; 15 isoforms in plants; and 2 isoforms in yeast (BMH1/2 and rad24/25), nematode, and Drosophila. They all form homo- or heterodimers and work as scaffold proteins to interact with target proteins on the motif with phosphorylated serine/threonine. Expression of 14-3-3 isoforms is tissue specific. The interacting partners of 14-3-3 proteins include metabolic enzymes, kinases, cell cycle effectors, apoptotic factors, receptors, and transcriptional regulators. Although 14-3-3 proteins are ubiquitously expressed in distinct tissues, an increasing number of studies indicate that 14-3-3 proteins are overexpressed in the brain and human malignancy. Dysregulation of 14-3-3 expression results in induction of neuronal disorders. Aberrant expression of 14-3-3 leads to modulation of cell proliferation, epithelial-mesenchymal transition, migration, and invasion in cancer cells. Development of 14-3-3 antagonists that interact with specific 14-3-3 ligands is a potential strategy for cancer therapy.
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