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Using the Intel® Software License Manager 6 Document Number: 251879-014US 1.2 Conventions and Symbols The following conventions are used in this document. Other proteins identified (e.g. Hsp70, 14-3-3, tubulin) have been associated with roles in maintenance of root architecture 30–32, 43, 44. Previous work in tomato roots identified Hsp70 as methylated 45 , and α- and β- tubulin were reported to be methylated in mammalian tissues 46 , however methylation of 14-3-3 like proteins has. Cocktail is a general purpose utility for macOS that lets you clean, repair and optimize your Mac. It is a powerful digital toolset that helps hundreds of thousands of Mac users around the world get the most out of their computers every day. The application serves up a perfect mix of maintenance tools and tweaks, all accessible through a clean. Background Transcriptional activator-like (TAL) effectors, formerly known as the AvrBs3/PthA protein family, are DNA-binding effectors broadly found in Xanthomonas spp. That transactivate host genes upon injection via the bacterial type three-secretion system. Biologically relevant targets of TAL effectors, i.e. Host genes whose induction is vital to establish a compatible interaction, have.

  • Authors:
    • Rongfei Chai
    • Huiling Fu
    • Zhaodi Zheng
    • Tingting Liu
    • Shuhua Ji
    • Guorong Li

  • Affiliations: Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan, Shandong 250014, P.R. China
  • Published online on:September 26, 2017
  • Pages: 8037-8044
  • Copyright: © Chai et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Resveratrol (RES), a polyphenolic compound present in grapes and red wine, has potential anticancer properties. The present study aimed to examine the effects of resveratrol and its underlying mechanism on hepatocellular carcinoma (HCC) cell lines HepG2, Bel‑7402 and SMMC‑7721. It was demonstrated that resveratrol inhibited the viability and proliferation of HCC cells assessed by MTT and EdU assays. TUNEL assay revealed that resveratrol induced cell apoptosis by increasing HCC apoptosis rate from 3±0.78% to 16±1.12% with upregulation of B‑cell lymphoma (Bcl)‑2 associated X, apoptosis regulator and cleaved‑poly (ADP‑Ribose) polymerase 1 (PARP), and downregulation of Bcl‑2, caspase‑3, caspase‑7 and PARP. As a sirtuin (SIRT) 1 activator, resveratrol elevated SIRT1 protein expression and its enzyme activity and decreased expression levels of phosphorylated (p)‑phosphoinositide‑3‑kinase (PI3K), p‑AKT Serine/Threonine Kinase 1 (AKT), and its downstream target p‑Forkhead Box O3a in HepG2 cells. Furthermore, inhibition of SIRT1 enzymatic activity by EX527 resulted in increased phosphorylation levels of PI3K and AKT. This demonstrated that resveratrol inhibited the PI3K/AKT pathway by SIRT1 activation. In addition to inhibition of cancer cell migration, tumor suppressor gene DLC1 Rho GTPase activating protein level was upregulated and its phosphorylation was enhanced by AKT with resveratrol treatment. These findings suggested that resveratrol inhibits proliferation and migration through SIRT1 mediated post‑translational modification of PI3K/AKT pathway in HCC cells.

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Hepatocellular carcinoma (HCC) is one of the majorleading causes of tumor-associated deaths, with high rates ofincidence and disease-related mortality and morbidity in the world(1). As it is still difficult tomake an early diagnosis for HCC, most of the patients are diagnosedat advanced stages. Despite the improvement of conventionaltherapies for HCC, including surgery, chemotherapy andradiotherapy, the length or quality of life of patients with HCC isstill poor. Therefore, it is urgent to develop a new preventivestrategy for liver cancer.

Resveratrol (RES, trans-3,5,4′-trihydroxystilbene)is a polyphenol compound derived from grapes, berries, peanuts andother sources, and it has inhibitory effects on several types ofcancer cell lines such as colon, lung and prostate and affectsdiverse molecular targets (2).Sirtuin 1 (SIRT1) has been reported to be a key target ofresveratrol in several tumor models (3,4).Whether SIRT1 as a tumor promoter or tumor suppressor remainscontroversial, it might depend on tumor type (5). Resveratrol suppresses tumor cellgrowth and metastasis in colorectal cancer cells by targeting SIRT1protein and regulating NF-κB signaling pathway (6). The forkhead box O transcriptionfactors (FoxOs) have emerged as critical transcriptional factors inregulating metabolism and stress responses and been considered asdownstream targets of SIRT1. FoxO1 translocated into nucleusincreases FoxO1-DNA binding and expression of proapoptotic gene Bim(7). SIRT1 might regulate cellapoptosis by deacetylating FoxOs protein.

The phosphatidylinositol 3′-kinase (PI3K)/AKTpathway plays an important role in cell survival and PI3K activityhas been linked to a variety of human cancers (8). AKT, a downstream kinase of PI3K,regulates many cellular proteins including metabolism, apoptosisand proliferation (9). PI3Kpathway phosphorylates FoxOs via activation of its downstreamkinase AKT (10). Inhibition ofPI3K pathway leads to dephosphorylation and nuclear translocationof active FoxOs, which induce cell cycle arrest and apoptosis(11). These indicate that FoxOsare important downstream effectors of PI3K/AKT pathway. Resveratrolhas been shown to inhibit activation of multiple survival pathwaysincluding PI3K/AKT pathway to induce apoptosis in various cancercells (9,12).

Deleted in liver cancer 1 (DLC1), a focal adhesionprotein, is identified as a putative tumor suppressor in HCC in1998 (13). It functions as aRhoGTPase activating protein (RhoGAP) (14). Activated protein kinase C (PKC) andprotein kinase D (PKD) stimulate the association between DLC1 and14-3-3 protein, which blocks DLC1 nucleocytoplasmic shuttling andinhibits RhoGAP activity of DLC1 (15). DLC1 activity could be regulated bypost-translational modification and it might be a substrate of AKT.Expression of DLC1 suppresses cell proliferation,anchorage-independent growth, tumorigenicity and invasiveness inHCC cells (16). DLC1 inhibitsRho-dependent stress fiber formation in fibroblasts and serves as atumor suppressor gene in human non-small cell lung carcinomas(17). Thus, we hypothesized thatAKT involved in regulation of DLC1 mediated cell motilityinhibition in HCC.

The purposes of the present study were to determinethe molecular mechanism of resveratrol affected proliferation andmigration through SIRT1 mediated post-translational modification ofPI3K/AKT signaling pathway in HCC cells.

Materials and methods

Cell culture

The human hepatocellular carcinoma (HCC) cell linesBel-7402, SMMC-7721, hepatoblastoma cells HepG2 (The HepG2 cellline was originally thought to be a hepatocellular carcinoma cellline but was later shown to be from an hepatoblastoma,PubMed=19751877), and human liver normal cell line HL-7702 wereobtained from the Cell Bank of Type Culture Collection of ChineseAcademy of Science (Shanghai, China). HepG2 cells were cultured inDMEM, other cells in RPMI 1640 medium. All the experiments wereperformed in medium containing 10% fetal bovine serum, 100 U/mlpenicillin and 100 µg/ml streptomycin, maintained at 37°C inhumidified atmosphere with 5% CO2.

Proliferation assay by MTT andEdU

MTT assay was used to assess cell viability. Thecells were seeded in 96-well plates at a density of1×104/well overnight and treated without or withresveratrol (Sigma, St. Louis, MO, USA) dissolved in 0.1% (v/v)DMSO at various concentrations for 24 h. Then cells were incubatedwith MTT solution for 4 h. The formazan crystals dissolved by 150µl DMSO, the solution was absorbed at 492 nm using enzyme-linkedimmunosorbent assay reader (Awareness, Palm City, FL, USA).

Cell proliferation was tested by EdU(5-ethynyl-2-deoxyuridine) incorporation assay kit (Ribobio,Guangzhou, China). Briefly, cells cultured in 96-well platesexposed to 50 µM EdU for 2 h at 37°C, and fixed in 4% formaldehyde.After permeabilization with 0.5% Triton-X, the cells were reactedwith 1xApollo reaction cocktail for 30 min, the DNA contents werestained with Hoechst 33342 and visualized under fluorescentmicroscope. Cells were counted in five selected arbitrarily fields,at least 300 cells were counted per well. EdU positive cells werecalculated with (EdU incorporated-in cells/Hoechst stained cells)×100%.

Apoptosis detection by TUNELassay

TUNEL staining was performed using an EdUTP TUNELcell detection kit (Ribobio, Gangzhou, China) according to themanufacturer's instructions. The cells cultured in 96-well plateswere treated without or with 100 µM resveratrol, fixed in 4%paraformaldehyde, permeabilized with 0.1% Triton X-100, washedtwice, incubated with TUNEL detecting liquid for 1 h at 37°C andobserved by a fluorescent microscope (Olympus, Tokyo, Japan) at 488nm excitation and 530 nm emission. TUNEL positive cells werecalculated as the number of apoptotic cells/DAPI stained cells×100%.

Western blotting andCo-immunoprecipitation analysis

The cells were lysed by RIPA (Beyotime, Shanghai,China). The inhibitor of SIRT1, EX-527 (Selleck Chemicals, Houston,TX, USA), was used to PI3K/AKT pathway. Proteins were separated bySDS-PAGE and transferred on membranes were incubated in primaryantibodies against SIRT1, p-AKT, AKT, p-PI3K, PI3K, PARP,Cleaved-PARP, p-FoxO3a, Caspase-3/-7, Bax, Bcl-2 and p53 (CST,Danvers, MA, USA) and FoxO1, FoxO3a (Santa Cruz Biotechnology, SanDiego, CA, USA) overnight at 4°C, followed by incubation withHRP-conjugated rabbit/mouse secondary antibodies (ZSGB-BIO,Beijing, China). Protein expressions were visualized ECL detectionsystem (Beyotime, Shanghai, China).

Immunoprecipitation (IP) was carried out usingPierce Classic Magnetic IP/Co-IP Kit (Thermo Scientific, Waltham,MA, USA) according to the manufacturer's protocol. The proteinlysates were incubated with DLC1 antibody (BD Biosciences, SanJose, CA, USA), and precipitated with Protein A/G Magnetic Agaroseat 4°C. The immunocomplex collected was washed, and theimmunoprecipitates were subjected to western blotting andphosphorylation signals were determined using phospho-AKT substrate(PAS) antibody (CST, Danvers, MA, USA).

Wound healing assay

Cells were seeded into 24-well plates(1.0×105 cells/well). Sterile pipette tip was used toproduce a wound line between cells after the cells grew to 80–90%confluence and allowed the cells migrated for 24 h. Images werecaptured and the relative distance traveled by the leading edgefrom 0 to 24 h was assessed using Image Pro Plus 6.0 software(n=5).

SIRT1 activity assays

SIRT1 activity was quantified with a SIRT1Fluorometric Assay Kit (Sigma, St. Louis, MO, USA) according to themanufacturer's protocol. Fluorescence intensities were measuredwith a microplate fluorometer (excitation wavelength=360 nm,emission wavelength=450 nm). Experimental values are represented asa percentage of control.

Statistical analysis

All of the assays were performed three timesindependently at least. Value presented as the means ± standarddeviation (SD) by GraphPad Prism software (GraphPad Software, CA,USA). Statistical analyses were performed using one-way ANOVA andStudent's t-test, *P<0.05, **P<0.01 were considered toindicate a statistically significant difference.


Effect of resveratrol on cellviability and proliferation

Three HCC cell viability was determined by MTTassay. The results showed that resveratrol inhibited cell viabilitywhen its concentrations were higher than 80 µM compared to normalHL-7702 cell (Fig. 1A). From80–200 µM, 100 µM resveratrol was selected as IC50 (half maximalinhibitory concentration) in the subsequent experiments. Itimplicated that resveratrol was able to reduce cancer cellviability in a dose-dependent manner. The percentage of EdUpositive cells was markedly reduced in HCC cells with 100 µMresveratrol treatment compared to the controls, whilenon-tumorigenic cell line HL-7702 had a slight reduction (Fig. 1B and C).

To understand the molecular basis of proliferationinhibition caused by resveratrol, proliferation regulation proteinPCNA (proliferating cell nuclear antigen) was evaluated in HepG2cells. The level of PCNA reduced after 100 µM resveratrol treatment(Fig. 1D). It was consistent withabove results of EdU assay. These findings demonstrate theanti-proliferative effect of resveratrol on HCC cells.

Resveratrol induced apoptosis viadecreasing phosphorylation of FoxO3a with suppressing PI3K/AKTpathway

TUNEL assay was assessed whether anti-proliferativeeffects of resveratrol against HCC cells are mediated viaapoptosis. Results showed that resveratrol increased apoptosis from4±0.83% to 13±1.32%, 3±0.78% to 14±0.72%, 5±0.33% to 16±1.12% inHepG2, Bel-7402 and SMMC-7721 cells (Fig. 2A).

Studies have reported that Bcl-2 could be a crucialtarget gene of PI3K/AKT signaling, whereas AKT has been shown tonegatively regulate the activity of proapoptotic members of theBcl-2 family (8). Next effects ofresveratrol on apoptosis-related proteins were further detected. Asshowed by Fig. 2B, resveratrolinhibited Bcl-2 expression and concomitant up-regulatedproapoptotic protein Bax, causing a significant decrease inBcl-2/Bax ratio. The apoptosis regulators were further detected andthe precursor forms of caspase-3/7 induced by resveratrol weredown-regulated obviously in HCC cells (Fig. 2B). The activation of caspases werealso related to another marker of apoptosis, proteolysis of the DNArepair enzyme PARP (18). Theresults indicated that precursor form PARP decreased as active formcleavage-PARP significantly enhanced (Fig. 2B).

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Resveratrol activated SIRT1 andinhibited SIRT1-mediated post-translational modification ofPI3K/AKT signaling

To investigate effects of resveratrol on the pathwayof PI3K/AKT/FoxO3a to induce cell apoptosis, western blotting wasperformed for their phosphorylation levels. Resveratrol inhibitedphosphorylation of PI3K and AKT without effect on total levels ofPI3K and AKT in HepG2 cells (Fig.3D), and inhibited FoxO3a phosphorylation in HCC cells with nototal FoxO3a change (Fig. 3D).These data indicated that resveratrol down-regulated p-FoxO3a levelwith reduction of phosphorylation level of PI3K/AKT.

We then examined whether resveratrol stimulatesexpression of SIRT1. Resveratrol up-regulated protein expression ofSIRT1 in HCC cells but not in normal liver HL-7702 cells (Fig. 3A). Deacetylation of FoxO proteinshas been shown to result from the activity of SIRT1 (19). It has been shown that SIRT1promotes transcription of FoxO target genes involved in stressresistance, while decreasing transcription of genes involved inapoptosis (20). Our result showedthat protein levels of FoxO1 and Ac-FoxO1 were significantlydecreased with resveratrol treatment compared with control(Fig. 3D). Up-regulation of SIRT1activated by resveratrol involved in deacetylation of FoxO1.

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To determine the relationship between SIRT1 activityand PI3K/AKT signaling pathway, the activity of intracellular SIRT1was analyzed after SIRT1 inhibitor EX527 was used. Consistent withits protein level, SIRT1 activity increased by resveratrol anddecreased after exposure to EX527 in HepG2 cells (Fig. 3B). Treatment of 1 µM EX527 enhancedp-PI3K, p-AKT and p-FoxO3a levels while slight effect on total PI3K(Fig. 3C). These results showedthat resveratrol suppressed post-translational modification ofSIRT1 mediated PI3K/AKT signaling.

Resveratrol enhanced phosphorylationof DLC1 by AKT and inhibited cell migration

As PI3K/AKT pathway is an important cell survivalcascade, DLC1 might be a substrate of AKT. In order to explorewhether AKT involved in regulation of tumor suppressor DLC1,phosphorylation on the biological activities of DLC1 wasdemonstrated in HCC cells. DLC1 protein expression elevated inthree kinds of HCC cells and no effect on HL-7702 cells byresveratrol (Fig. 4A). An antibodyagainst PAS (phospho-AKT substrate) was employed to detectphosphorylation of DLC1. Immunoprecipitation result demonstratedthat DLC1 phosphorylation level was enhanced by resveratrol inHepG2 cells (Fig. 4B). Therelevance and functional effect of AKT mediated phosphorylation ofDLC1 remain unclear and await further investigation.

Wound healing assay measures the ability of cells tomigrate into an area of a cell culture plate denuded of cells(wound). Our result showed that resveratrol inhibited the woundclosures from 32.5 to 11.5% in HepG2 cells by 24 h treatment(Fig. 4C). The findings revealedthat up-regulation of DLC1 and its phosphorylation level viaresveratrol treatment might cause motility inhibition in cancercells.


Although the capacity of resveratrol to preventcancer development has been studied for many years, its mechanismunderlying remains to be fully elucidated. Proliferating cellnuclear antigen (PCNA) is a critical event in growth regulation ofcancer cells (21). Theanti-metastatic effect of resveratrol was associated withrestriction of invasion, mobility, adhesion, and MMP expression incolon carcinoma (22). Here, wefound that resveratrol inhibited the viability and proliferation byMTT and EdU assays and suppressed expression of PCNA accompanyingproliferation inhibition in HCC cells.

The PI3K/AKT signaling is a critical pathway in cellproliferation, survival, neovascularization and tumor growth(23). AKT is an importantdownstream target kinase of PI3K signaling pathway. Activated AKTcan inhibit release of cytochrome c and apoptosis factor, therebyinhibiting apoptosis and promote the growth of cancer cells(8). Resveratrol has been shown toinhibit constitutive activation of PI3K/AKT pathway to induceapoptosis in several types of cancer cells (12,24).FoxO3a is the downstream targets of AKT and AKT can promote FoxO3aphosphorylation, leading to FoxO3a translocation from the nucleusto the cytoplasm, which de-activates FoxO3a; conversely, inhibitionof AKT promotes de-phosphorylation of FoxO3a, resulting in nucleartranslocation of FoxO3a (25). Ourresults showed that resveratrol resulted in significant inhibitionin constitutively elevated levels of phosphorylated PI3K/AKT andreduced phosphorylated FoxO3a significantly in HepG2 cells. AKTinhibits apoptosis through multiple targets, including Bcl-2 familyand caspase proteases (8). Bcl-2members are well characterized as regulators of apoptosis, such asBax and Bim. The ratio of Bcl-2/Bax protein regarded as a drivingforce for apoptosis in cancer cells (26). Caspases are a family of cell deathproteases triggered in response to proapoptotic signals and play anessential role in the execution phase of apoptosis (27). TUNEL assays are used to detect DNAfragmentation from apoptosis. In the present study, resveratrolresulted in an increase of green fluorescence signal which wasindicative of apoptosis. Also, resveratrol caused a significantdown-modulation of Bcl-2/Bax ratio and activated caspase-3,caspase-7, PARP and induced the cleavage-PARP in HCC cells. Itsuggested that the apoptosis of HCC cells induced by resveratrolmight act through the mitochondrial pathways.

SIRT1 plays a key role in both cell death andsurvival with p53 family members, FoxOs and the nuclear factor-κBfamily (28). Furthermore,resveratrol suppresses the proliferation of gastric cancer cells ina SIRT1-dependent manner in vitro and in vivo(29). We showed that resveratrolsignificantly increased SIRT1 expression in HCC cells. As anicotinamide adenine dinucleotide-dependent protein deactylase,SIRT1 is known to be directly involved in the acetylation of FoxOsand expression of proapoptotic protein Bim (19). FoxO1 has emerged as an importantprotein that modulates the expression of apoptosis-related genes incancer cells (7). SIRT1 knockdownenhanced Ac-FoxO1 expression to block reactive oxygenspecies-induced apoptosis in mouse embryonic stem cells (30). Our results showed that resveratrolsignificantly decreased expressions of FoxO1 and Ac-FoxO1 withactivation of SIRT1 by resveratrol.

SIRT1 has been also implicated as a negativeregulator for the PI3K/AKT pathway by deacetylating the tumorsuppressor PTEN (31) and bydown-regulation of both AKT and phosphorylation levels to inhibitthe PI3K/AKT pathway in glioblastoma cell (32). The regulation of PI3K/AKT pathwayby SIRT1 may provide a potential mechanism in tumorigenesis, andSIRT1 inhibitor EX527 was used to evaluate the underlyingmechanism. We found that resveratrol up-regulated SIRT1 level todecrease PI3K and AKT phosphorylation and the phosphorylation ofPI3K and AKT became significantly higher when SIRT1 was inhibitedin HepG2 cells. It indicated that the inhibition of PI3K/AKTpathway by resveratrol is mediated by up-regulation of SIRT1.

DLC1 is a Rho GTPase-activating protein (RhoGAP) andfrequently deleted and underexpressed in cancers (14). Restoration of DLC1 gene expressioninduces apoptosis and inhibits both cell growth and tumorigenicityin HCC cells (33). Our previousresults has been shown that DLC1 is a multifunctional protein whichinteracts with tensin, talin, FAK in focal adhesion (34,35).DLC1 expression could significantly suppress Rho-dependent actinstress fiber formation in hepatocellular carcinoma and fibroblastcell lines (16). Cell migrationis tightly regulated by the activity of Rho proteins through actincytoskeletal rearrangements (36).In addition, DLC1 overexpression inhibited cell migration byinduced disassembly of stress fibers and extensive membraneprotrusions around cells on laminin-1 in HCC (37). Our result showed that resveratrolsignificantly up-regulated expression of DLC1 protein and inhibitedthe migration ratio from 32.5 to 11.5% in HCC cells, indicatingthat induced DLC1 level was associated with tumor suppressioneffect. The post-translational modification of DLC1 has garneredmuch attention as the important regulatory mechanism of DLC1activity, and kinases such as AKT, PKC and PKD have been shown tophosphorylate DLC1 at different residues and regulate itsbiological activities via RhoGAP-dependent as well asRhoGAP-independent pathways (15,38).Phosphorylation of DLC1 by PKA contributes to enhance RhoGAPactivity and promotes activation of DLC1, which suppresses hepatomacell growth, motility and metastasis both in vitro and invivo models (39). Toelucidate whether AKT could phosphorylate DLC1, an antibody againstPAS (phospho-AKT substrate) was employed to detect phosphorylationof DLC1. Our findings showed that DLC1 was directly phosphorylatedby AKT in HepG2 cells. These results suggested that DLC1 as a tumorsuppressor was up-regulated by resveratrol and itspost-translational modification was mediated by PI3K/AKT signaling.Although previous studies have characterized functional effects ofthe identified phosphorylated residues of DLC1 (40), the physiological stimuli of thesephosphorylations remain unclear. Future work are warrant to clarifyhow DLC1 regulated by its domains and phosphorylation as well asprecise downstream mechanisms through post-translationalmodification of DLC1 acts as a tumor suppressor.

Taken together, our findings suggested thatresveratrol activated SIRT1 to induce liver cancer cell apoptosisand to inhibit migration through SIRT1 mediated post-translationalregulation of PI3K/AKT signaling and phosphorylation level ofFoxO3a and DLC1 and deacetylation of FoxO1 leading to tumorsuppression in HCC cells.


This work was supported by the National NaturalScience Foundation of China (Grant No. 31672377), the Major KeyScience and Technology Project of Shandong Province(2015ZDJS04003), the Key Program of Shandong Provincial NaturalScience Foundation of China (ZR2013CZ002), Science and TechnologyProgram of Jinan (201202033).



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Volume 16 Issue 6

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Print ISSN: 1791-2997
Online ISSN:1791-3004

Cartoon diagram of Human 14-3-3 protein beta PDB entry 2bq0[1]
Available protein structures:
Pfamstructures / ECOD
PDBsumstructure summary

14-3-3 proteins are a family of conserved regulatory molecules that are expressed in all eukaryotic cells. 14-3-3 proteins have the ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. More than 200 signaling proteins have been reported as 14-3-3 ligands.

Elevated amounts of 14-3-3 proteins in cerebrospinal fluid may be a sign of Creutzfeldt–Jakob disease.[2]

Molecular structure of a 14-3-3 protein dimer bound to a peptide.


There are seven genes that encode seven distinct 14-3-3 proteins in most mammals (See Human genes below) and 13-15 genes in many higher plants, though typically in fungi they are present only in pairs. Protists have at least one. Eukaryotes can tolerate the loss of a single 14-3-3 gene if multiple genes are expressed, however deletion of all 14-3-3s (as experimentally determined in yeast) results in death.[citation needed]

14-3-3 proteins are structurally similar to the Tetratrico Peptide Repeat (TPR) superfamily, which generally have 9 or 10 alpha helices, and usually form homo- and/or hetero-dimer interactions along their amino-termini helices. These proteins contain a number of known common modification domains, including regions for divalent cation interaction, phosphorylation & acetylation, and proteolytic cleavage, among others established and predicted.[3]

14-3-3 binds to peptides. There are common recognition motifs for 14-3-3 proteins that contain a phosphorylated serine or threonine residue, although binding to non-phosphorylated ligands has also been reported. This interaction occurs along a so-called binding groove or cleft that is amphipathic in nature. To date, the crystal structures of six classes of these proteins have been resolved and deposited in the public domain.[citation needed]

14-3-3 recognition motifs[4]
Non-phos (ATP)
All entrys are in regular expression format. Newlines are added in 'or' cases for readability. Phosphorylation sites are in bold.

The motif sites are way more diverse than the patterns here suggest. For an example with a modern recognizer using an artificial neural network, see the cited article.[5]

Discovery and naming[edit]

14-3-3 proteins were initially found in brain tissue in 1967 and purified using chromatography and gel electrophoresis. In bovine brain samples, 14-3-3 proteins were located in the 14th fraction eluting from a DEAE-cellulose column and in position 3.3 on a starch electrophoresis gel.[6]

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14-3-3 proteins play an isoform-specific role in class switch recombination. They are believed to interact with the protein Activation-Induced (Cytidine) Deaminase in mediating class switch recombination.[7]

Phosphorylation of Cdc25C by CDS1 and CHEK1 creates a binding site for the 14-3-3 family of phosphoserine binding proteins. Binding of 14-3-3 has little effect on Cdc25C activity, and it is believed that 14-3-3 regulates Cdc25C by sequestering it to the cytoplasm, thereby preventing the interactions with CycB-Cdk1 that are localized to the nucleus at the G2/M transition.[8]

The eta isoform is reported to be a biomarker (in synovial fluid) for rheumatoid arthritis.[9]

14-3-3 regulating cell-signalling[edit]

  • Bad – see Bcl-2
  • SOS1[10] – see RSK

Human genes[edit]

  • YWHAB – '14-3-3 beta'
  • YWHAE – '14-3-3 epsilon'
  • YWHAG – '14-3-3 gamma'
  • YWHAH – '14-3-3 eta'
  • YWHAQ – '14-3-3 tau'
  • YWHAZ – '14-3-3 zeta'
  • SFN or YWHAS – '14-3-3 sigma' (Stratifin)

The 14-3-3 proteins alpha and delta (YWHAA and YWHAD) are phosphorylated forms of YWHAB and YWHAZ, respectively.

In plants[edit]

Presence of large gene families of 14-3-3 proteins in the Viridiplantae kingdom reflects their essential role in plant physiology. A phylogenetic analysis of 27 plant species clustered the 14-3-3 proteins into four groups.

14-3-3 proteins activate the auto-inhibited plasma membrane P-type H+ ATPases. They bind the ATPases' C-terminus at a conserved threonine.[11]


  1. ^Yang, X.; Lee, W. H.; Sobott, F.; Papagrigoriou, E.; Robinson, C. V.; Grossmann, J. G.; Sundstrom, M.; Doyle, D. A.; Elkins, J. M. (2006). 'Structural basis for protein-protein interactions in the 14-3-3 protein family'. Proc. Natl. Acad. Sci. U.S.A. 103 (46): 17237–17242. Bibcode:2006PNAS..10317237Y. doi:10.1073/pnas.0605779103. PMC1859916. PMID17085597.
  2. ^Takahashi H, Iwata T, Kitagawa Y, Takahashi RH, Sato Y, Wakabayashi H, Takashima M, Kido H, Nagashima K, Kenney K, Gibbs CJ, Kurata T (November 1999). 'Increased levels of epsilon and gamma isoforms of 14-3-3 proteins in cerebrospinal fluid in patients with Creutzfeldt-Jakob disease'. Clinical and Diagnostic Laboratory Immunology. 6 (6): 983–5. doi:10.1128/CDLI.6.6.983-985.1999. PMC95810. PMID10548598.
  3. ^Bridges D, Moorhead GB (August 2005). '14-3-3 proteins: a number of functions for a numbered protein'. Science's STKE. 2005 (296): re10. doi:10.1126/stke.2962005re10. PMID16091624. S2CID5795342.
  4. ^'ELM search: '14-3-3''. Eukaryotic Linear Motif resource. Retrieved 16 May 2019.
  5. ^Madeira F, Tinti M, Murugesan G, Berrett E, Stafford M, Toth R, Cole C, MacKintosh C, Barton GJ (July 2015). '14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides'. Bioinformatics. 31 (14): 2276–83. doi:10.1093/bioinformatics/btv133. PMC4495292. PMID25735772.
  6. ^Aitken, A (2006). '14-3-3 proteins: a historic overview'. Semin Cancer Biol. 50 (6): 993–1010. doi:10.1023/A:1021261931561. PMID16678438. S2CID41949194.
  7. ^Xu Z, Zan H, Pone EJ, Mai T, Casali P (June 2012). 'Immunoglobulin class-switch DNA recombination: induction, targeting and beyond'. Nat Rev Immunol. 12 (7): 517–31. doi:10.1038/nri3216. PMC3545482. PMID22728528.
  8. ^Cann KL, Hicks GG (December 2007). 'Regulation of the cellular DNA double-strand break response'. Biochemistry and Cell Biology. 85 (6): 663–74. doi:10.1139/O07-135. PMID18059525.
  9. ^Detection of high levels of 2 specific isoforms of 14-3-3 proteins in synovial fluid from patients with joint inflammation.
  10. ^Saha M, Carriere A, Cheerathodi M, Zhang X, Lavoie G, Rush J, Roux PP, Ballif BA (October 2012). 'RSK phosphorylates SOS1 creating 14-3-3-docking sites and negatively regulating MAPK activation'. The Biochemical Journal. 447 (1): 159–66. doi:10.1042/BJ20120938. PMC4198020. PMID22827337.
  11. ^Jahn TP, Schulz A, Taipalensuu J, Palmgren MG (February 2002). 'Post-translational modification of plant plasma membrane H(+)-ATPase as a requirement for functional complementation of a yeast transport mutant'. The Journal of Biological Chemistry. 277 (8): 6353–8. doi:10.1074/jbc.M109637200. PMID11744700.

Further reading[edit]

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  • Moore BW, Perez VJ (1967). FD Carlson (ed.). Physiological and Biochemical Aspects of Nervous Integration. Prentice-Hall, Inc., The Marine Biological Laboratory, Woods Hole, MA. pp. 343–359.
  • Mhawech P (April 2005). '14-3-3 proteins--an update'. Cell Research. 15 (4): 228–36. doi:10.1038/ PMID15857577.
  • Steinacker P, Aitken A, Otto M (September 2011). '14-3-3 proteins in neurodegeneration'. Seminars in Cell & Developmental Biology. 22 (7): 696–704. doi:10.1016/j.semcdb.2011.08.005. PMID21920445.

External links[edit]

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  • Eukaryotic Linear Motif resource motif class LIG_14-3-3_1
  • Eukaryotic Linear Motif resource motif class LIG_14-3-3_2
  • Eukaryotic Linear Motif resource motif class LIG_14-3-3_3
  • 14-3-3+Protein at the US National Library of Medicine Medical Subject Headings (MeSH)

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