AFDN (afadin, adherens junction formation factor)

2018-12-01   Jean Loup Huret , Philippe Dessen (JLH); UMR 1170 INSERM, Gustave Roussy, 114 rue Edouard Vaillant, 94805 Villejuif 94805, France (PHD)


Atlas Image
Figure 1 Localization of AFDN (also called AF6 or MLLT4) - Courtesy Mariano Rocchi.


Afadin, the protein coded by AFDN (6q27), also known as AF6 or MLLT4, is a cytoskeletal and junction-associated protein that links nectins, transmembrane proteins, to the F-actin (actin cytoskeleton) in a type of cell-cell junctions: the adherens junctions (AJs). Afadin plays an important role in AJs integrity and apical-basal polarity. There is growing evidence of its role in carcinogenesis.



The AFDN gene, located on 6q27, has a genomic size of 145,030 bases and encodes for several transcripts on the position chr6:167826991-167972020 in the + strand.


According to RefSeq, there are 3 transcripts (see below) and an anti-sens (NR_027906)
AFDN at chr6:167826991-167972020 - (NM_001207008) afadin isoform 1: 7,459 bp
AFDN at chr6:167826991-167965113 - (NM_001040000) afadin isoform 2: 7,629 bp
AFDN at chr6:167827635-167972020 - (NM_001291964) afadin isoform 4: 7,750 bp PSEUDOGENE


Atlas Image
Figure 2 Afadin protein


The AFDN gene codes for the afadin protein.
From N-term to C-term, afadin possess two Ras-association (RA) domains, a forkhead-associated (FHA) domain, a DIL domain (responsible for actin stress fiber formation (Saito et al., 2015)), a PDZ domain (responsible for binding the cytoplasmic C-terminus of nectins; where TJP1 (ZO-1) also binds (Kuriyama et al., 1996)), three proline-rich (PR) domains, one of which interacts with USP9X (FAM): (aa 1130-1612) (Hock et al., 199; Taya et al., 1998), and an F-actin-binding domain (see Figure 2).
Phosphorylation sites: KERQRLFSQG (aa 1792-1801 according to UniProt). There is an AKT phosphorylation site at Ser1718 in l-Afadin (large variant, see below). Phosphorylation of l-Afadin by AKT at Ser1718 promotes nuclear localization, which enhances migration and perturbs cell to cell adhesion (Ellol et al. 2014).
Afadin has many splice variants, in particular two of them: the larger l-afadin and the smaller s-afadin. s-Afadin lacks the F-actin-binding domain and the third proline-rich domain. These variants are made of 1612 to 1816 amino acids (aa) according to UniProt, or 1655 and 1829 aa according to others (see Figure 2).
l-Afadin links nectins to actin filaments (F-actin). Through its PDZ domain, l-Afadin binds to the nectin conserved motif of four amino acid residues (Glu/Ala-X-Tyr-Val) (except for nectin-4), and F-actin through its F-actin-binding domain. Afadin binds nectins ( NECTIN1, NECTIN2, NECTIN3, NECTIN4), but not nectin-like molecules (Necls: CADM1, CADM2, CADM3, CADM4, PVR) (Review in Ogita and Takai, 2006).
Afadin forms homodimer.
The 3 main isoforms produced by alternative splicing are:
Canonic sequence (Isoform 4, identifier: P55196-4). 1,824 amino acids; 206,804 Da.
s-afadin (Isoform 1, identifier: P55196-2); 1,612 aa; 182,000 Da. Compared to the canonical sequence, are missing aa: 139, 393-407, 1605-1628, 1629-1824.
l-afadin (Isoform 2, identifier: P55196-1); 1,816 aa; 205,605 Da. Compared to the canonical sequence, are missing aa: 139, 393-407, 1048-1048, 1747-1824.


L-afadin is widely expressed in epithelial cells, while s-afadin expression is restricted to the brain (Buchert et al., 2007)


Afadin is mainly located at the cell junctions named adherens junctions. s-afadin is able to localize both to the plasma membrane or to the nucleus while l-afadin was said to be unable to localize to the nucleus (Buchert et al., 2007).
Atlas Image
Figure 3 Nectin/Afadin: nectin activations.


Epithelial cells contain three types of cell-cell junction: tight junctions, localized in the apex of the cells, adherens junctions (AJs), and desmosomes.
Adherens junctions: two types of cell adhesion molecules (CAMs), cadherins and nectins, interact with actin filaments (F-actin). They interact through their cytoplasmic domain to form adherens junctions.
Cadherins and nectins: In epithelial cells, β-catenin ( CTNNB1) directly binds to cadherins and links it to the actin cytoskeleton through &alpha-catenin (CTNNA1, CTNNA2, CTNNA3), while nectins are linked to the actin cytoskeleton through afadin (Tachibana et al., 2000; Ogita and Takai, 2006) (see Figure 4).
Nectins first activate SRC. Activated SRC then activates/phosphorylates FARP2, and VAV2. SRC also activates RABGEF1 (also known as Rap1) through CRK and RAPGEF1 (C3G).
Activated RABGEF1 (Rap1) also activates FARP2, which activates CDC42 40012. Activated CDC42 induces the activation of VAV2, followed by the activation of RAC1 (RAC) (see Figure 3).
Activated RABGEF1 (Rap1) is essential for down-regulation of Rho signaling and actin stress fiber dissolution (Birukova et al., 2013).
Some of the downstream effectors for VAV2 and RAC1 are actin filaments (F-actin)-binding proteins.
Nectins associate with cadherins through the interaction of afadin with &alpha-catenin and: a ponsin ( SORBS1) - vinculin (VCL) unit, an SSX2IP (ADIP) - a-actinin ( ACTN1, ACTN2 and ACTN3) unit, and a LMO7 - a-actinin unit (see Figure 4). RABGEF1 (Rap1) activates afadin to interacts with CTNND1 (p120 catenin) and strengthens its binding to E-cadherin ( CDH1), which results in reduced E-cadherin endocytosis (Bégay-Müller et al., 2002; Kooistra et al. 2007; Sakisaka et al., 2007 ; Birukova et al., 2013; Takeichi 2014)
Tight junctions/role of ZO-1 Afadin also associates transiently with tight junction protein TJP1 (ZO-1). ZO-1 is a member of F-actin-binding ZO proteins, which bind CAMs of tight junctions (TJs) such as claudins, occludin ( OCLN), and junctional adhesion molecules (JAMs) and link them to the actin cytoskeleton (Sakisaka et al., 2007) (see Figure 4). Occludin, EPHA2, a transmembrane tyrosine kinase receptor, and afadin also cooperate in tight junction organization (Perez White et al., 2017). Afadin is therefore a peripheral component of tight junctions in epithelial cells. The binding of claudins to tight junction proteins (or zonula occludens ZOs) and afadin and other proteins constitutes a step in cellular signal transduction (Zhang et al., 2018).
Apico-basal polarization/role of Par-3: PARD3 (Par-3), Par-6 and atypical protein kinase C (PRKCI) are required for apico-basal polarization of epithelial cells. Nectin-1 and nectin-3, but not nectin-2, bind the PDZ domain of Par-3 (Rakotomamonjy et al., 2017)
1- Par-3 regulates association of afadin with trans-interacting nectin and the formation of AJs;
2- Par-3 regulates E-cadherin-induced activation of Rac and formation of AJs;
3- Par-3 and afadin cooperatively regulate nectin-induced formation of TJs (Sakisaka et al 2007)
Migration Afadin is localized at cell-cell contact sites in mesangial cells. Afadin forms a complex with CTNNB1 (β-catenin) in cultured mesangial cells and Afadin regulates migratory polarity (Tsurumi et al., 2016). Afadin is required for the maintenance of the radial glial scaffold for neuronal migration during cortical development (Yamamoto et al., 2015b).
Mitotic spindle orientation Afadin is required for mitotic spindle orientation and correct epithelial morphogenesis (Carminati et al., 2016). Afadin orients the mitotic spindle and is required for lumen continuity in developing renal tubules by orienting the mitotic spindle during cell division (Gao et al., 2017). Afadin controls cell polarization and mitotic spindle orientation in developing cortical radial glia (Rakotomamonjy et al., 2017).
Actin polymerization/profilin profilin plays an important role in actin polymerization. Profilin 1 and Profilin 2 (PFN1 and PFN2) are afadin-binding protein (Boettner B et al., 2000)
MAPK cascade Afadin interacts with HRAS, KRAS, and NRAS GTPases and the Ras-related RAP1A leading to MAPK activation (Yamamoto et al., 1997). MRAS, whose GTP/GDP cycle is sensitive to the Ras GEFs, SOS1 42355, and RASGRF1 43453 (GRF1) and to RASA1 (p120 RASGAP) interacts with Afadin (Quilliam et al. 1999). RRAS, RRAS2 also interact with Afadin (Linnemann et al., 1999). Afadin also acts downstream of EGFR-Ras and provides a link from EGFR to cytoskeletal elements in the cell motility process (Gaengel and Mlodzik 2003).
Ubiquitination Afadin is a substrate of the USP9X deubiquitinating enzyme. USP9X can release ubiquitin from Afadin (Taya et al., 1998).
Ephrin receptors Eph receptor tyrosine kinases are membrane-bound proteins implicated in cell migration and intercellular communication during embryonic development, regulating cell pattern formation during organogenesis. EPHA7, EPHB2, EPHB3, and EPHB6 interact with Afadin. Afadin is phosphorylated specifically by EPHB3 and EPHB2 (Hock et al., 1998)
Atlas Image
Figure 4 Afadin in adherens junctions ( AJ) and tight junctions ( TJ)


Homologs of the AFDN gene: The AFDN gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, C.elegans, and frog (Orthologs from Annotation Pipeline: 201 organisms have orthologs with human gene AFDN).

Implicated in

Top note
AFDN is implicated in a few translocations and/or fusion genes, in particular in the t(6;11) seen in leukemia (see Figure 5).
Down regulation of afadin appears to drive to carcinogenesis in a number of cancer types.
Entity name
t(6;11)(q27;q23)/AML --> KMT2A/AFDN
Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) of children and adults. Translocation t(6;11) represent about 5% of acute leukemia with 11q23/KMT2A rearrangement and is more frequent in AML than in ALL (Prasad et al., 1993; review in Huret 2018).
The prognosis is poor.
may be overlooked; The t(6;11) is the sole abnormality in most cases, but may be accompanied with, +8, +19 and+21.
Hybrid gene
Fus prot
NH2-term KMT2A (with the AT hook and DNA binding motifs) is fused to most of AFDN. KMT2A/AFDN, through constitutive self-association and in cooperation with the histone-methyltransferase DOT1L, activates aberrant gene expression (Deshpande et al., 2013). KMT2A/AFDN directly upregulates BHLHE41 (SHARP1) by DOT1L. SHARP1 binds to transcriptionally active chromatin. Suppression of SHARP1 induces robust apoptosis. The circadian clock transcription factor SHARP1 as an oncogenic target in KMT2A/AFDN cells (Numata et al., 2018). AFDN is expressed in the cytoplasm of normal cells and controls RAS levels. By contrast, in KMT2A/AFDN localize in the nucleus, leading to aberrant activation of RAS and of its downstream targets (Joh et al. 1997).
Entity name
Breast carcinoma
In breast cancer, loss of afadin protein expression induces cell migration and cell invasion (Yamamoto et al., 2015a), and is associated with adverse prognosis (Letessier et al., 2007; Fournier et al., 2011). Afadin loss of expression (in 15% of breast carcinoma) is also associated with an increased risk of metastatic relapse (Fournier et al., 2011).
The nuclear localization of L-Afadin, regulated by phosphorylation at Ser1718 by the Akt pathway, is clinically relevant for breast cancer progression (Ellol et al. 2014).
The fusion gene AFDN/ HMGCLL1 has also been found in breast adenocarcinoma (Hu et al. 2018).
Entity name
Uterus cancer
Afadin expression was significantly associated with myometrial invasion and high histological grade in uterine corpus endometrial carcinoma (Yamamoto et al., 2015a).
The fusion gene ATG5/AFDN has been found in uterine carcinosarcoma (Hu et al. 2018).
Entity name
Ovarian serous cystadenocarcinoma
The fusion gene AFDN/ UNC93A has been found in ovarian serous cystadenocarcinoma (Yoshihara et al., 2015).
Entity name
Prostate adenocarcinoma
The fusion gene AFDN/ EXOC6B has been found in prostate adenocarcinoma (Yoshihara et al., 2015).
The fusion gene AFDN/ SYK has also been found in prostate adenocarcinoma (Hu et al. 2018).
Entity name
Clear cell renal cell carcinoma
The fusion gene AFDN/ TTLL2 has been found in clear cell renal cell carcinoma (Hu et al. 2018).
Entity name
Colon cancer
Lower expression of CFTR and/or afadin is correlated with a poor prognosis in colon cancer (Sun et al., 2014). Loss of afadin induces cell migration and cell invasion (Yamamoto et al., 2015a).
Entity name
Pancreatic cancer.
Afadin is expressed at low levels in pancreatic cancer. Depletion of Afadin promotes proliferation through upregulation of the expression of SNAIL proteins, and this requires the nuclear localization of afadin (Xu et al., 2015).
Entity name
Gastric cancer
Helicobacter pylori disrupts cell-cell junctions through down regulation of afadin and induces epithelial to mesenchymal transition (EMT) of gastric cells, leading to the acquisition of an aggressive phenotype, which can contribute to gastric carcinogenesis (Marques et al., 2018).
AFDN antisense RNA 1 ( AFDN-AS1) is significantly downregulated in gastric cancer and a predictor of a poor prognosis (Lai et al., 2017).

Entity name
Low grade glioma
The fusion gene AFDN/ SASH1 has been found in low grade glioma (Yoshihara et al., 2015).
Entity name
High expression of CLDN2 (claudin2) induces high expression of afadin, which results in silencing of the MAPK signaling pathway and inhibits the metastasis phenotype in osteosarcoma cells (Zhang et al., 2018).
Entity name
Lung squamous cell carcinoma
The fusion gene AFDN/ GMDS has been found in squamous cell carcinoma of the lung (Hu et al. 2018).
Entity name
Neurone synapse and nucleus
Afadin signaling at synapses contributes to activity-dependent spine morphogenic activity. Following stimulation by 17β-estradiol, afadin locates to both synapses and the nucleus. Accumulation of afadin in the nucleus induces phosphorylation of kinases MAPK3 / MAPK1 (pERK1/2), phosphorylation of RPS6KA1 (p90RSK) that can directly phosphorylate histone H3 at serine 10 (H3S10p.). This in turn contributes to long term alterations in synapse structure (VanLeeuwen et al., 2014; Sellers et al., 2018).


Atlas Image
Figure 5 ADFN fusion partners.


Pubmed IDLast YearTitleAuthors
120677212002The LIM domain protein Lmo2 binds to AF6, a translocation partner of the MLL oncogene.Bégay-Müller V et al
109220602000The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin.Boettner B et al
170138122007AF6/s-afadin is a dual residency protein and localizes to a novel subnuclear compartment.Buchert M et al
267516422016Concomitant binding of Afadin to LGN and F-actin directs planar spindle orientation.Carminati M et al
233619072013Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l.Deshpande AJ et al
242699532014The adherens junction protein afadin is an AKT substrate that regulates breast cancer cell migration.Elloul S et al
214789122011Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth.Fournier G et al
255345542015Crystal structure of afadin PDZ domain-nectin-3 complex shows the structural plasticity of the ligand-binding site.Fujiwara Y et al
145077822003Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6.Gaengel K et al
288601152017Afadin orients cell division to position the tubule lumen in developing renal tubules.Gao L et al
97075521998PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.Hock B et al
290999512018TumorFusions: an integrative resource for cancer-associated transcript fusions.Hu X et al
93495011997Chimeric MLL products with a Ras binding cytoplasmic protein AF6 involved in t(6;11) (q27;q23) leukemia localize in the nucleus.Joh T et al
171829002007Rap1: a key regulator in cell-cell junction formation.Kooistra MR et al
85576591996Identification of AF-6 and canoe as putative targets for Ras.Kuriyama M et al
289270282017Decreased expression of the long non-coding RNA MLLT4 antisense RNA 1 is a potential biomarker and an indicator of a poor prognosis for gastric cancer.Lai Y et al
168195132007Correlated break at PARK2/FRA6E and loss of AF-6/Afadin protein expression are associated with poor outcome in breast cancer.Letessier A et al
222118082012The effector and scaffolding proteins AF6 and MUPP1 interact with connexin36 and localize at gap junctions that form electrical synapses in rodent brain.Li X et al
102241251999Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily.Linnemann T et al
168826942006Regulation of epithelial wound closure and intercellular adhesion by interaction of AF6 with actin cytoskeleton.Lorger M et al
246958512014MLL-AF6 fusion oncogene sequesters AF6 into the nucleus to trigger RAS activation in myeloid leukemia.Manara E et al
234812062013Afadin/AF-6 and canoe: roles in cell adhesion and beyond.Mandai K et al
304736882018Afadin Downregulation by Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Gastric Cells.Marques MS et al
42699531973Newer antibiotics.Neu HC et al
296924082018The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia.Numata A et al
167543282006Nectins and nectin-like molecules: roles in cell adhesion, polarization, movement, and proliferation.Ogita H et al
278154082017EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions.Perez White BE et al
76678781995AF-6/cno: neither a kinesin nor a myosin, but a bit of both.Ponting CP et al
82426161993Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation.Prasad R et al
104461491999M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6.Quilliam LA et al
284828672017Afadin controls cell polarization and mitotic spindle orientation in developing cortical radial glia.Rakotomamonjy J et al
257122702015Afadin regulates RhoA/Rho-associated protein kinase signaling to control formation of actin stress fibers in kidney podocytes.Saito K et al
179422952007The roles of nectins in cell adhesions: cooperation with other cell adhesion molecules and growth factor receptors.Sakisaka T et al
302685212018Cyto-nuclear shuttling of afadin is required for rapid estradiol-mediated modifications of histone H3.Sellers KJ et al
243738472014Disrupted interaction between CFTR and AF-6/afadin aggravates malignant phenotypes of colon cancer.Sun TT et al
109740032000Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins.Tachibana K et al
248240682014Dynamic contacts: rearranging adherens junctions to drive epithelial remodelling.Takeichi M et al
97226161998The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.Taya S et al
265682952016Afadin is localized at cell-cell contact sites in mesangial cells and regulates migratory polarity.Tsurumi H et al
245673312014Coordinated nuclear and synaptic shuttling of afadin promotes spine plasticity and histone modifications.VanLeeuwen JE et al
260131252015Loss of polarity protein AF6 promotes pancreatic cancer metastasis by inducing Snail expression.Xu Y et al
259888342015Impairment of radial glial scaffold-dependent neuronal migration and formation of double cortex by genetic ablation of afadin.Yamamoto H et al
93482941997The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells.Yamamoto T et al
255005442015The landscape and therapeutic relevance of cancer-associated transcript fusions.Yoshihara K et al
303494222018CLDN2 inhibits the metastasis of osteosarcoma cells via down-regulating the afadin/ERK signaling pathway.Zhang X et al

Other Information

Locus ID:

NCBI: 4301
MIM: 159559
HGNC: 7137
Ensembl: ENSG00000130396


dbSNP: 4301
ClinVar: 4301
TCGA: ENSG00000130396


Gene IDTranscript IDUniprot

Expression (GTEx)



PathwaySourceExternal ID
Adherens junctionKEGGko04520
Tight junctionKEGGko04530
Leukocyte transendothelial migrationKEGGko04670
Adherens junctionKEGGhsa04520
Tight junctionKEGGhsa04530
Leukocyte transendothelial migrationKEGGhsa04670
Ras signaling pathwayKEGGhsa04014
Rap1 signaling pathwayKEGGhsa04015
Rap1 signaling pathwayKEGGko04015
cAMP signaling pathwayKEGGhsa04024
cAMP signaling pathwayKEGGko04024
Cell-Cell communicationREACTOMER-HSA-1500931
Cell junction organizationREACTOMER-HSA-446728
Cell-cell junction organizationREACTOMER-HSA-421270
Adherens junctions interactionsREACTOMER-HSA-418990

Protein levels (Protein atlas)

Not detected


Pubmed IDYearTitleCitations
191767532009Junctional adhesion molecule A interacts with Afadin and PDZ-GEF2 to activate Rap1A, regulate beta1 integrin levels, and enhance cell migration.74
203796142010Personalized smoking cessation: interactions between nicotine dose, dependence and quit-success genotype score.62
238851232013JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function.44
97226161998The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.35
204137832010Role of afadin in vascular endothelial growth factor- and sphingosine 1-phosphate-induced angiogenesis.32
168195132007Correlated break at PARK2/FRA6E and loss of AF-6/Afadin protein expression are associated with poor outcome in breast cancer.30
128081052003The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain.29
243738472014Disrupted interaction between CFTR and AF-6/afadin aggravates malignant phenotypes of colon cancer.27
217323592012Afadin controls p120-catenin-ZO-1 interactions leading to endothelial barrier enhancement by oxidized phospholipids.25
160516022005AF6 negatively regulates Rap1-induced cell adhesion.23


Jean Loup Huret ; Philippe Dessen

AFDN (afadin, adherens junction formation factor)

Atlas Genet Cytogenet Oncol Haematol. 2018-12-01

Online version:

Historical Card

1997-12-01 AFDN (afadin, adherens junction formation factor) by  Jean-Loup Huret 

Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France