Alternative titles; symbolsMYELOID/LYMPHOID OR MIXED LINEAGE LEUKEMIA, TRANSLOCATED TO, 4; MLLT4MIXED LINEAGE LEUKEMIA, TRANSLOCATED TO, 4ALL1-FUSED GENE FROM CH...
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HGNC Approved Gene Symbol: AFDN
Cytogenetic location: 6q27 Genomic coordinates (GRCh38): 6:167,826,563-167,972,022 (from NCBI)
AF6 is a Ras (see HRAS; 190020) target that regulates cell-cell adhesions downstream of Ras activation. It is fused with MLL (159555) in leukemias caused by t(6;11) translocations (Taya et al., 1998).
▼ Cloning and Expression
Most acute leukemias in infancy and at least 5% of acute lymphoblastic leukemias and acute myeloid leukemias of older children and adults show abnormalities of chromosome band 11q23. In these cases, translocation results in fusion of a gene at 11q23, variously called ALL1, MLL, and the human homolog of Drosophila 'trithorax' (159555), with part of a gene on chromosome 4 (159557), chromosome 9 (159558), or chromosome 19 (159556). Prasad et al. (1993) described the cloning and characterization of the 'partner gene' involved in a fourth common translocation involving 11q23, t(6;11)(q27;q23). The gene, designated AF6 by them, was found to be expressed in a variety of cell types and to encode a protein of 1,612 amino acids. The protein contains short stretches rich in proline, charged amino acids, serines, or glutamines. In addition, the AF6 protein contains the GLGF motif shared with several proteins of vertebrates and invertebrates thought to be involved in signal transduction at special cell-cell junctions.
Saito et al. (1998) identified 2 splice variants of AF6 that use an alternative 3-prime splice site in exon 28 and contain additional downstream exons compared with the AF6 cDNA reported by Prasad et al. (1993). The deduced proteins contain 1,817 and 1,744 amino acids and have different C termini. Saito et al. (1998) noted that the AF6 protein has 2 distinct features: a Dlg (see 601014) homology repeat (DHR)/GLGF domain and a Ras-interacting domain.
By Western blot analysis, Boettner et al. (2000) detected AF6 proteins of about 195 and 180 kD in human lung, kidney, testis, ovary, pancreas, but not in spleen and heart. AF6 localized to areas of cell-cell junctions in confluent cultures of canine kidney cells and MCF7 human breast carcinoma cells. AF6 colocalized with ZO1 (TJP1; 601009) at the apical surface of MCF7 cells, but not with beta-catenin (see 116806) in basal surfaces. MCF7 cells showed increasing AF6 intensity and membrane localization as they established full adherens potential with neighboring cells. In completely confluent cell layers, AF6 staining increased in the cytoplasm.
▼ Gene Function
Using protein pull-down assays with in vitro translated cDNAs, Kuriyama et al. (1996) found that an N-terminal domain of bovine Af6 interacted with Hras bound to a nonhydrolyzable GTP analog and much more weakly with GDP-Hras, Rras (165090), Rala (179550), and Rhoa (ARHA; 165390). Mutation analysis indicated that the GTP-Ras-interacting domain was between amino acids 36 and 206 of bovine Af6.
Taya et al. (1998) showed that Af6 interacted with Fam (USP9X; 300072), a deubiquitinating enzyme, in bovine brain cytosol. Af6 interacted with Fam in a ratio of about 1:10 in canine kidney cells. Fam accumulated at cell-cell contact sites in canine kidney cells and partially colocalized with Af6. Af6 was ubiquitinated in intact cells, and Fam prevented ubiquitination of Af6.
By mutation analysis, Boettner et al. (2000) determined that the first of 2 N-terminal Ras-interacting domains in human AF6 bound HRAS, KRAS (KRAS2; 190070), and NRAS (164790); the second Ras-binding domain did not bind these Ras proteins. RAP1A (179520) also associated with this motif. HRAS and RAP1A interacted with full-length AF6 in vivo in cotransfected COS cells, and a fraction of RAP1A colocalized with AF6 at the plasma membrane. Using a yeast 2-hybrid screen of Jurkat human T cells, Boettner et al. (2000) found that residues 912 to 1,612 of AF6 interacted with profilin-1 (PFN1; 176610) and -2 (PFN2; 176590); the interaction between AF6 and profilin-1 was more pronounced. A direct interaction between the 2 proteins was confirmed by coimmunoprecipitation of profilin-1 from AF6-transfected COS cells. Since profilin participates in cortical actin assembly, Boettner et al. (2000) hypothesized that AF6 may modulate actin modeling by profilin.
Begay-Muller et al. (2002) found that rat Af6 interacted with amino acids 27 to 158 of human LMO2 (180385), a gene expressed in myeloid and erythroid precursor cells. They suggested that the chimeric MLL/AF6 protein may cause leukemias through the interaction of the AF6 portion of the protein with LMO proteins at hematopoietic gene regulatory complexes.
Zhang et al. (2005) found that knockdown of AF6 by RNA interference in Jurkat cells enhanced RAP1-induced integrin (see ITGB1; 135630)-mediated cell adhesion. Overexpression of AF6 inhibited RAP1-induced cell adhesion, but AF6 did not inhibit Mn(2+)-induced cell adhesion, where integrins are activated directly by divalent cations. Full-length AF6 was much more efficient in inhibiting cell adhesion than the isolated Ras association domain of AF6. Expression of AF6 increased endogenous RAP1-GTP levels and appeared to stabilize RAP1 in the GTP-bound state. Zhang et al. (2005) concluded that AF6 inhibits RAP1-mediated cell adhesion by sequestering RAP1-GTP in an unproductive complex, thus preventing interaction of RAP1 with effectors that mediate adhesion and with RAP GTPase-activating proteins.
▼ Gene Structure
Saito et al. (1998) determined that the MLLT4 gene contains 32 exons and spans about 140 kb.
By FISH, Saha et al. (1995) mapped the AF6 gene to chromosome 6q27, distal to the critically deleted region associated with ovarian malignancies (167000). AF6 is therefore distinct from and lies telomeric to that region.
Prasad et al. (1993) identified AF6 as the partner of MLL in a common translocation, t(6;11)(q27;q23), associated with leukemia. Using RACE-PCR, Saha et al. (1995) confirmed the breakpoint in AF6.
The t(6;11)(q27;q23) translocation results in a chimeric MLL/AF6 protein with a calculated molecular mass of 325 kD. In the chimeric protein, the N-terminal portion of MLL, including 3 AT hook motifs, is fused to all of AF6 except the first 35 amino acids, leaving the Ras-interacting domain and the DHR motif of AF6 intact. By Western blot analysis of transfected COS cells and a human cell line with the t(6;11)(q27;q23) translocation, Joh et al. (1997) found that the MLL/AF6 fusion protein had an apparent molecular mass of 360 kD. Immunolocalization and cell fractionation followed by Western blot analysis indicated that MLL/AF6 was targeted to the nucleus, whereas AF6 itself was cytoplasmic. Mutation analysis indicted that the region of MLL containing AT hook motifs was responsible for the nuclear localization of the chimeric protein.
▼ Molecular Genetics
Saito et al. (1998) identified an insertion/deletion polymorphism in AF6 that determines the presence or absence of a 21-bp exon, which they designated exon 14a. Insertion of exon 14a results in a protein with 7 additional amino acids between the Ras-interacting domain and the DHR motif. Saito et al. (1998) estimated that the insertion has a frequency of 0.35.