Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, a yeast two-hybrid screening was carried out for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibits a strong structural homology with human AF-6, rat Afadin and Drosophila Canoe and possesses both a Ras-associating (RA) domain and a PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associates with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras (Watari, 1998).

Reciprocal chromosome translocations involving 11q23 are frequently associated with acute leukemias, with the t(4;11) translocation predominating among acute lymphoblastic leukemias, and the t(9;11), t(11;19) and t(6;11) translocations most common among acute myeloid leukemias. In each of these translocations the ALL-1 gene, located at 11q23 and constituting the human homolog of Drosophila trithorax, fuses to a specific gene on the partner chromosome to produce a chimeric protein. The partner gene from chromosome 6 (AF-6) is expressed in a variety of cell types and encodes a protein of 1612 amino acids. The protein contains short stretches rich in prolines, charged amino acids, serines, or glutamines. In addition, the AF-6 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 (Prasad, 1993).

The dynamic rearrangement of cell-cell junctions (such as tight junctions and adherens junctions) is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions contain associated proteins such as occludin and its associated ZO-1 and ZO-2 and adherens junctions contain associated adhesion proteins such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. The ALL-1 fusion partner from chromosome 6 (AF-6) has been identified as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes, such as cell-cell contact sites. The roles of Ras and AF-6 were investigated in the regulation of cell-cell contacts and it was found that AF-6 accumulates at the cell-cell contact sites of polarized MDCKII epithelial cells and has a distribution similar to that of ZO-1 but somewhat different from that of catenins. Immunoelectron microscopy reveales a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacts with ZO-1 in vitro. ZO-1 interacts with the Ras-binding domain of AF-6, and this interaction is inhibited by activated Ras. AF-6 accumulates with ZO-1 at the cell-cell contact sites in cells lacking tight junctions, such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 fibroblasts results in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells, and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras (Yamamoto, 1997).

In infantile leukemias and therapy-related leukemias, the MLL gene is frequently found to be disrupted and fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL as a result of 11q23 translocations. The N-terminal portion common to various chimeric MLL products, as well as to MLL-LTG9 and MLL-LTG19, localizes in the nuclei, and therefore might play an important role in leukemogenesis. In the present study, MLL-AF6 chimeric products found in the t(6;11)(q27;q23) translocation were analysed, since AF6, a Ras-binding protein, exhibits a different subcellular localization from that of LTG9/AF9 and LTG19/ENL. Immunofluorescence staining data and cell fractionation analyses demonstrate that MLL-AF6 chimeric products localize in the nuclei despite the fact that AF6 itself localizes in the cytoplasm, confirming the importance of the nuclear localization of chimeric MLL products. The region in the N-terminal portion of MLL responsible for this nuclear localization is a region containing AT-hook motifs (Joh, 1997).

The Drosophila fat facets and canoe genes regulate non-neural cell fate decisions during ommatidium formation. The FAM (Fat facets in mouse) de-ubiquitinating enzyme regulates the function of AF-6 (mammalian Canoe homolog) in the MDCK epithelial cell line. The expression of the FAM and AF-6 proteins overlaps extensively in the mouse eye from embryogenesis to maturity, especially in the non-neural epithelia including the retinal pigment epithelium, subcapsular epithelium of the lens and corneal epithelium. Expression is not limited to the epithelia however, because FAM and AF-6 also co-localize during lens fiber development as well as in sub-populations of the neural retina (Kanai-Azuma, 2000).

Par-3 is a cell-polarity protein that regulates the formation of tight junctions (TJs) in epithelial cells, where claudin is a major cell-cell adhesion molecule (CAM). TJs are formed at the apical side of adherens junctions (AJs), where E-cadherin and nectin are major CAMs. Nectin first forms cell-cell adhesions, and then recruits cadherin to nectin-based cell-cell adhesion sites to form AJs and subsequently recruits claudin to the apical side of AJs to form TJs. The cytoplasmic tail of nectin binds afadin and Par-3. Afadin regulates the formation of AJs and TJs cooperatively with nectin. This paper deals with the role of Par-3 in the formation of these junctions by using Par-3-knockdown MDCK cells. Par-3 is necessary for the formation of AJs and TJs but was not necessary for nectin-based cell-cell adhesion. Par-3 promotes the association of afadin with nectin, whereas afadin is not necessary for the association of Par-3 with nectin. However, the association of afadin with nectin alone is not sufficient for the formation of AJs or TJs, and Par-3 and afadin cooperatively regulates it. This paper describes these novel roles of Par-3 in the formation of junctional complexes (Ooshio, 2007).

Junctional adhesion molecule-A (JAM-A) is a transmembrane tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. JAM-A regulates cell migration by dimerization of the membrane-distal immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding motif. Disruption of dimerization results in decreased epithelial cell migration secondary to diminished levels of beta1 integrin and active Rap1. This study reports that JAM-A is physically and functionally associated with the PDZ domain-containing molecules Afadin and PDZ-guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both Afadin and PDZ-GEF2 colocalize and coimmunoprecipitate with JAM-A. Furthermore, association of PDZ-GEF2 with Afadin is dependent on the expression of JAM-A. Loss of JAM-A, Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreases cellular levels of activated Rap1, beta1 integrin protein, and epithelial cell migration. The functional effects observed are secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, results in decreased beta1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with Afadin and PDZ-GEF2 that activates Rap1A, which regulates beta1 integrin levels and cell migration (Severson, 2009).