The Ras Target AF-6 Interacts with ZO-1 and Serves as a Peripheral Component of Tight Junctions in Epithelial Cells

Abstract

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 are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) 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. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated 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 cells resulted 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.

Different behavior of l-Afadin and Neurabin-II during the formation and destruction of cell – cell adherens junction

“…In contrast to our results, another group has reported that the AF-6 protein (s-afadin) binds to Factin, is present in MDCK cells and is localized at ZO by interacting with ZO-1 in MDCK cells (Yamamoto et al, 1997). We ®rst examined here which afadin is expressed in MDCK cells and then where the expressed protein is localized in this cell line.…”

“…No maltose-binding protein (MBP)-fusion protein of the N-terminal, middle or C-terminal fragment of l-afadin bound to a glutathione Stransferase (GST)-fusion protein of the N-terminal fragment of ZO-1 immobilized on glutathione-Sepharose (data not shown), in contrast to the result reported by Yamamoto et al (1997). No MBP fusion protein of l-afadin bound to a GST-fusion protein of the cytoplasmic region of occludin (data not shown).…”

“…It has been shown that the AF-6 protein (s-afadin) is expressed in MDCK cells (Yamamoto et al, 1997), but we have shown here that l-afadin is mainly expressed in this cell type. This inconsistency is just due to the fact that when Yamamoto et al (1997) reported the paper, l-afadin was not found and that they used an Ab against the AF-6 protein (s-afadin) which might recognize l-afadin.…”

“…This inconsistency is just due to the fact that when Yamamoto et al (1997) reported the paper, l-afadin was not found and that they used an Ab against the AF-6 protein (s-afadin) which might recognize l-afadin. Yamamoto et al (1997) have moreover shown that the AF-6 protein (s-afadin) is localized at ZO.…”

“…This inconsistency is just due to the fact that when Yamamoto et al (1997) reported the paper, l-afadin was not found and that they used an Ab against the AF-6 protein (s-afadin) which might recognize l-afadin. Yamamoto et al (1997) have moreover shown that the AF-6 protein (s-afadin) is localized at ZO. However, it is practically di cult to conclude whether a certain protein is localized at ZA or ZO in cultured MDCK cells, because these two structures are not well separated in MDCK cells, in contrast to those in intestine absorptive epithelial cells where they are well separated.…”