The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin

The AF-6 protein is a multidomain protein that contains two potential Ras-b inding domains within its N terminus. Because of this feature, AF-6 has bee n isolated in both two-hybrid and biochemical approaches and is postulated to be a potential Ras-effector protein, Herein, we show that it is specific ally the first Ras-binding domain of AF-6 that mediates this interaction an d that the Ras-related Rap1A protein can associate with this motif even mor e efficiently than the oncogenic Ha-, K-, and N-Ras GTPases. We further dem onstrate that both Ras and Rap1 interact with full-length AF-6 in vivo in m ammalian cells and that a fraction of Rap1 colocalizes with AF-6 at the mem brane. Dominant active Rap1A, in contrast to Ras, when introduced into epit helial MDCK and MCF-7 cells, does not perturb AF-6-specific residency in ce ll-cell adhesion complexes. In a pursuit to gain further understanding of t he role of AF-6 in junctions, we identified profilin as an AF-6-binding pro tein. Profilin activates monomeric actin units for subsequent polymerizatio n steps at barbed ends of actin filaments and has been shown to participate in cortical actin assembly. To our knowledge, AF-6 is the only integral co mponent in cell-cell junctions discovered thus far that interacts with prof ilin and thus could modulate actin modeling proximal to adhesion complexes.