Ponsin/SH3P12: An 1-afadin- and vinculin-binding protein localized at cell-cell and cell-matrix adherens junctions

We recently isolated a novel actin filament (F-actin)-binding protein, afad in, that has two isoforms, 1- and s-afadins. 1-Afadin is ubiquitously expre ssed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell-cell adherens junction (AJ) in nonepithelial cells, whereas s- afadin is abundantly expressed in neural tissue. 1-Afadin has one PDZ domai n, three proline-rich regions, and one F-actin-binding domain, whereas s-af adin lacks the third proline-rich region and the F-actin-binding domain. To understand the molecular mechanism of the specific localization of 1-afadi n at ZA in epithelial cells and at cell-cell AJ in nonepithelial cells, we attempted here to identify an 1-afadin-binding protein(s) and isolated a pr otein, named ponsin. Ponsin had many splicing variants and the primary stru ctures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of 1-afadin bound to the region of ponsin con taining the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at eel-matrix AJ in both types of cells. Ponsin furthermore dire ctly bound vinculin, an F-actin-binding protein localized at ZA in epitheli al cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline -rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. 1-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly for med a ternary complex. These results indicate that ponsin is an 1-afadin- a nd vinculin-binding protein localized at ZA in epithelial cells, at cell-ce ll AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells.