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© The Rockefeller University Press, 0021-9525/1999//1001 $5.00
The Journal of Cell Biology, Volume 144, Number 5, , 1999 1001-1018

Regular Articles

Ponsin/SH3P12: An l-Afadin– and Vinculin-binding Protein Localized at Cell–Cell and Cell–Matrix Adherens Junctions



Kenji Mandai*, Hiroyuki Nakanishi*, Ayako Satoh*, Kenichi Takahashi*, Keiko Satoh*, Hideo Nishioka*, Akira Mizoguchi{ddagger}, and Yoshimi Takai*,§

* Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan; {ddagger} Department of Anatomy and Neurobiology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan; and § Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565-0871, Japan

We recently isolated a novel actin filament (F-actin)–binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed 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. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin–binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin–binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell–cell AJ in nonepithelial cells, we attempted here to identify an l-afadin–binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures 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 l-afadin bound to the region of ponsin containing 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 cell–matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin–binding protein localized at ZA in epithelial 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. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin– and vinculin-binding protein localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells.

Key Words: ponsin • l-afadin • vinculin • zonula adherens • cell–matrix adherens junction

Abbreviations used in this paper: aa, amino acid; AJ, adherens junction; F-actin, actin filament; FISH, fluorescence in situ hybridization; GST, glutathione S-transferase; His6, six histidine residues; MBP, maltose-binding protein; MDCK cells, Madin-Darby canine kidney cells; SH3, Src homology 3; ZA, zonula adherens; ZO, zonula occludens.


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