In this study, we isolated an l-afadin– and vinculin-binding protein, named ponsin, which was ubiquitously expressed and colocalized with vinculin at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells (Fig. ). We first purified three 35S-l-afadin–binding proteins, p95, p93, and p70, from the AJ-enriched fraction of rat liver. We then analyzed the peptide maps and sequences of these proteins and found that they were related to each other. Based on their peptide sequences, we obtained the mouse cDNAs of ponsin-1 and -2. Ponsin-1 was likely a mouse counterpart of rat p93. However, ponsin-2 did not show the same mobility to that of p95 or p70 on SDS-PAGE. During the molecular cloning of ponsin, we obtained at least 12 independent clones, all of which had the similar restriction maps. Northern blot analysis indicated that ponsin had several mRNA bands in heart, brain, liver, and skeletal muscle. When the aa sequences of ponsin-1 and -2 were aligned together with that of SH3P12, they were highly homologous but their differences were clustered and not distributed throughout the sequences. Moreover, the FISH analysis indicated that ponsin-1 and -2 were located at the same locus (data not shown). We have determined neither the genetic locus of SH3P12 nor the clones other than ponsin-1 and -2, but ponsin/SH3P12 had many splicing variants, and all of these variants might be derived from the same gene. The exact relationship of the 35S-l-afadin–binding proteins (p95 and p70) and ponsin-1 and -2 has not been clarified here, but there are two possibilities: (a) p95 and p70 are splicing variants of ponsin other than ponsin-1 and -2, and (b) they are posttranslationally modified forms of ponsin-1 or -2, such as the phosphorylated or proteolytic form. The physiological significance of the presence of many splicing variants of ponsin is unknown at present.
Schematic diagram of ZO, ZA, and cell–matrix AJ.
We presented the evidence that l-afadin bound to ponsin by four different methods: (a) blot overlay, (b) affinity chromatography, (c) immunoprecipitation, and (d) immunofluorescence and immunoelectron microscopy. Blot overlay analysis indicated that ponsin-2 appeared to bind l-afadin more than ponsin-1, but their exact binding affinities remain to be clarified. We moreover showed that the binding regions of l-afadin and ponsin were the third proline-rich region and the region containing the second and third SH3 domains, respectively, consistent with the earlier observation that proline-rich sequences interact with either profilin or SH3 domains (Tanaka and Shibata, 1985
; Yu et al., 1994
). Affinity chromatography analysis on each SH3 domain indicated that the third SH3 domain bound to l-afadin, whereas neither the first nor second SH3 domain bound, suggesting that the third SH3 domain is the minimal l-afadin–binding region. However, the l-afadin–binding activity of the third SH3 domain alone was less than that of the region containing the second and third SH3 domains. It remains to be clarified why the l-afadin–binding activity of ponsin is reduced when the second and third SH3 domains are separated from each other, but the highly ordered structure of the region containing the second and third SH3 domains may be necessary for the more efficient binding of ponsin to l-afadin. In contrast to l-afadin, s-afadin, which has the first and second proline-rich regions but lacks the third proline-rich region, hardly bound to ponsin. Neurabin-II, which also has three proline-rich regions (Satoh et al., 1998
), did not bind to ponsin either (data not shown). These results suggest that the SH3 domains of ponsin recognize a specific proline-rich region.
Computer analysis indicated that ponsin-1 and -2 had three SH3 domains and two sorbin-like regions, but no transmembrane region or other known signaling domains. However, ponsin was localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells (Fig. ). These results suggest that there is another protein that determines the specific localization of l-afadin at these specific sites of cells. Along this line, we found here that ponsin-1 and -2 bound vinculin, which interacts with α-catenin at cell–cell AJ (Watabe-Uchida et al., 1998
; Weiss et al., 1998
) and with talin at cell–matrix AJ (Burridge and Mangeat, 1984
). α-Catenin interacts with cadherin through β-catenin (Aberle et al., 1994
), whereas talin directly interacts with integrin (Horwitz et al., 1986
). We presented here the evidence that ponsin directly bound vinculin by four different methods: (a) blot overlay, (b) affinity chromatography, (c) immunoprecipitation, and (d) immunofluorescence and immunoelectron microscopy. Moreover, we showed that the binding regions of ponsin and vinculin were the region containing the first and second SH3 domains and the proline-rich region, respectively. The proline-rich region of vinculin is divided into at least two proline-rich sequences, the first of which binds to vasodilator-stimulated phosphoprotein (VASP) (Brindle et al., 1996
; Niebuhr et al., 1997
). We have not determined which proline-rich sequence is responsible for the binding to ponsin. Blot overlay and affinity chromatography analyses on each SH3 domain indicated that the second SH3 domain bound to vinculin, whereas neither the first nor third SH3 domain bound, suggesting that the second SH3 domain is the minimal vinculin-binding region. However, the vinculin-binding activities of the second SH3 domain alone and the region containing the second and third SH3 domains were less than that of the region containing the first and second SH3 domains. It remains to be clarified why the vinculin-binding activity of ponsin is reduced when the first and second SH3 domains are separated from each other. The highly ordered structure of the region containing the first and second SH3 domains may be necessary for the more efficient binding of ponsin to vinculin.
The result (the l-afadin– and vinculin-binding regions of ponsin were partly overlapped) suggested that l-afadin and vinculin bind to ponsin in a competitive manner and that these three proteins do not form a ternary complex. Consistently, we found that l-afadin and vinculin bound to ponsin in a competitive manner and that the affinities of l-afadin and vinculin to ponsin were apparently similar. We found here by immunoprecipitation and affinity chromatography analyses that ponsin formed a binary complex with either l-afadin or vinculin, but hardly formed a ternary complex with l-afadin and vinculin. This result suggests that ponsin does not serve as a direct linker between l-afadin and vinculin. The physiological significance of the result that ponsin forms a binary complex with either l-afadin or vinculin at the same time but not a ternary complex with the two F-actin–binding proteins is currently unknown, but ponsin may independently regulate the function of each protein, or may regulate the linkage between l-afadin and vinculin in cooperation with another still unidentified factor. If the latter is the case, l-afadin may be localized at ZA in epithelial cells and at cell–cell AJ in nonepithelial cells by interacting with the cadherin-catenin system through the ponsin-vinculin system (Fig. ).
ZO and ZA in the junctional complex of polarized epithelial cells are closely aligned from the apical side to the basal side, suggesting that these two junction structures have their molecular interactions. ZO is also linked to the actin cytoskeleton. At ZO, multiple integral membrane proteins with four transmembrane regions, the claudin family members and occludin, constitute tight junction strands (Furuse et al., 1993
; Ando-Akatsuka et al., 1996
). The cytoplasmic region of occludin binds ZO-1 (Furuse et al., 1994
), which interacts with F-actin (Itoh et al., 1997
). The cytoplasmic regions of the claudin family members may also directly or indirectly bind ZO-1 (Furuse et al., 1998
). Evidence is accumulating that the cadherin-catenin system plays essential roles for the assembly of the junctional complex (Gumbiner and Simons, 1986
; Gumbiner et al., 1988
; Watabe et al., 1994
). It has recently been shown by use of an α-catenin–deficient colon carcinoma cell line that the binding of vinculin to α-catenin is required for the organization of ZO (Watabe-Uchida et al., 1998
). It has furthermore been shown that the junctional organization is impaired in vinculin-null F9 cells (Watabe-Uchida et al., 1998
). It should be noted from the present and previous (Mandai et al., 1997
) results that l-afadin and ponsin as well as vinculin are more highly concentrated at ZA than cadherin. The afadin-ponsin system may be involved in the assembly of the junctional complex by interacting with vinculin.
We recently found that l-afadin and E-cadherin showed different behavior during the formation and destruction of cell–cell AJ in MDCK and L cells (Sakisaka et al., 1999
). Dissociation of MDCK cells by culturing the cells at 2 μM Ca2+
caused rapid endocytosis of E-cadherin, but not that of l-afadin or ZO-1. Addition of phorbol 12-myristate 13-acetate to these dissociated cells formed a ZO-like structure where ZO-1 and l-afadin, but not E-cadherin, accumulated. Even in cadherin-deficient L cells, l-afadin was mainly localized at cell–cell contacts, whereas ZO-1 was mainly localized at the tip area of cell processes. l-Afadin did not directly bind to α-, β-catenin, E-cadherin, ZO-1, or occludin. All of the results thus far available suggest that there is an integral membrane protein that is specifically localized at ZA in epithelial cells and at cell–cell AJ in nonepithelial cells and interacts with the afadin–ponsin– vinculin system (Fig. ). It would be of crucial importance to identify this protein for our understanding of the physiological significance of l-afadin and ponsin.
Ponsin-1 and -2 were splicing variants of SH3P12. SH3P12 was suddenly renamed CAP in the GenBank database during the revision of this manuscript. CAP was originally isolated as a c-Cbl–binding protein (Ribon et al., 1998a
). c-Cbl is a proto-oncogene product involved in T cell antigen receptor–mediated signaling (Thien and Langdon, 1998
). In this CAP paper, no nucleotide or amino acid sequence information was available. Homology searches of DNA and protein databases furthermore revealed another protein structurally related to ponsin/ SH3P12, ArgBP2. ArgBP2 was isolated as an Arg- and Abl-binding protein (Wang et al., 1997
). Arg and Abl represent the members of the Aberson family of tyrosine kinase. During the revision of this manuscript, Kioka et al. (1999)
isolated a protein structurally related to ponsin/ SH3P12, named vinexin, as a vinculin-binding protein. All of these proteins have three SH3 domains and one or two sorbin-like regions. These results suggest that ponsin/ SH3P12/CAP, ArgBP2, and vinexin constitute a family. Structural comparison of these proteins suggest that ponsin/SH3P12/CAP, ArgBP2, and vinexin are derived from three different genes and constitute three subfamilies. Vinexin as well as ponsin/SH3P12 is localized at both cell– cell and cell–matrix AJs (Kioka et al., 1999
), whereas both SH3P12/CAP and ArgBP2 are associated with actin stress fibers (Wang et al., 1997
; Ribon et al., 1998b
). Vinexin enhances the actin cytoskeleton reorganization and cell spreading (Kioka et al., 1999
). SH3P12/CAP enhances actin stress fiber formation and focal adhesions and is associated with signaling molecules such as the insulin receptor, focal adhesion kinase (FAK), and SOS, a Ras small G protein exchanger (Ribon et al., 1998a
,b). Thus, the members of the family that has three SH3 domains and one or two sorbin-like regions show similar and different subcellular localization and association with other proteins, suggesting their related but diverse functions.