Two actions of frabin: direct activation of Cdc42 and indirect activation of Rac.

Frabin is an actin filament-binding protein which shows GDP/GTP exchange activity specific for Cdc42 small G protein and induces filopodium-like microspike formation and c-Jun N-terminal kinase (JNK) activation presumably through the activation of Cdc42. Frabin has one actin filament-binding (FAB) domain, one Dbl homology (DH) domain, first pleckstrin homology (PH) domain adjacent to the DH domain, one cysteine-rich FYVE domain, and second PH domain from the N-terminus to the C-terminus in this order. Different domains of frabin are involved in the microspike formation and the JNK activation, and the association of frabin with the actin cytoskeleton through the FAB domain is necessary for the microspike formation, but not for the JNK activation. We have found here that frabin induces the formation of not only filopodium-like microspikes but also lamellipodium-like structures in NIH3T3 and L fibroblasts. We have analysed the mechanism of frabin in these two actions and found that frabin induces filopodium-like microspike formation through the direct activation of Cdc42 and lamellipodium-like structure formation through the Cdc42-independent indirect activation of Rac small G protein. The FAB domain of frabin in addition to the DH domain and the first PH domain is necessary for the filopodium-like microspike formation, but not for the lamellipodium-like structure formation. The FYVE domain and the second PH domain in addition to the DH domain and the first PH domain are necessary for the lamellipodium-like structure formation. We show here these two actions of frabin in the regulation of cell morphology.

Nectin-3, a new member of immunoglobulin-like cell adhesion molecules that shows homophilic and heterophilic cell-cell adhesion activities.

We have isolated a novel cell-cell adhesion system localized at cadherin-based adherens junctions (AJs). This system consists of at least nectin, a Ca(2+)-independent immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein, that connects nectin to the actin cytoskeleton. Nectin constitutes a family consisting of two members, nectin-1 and -2. We have isolated here a third member of the nectin family and named it nectin-3. Nectin-3 has three splicing variants, nectin-3alpha (biggest), -3beta (middle), and -3gamma (smallest). Like nectin-1 and -2, nectin-3alpha consists of three extracellular immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic region with the C-terminal consensus motif for binding to the PDZ domain. Nectin-3alpha formed a cis-homo-dimer and showed Ca(2+)-independent trans-homo-interaction to cause homophilic cell-cell adhesion. Nectin-3alpha furthermore showed trans-hetero-interaction with nectin-1 or -2 but did not form a cis-hetero-dimer with nectin-1 or -2. Nectin-1 did not show trans-hetero-interaction with nectin-2. The affinity of trans-hetero-interaction of nectin-3alpha with nectin-1 or -2 was higher than that of trans-homo-interaction of nectin-1, -2, or -3alpha. Nectin-2 and -3 were ubiquitously expressed, whereas nectin-1 was abundantly expressed in brain. Nectin-3alpha was colocalized with nectin-2 at cadherin-based AJs and interacted with afadin. These results indicate that the nectin family consists of at least three members, nectin-1, -2, and -3, all of which show homophilic and heterophilic cell-cell adhesion activities and are localized at cadherin-based AJs.

Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction.

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, a homophilic cell adhesion molecule, and afadin, an actin filament-binding protein, which connects nectin to the actin cytoskeleton. Here we studied a mechanism of cell-cell adhesion of the nectin-afadin system by use of a cadherin-deficient L cell line stably expressing the intact form of mouse nectin-2alpha, a truncated form of nectin-2alpha incapable of interacting with afadin (nectin-2alpha-DeltaC), or a point-mutated form of nectin-2alpha capable of interacting with afadin and a cadherin-expressing EL cell line, which transiently expressed the point-mutated form of nectin-2alpha. We found that the interaction of nectin-2alpha with afadin was necessary for their clustering at cell-cell contact sites. However, nectin-2alpha-DeltaC showed cis dimerization and trans interaction, both of which did not require the interaction of nectin-2alpha with afadin. We have previously shown in EL cells that the interaction of nectin-1 with afadin is necessary for its recruitment to adherens junctions. We found that the trans interaction of nectin-2alpha was furthermore necessary for this recruitment. On the basis of these observations, we propose a model for the mechanism of cell-cell adhesion of nectin and roles of afadin in this mechanism.

Association of frabin with the actin cytoskeleton is essential for microspike formation through activation of Cdc42 small G protein.

We have recently isolated a novel actin filament-binding protein, named frabin. Frabin has one actin filament-binding domain (ABD), one Dbl homology domain (DHD), first pleckstrin homology domains (PHD) adjacent to DHD, one cysteine rich-domain (CRD), and second PHD from the N terminus to the C terminus in this order. Full-length frabin induces microspike formation and c-Jun N-terminal kinase (JNK) activation. We found here that the fragment of frabin containing DHD and first PHD stimulated guanine nucleotide exchange of Cdc42Hs small G protein, but not that of RhoA or Rac1 small G protein. However, this fragment of frabin did not induce microspike formation, and ABD was additionally necessary for microspike formation. Frabin having ABD was associated with the actin cytoskeleton, whereas frabin lacking ABD was diffusely distributed in the cytoplasm. In contrast, ABD was not necessary for JNK activation but CRD and second PHD were additionally necessary for this activation. These results indicate that the association of frabin with the actin cytoskeleton is essential for microspike formation but not for JNK activation and that different domains of frabin are involved in microspike formation and JNK activation through Cdc42 activation.