Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG

Nectins, Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, initiate cell-cell adhesion by their trans interactions and recruit cadherins to cooperatively form adherens junctions (AJs). In addition, the trans interactions of nectins induce the activation of Cdc42 and Rac small G proteins, which increases the velocity of the formation of AJs. We examined here how nectins induce the activation of Cdc42 in MDCK epithelial cells and L fibroblasts. Nectins recruited and activated c-Src at the nectin-based cell-cell adhesion sites. FRG, a GDP/GTP exchange factor specific for Cdc42, was then recruited there, tyrosine phosphorylated by c-Src, and activated, causing an increase in the GTP-bound active form of Cdc42. Inhibition of the nectin-induced activation of c-Src suppressed the nectin-induced activation of FRG and Cdc42. Inhibition of the nectin-induced activation of FRG or depletion of FRG by RNA interference suppressed the nectin-induced activation of Cdc42. These results indicate that nectins induce the activation of Cdc42 through c-Src and FRG locally at the nectin-based cell-cell adhesion sites.     

Involvement of the c-Src-Crk-C3G-Rap1 signaling in the nectin-induced activation of Cdc42 and formation of adherens junctions

Nectins, Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, induce the activation of Cdc42 and Rac small G proteins, enhancing the formation of cadherin-based adherens junctions (AJs) and claudin-based tight junctions. Nectins recruit and activate c-Src at the nectin-based cell-cell contact sites. c-Src then activates Cdc42 through FRG, a Cdc42-GDP/GTP exchange factor. We showed here that Rap1 small G protein was involved in the nectin-induced activation of Cdc42 and formation of AJs. Rap1 was recruited to the nectin-based cell-cell contact sites and locally activated through the c-Src-Crk-C3G signaling there. The activation of either c-Src or Rap1 alone was insufficient for and the activation of both molecules was essential for the activation of FRG. The activation of Rap1 was not necessary for the c-Src-mediated phosphorylation or recruitment of FRG. The inhibition of the Crk, C3G, or Rap1 signaling reduced the formation of AJs. These results indicate that Rap1 is activated by nectins through the c-Src-Crk-C3G signaling and involved in the nectin-induced, c-Src- and FRG-mediated activation of Cdc42 and formation of AJs.     

A novel role of nectins in inhibition of the E-cadherin-induced activation of Rac and formation of cell-cell adherens junctions

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules. The trans-interactions of nectins recruit cadherins to the nectin-based cell-cell adhesion, resulting in formation of cell-cell adherens junctions (AJs) in epithelial cells and fibroblasts. The trans-interaction of E-cadherin induces activation of Rac small G protein, whereas the trans-interactions of nectins induce activation of not only Rac but also Cdc42 small G protein. We showed by the fluorescent resonance energy transfer (FRET) imaging that the trans-interaction of E-cadherin induced dynamic activation and inactivation of Rac, which led to dynamic formation and retraction of lamellipodia. Moreover, we found here that the nectins, which did not trans-interact with other nectins (non-trans-interacting nectins), inhibited the E-cadherin-induced activation of Rac and reduced the velocity of the formation of the E-cadherin-based cell-cell AJs. The inhibitory effect of non-trans-interacting nectins was suppressed by the activation of Cdc42 induced by the trans-interactions of nectins. These results indicate a novel role of nectins in regulation of the E-cadherin-induced activation of Rac and formation of cell-cell AJs.     

Trans-interactions of nectins induce formation of filopodia and Lamellipodia through the respective activation of Cdc42 and Rac small G proteins

Nectins and afadin constitute a novel cell-cell adhesion system that plays a cooperative role with cadherins in the organization of adherens junctions (AJs). Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, and afadin is a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton. Rac and Cdc42 small G proteins have been implicated in the organization of AJs, but their modes of action remain unknown. The trans-interaction of E-cadherin has recently been shown to induce the activation of Rac, but not that of Cdc42. We show here that the trans-interactions of nectins induce the formation of filopodia and lamellipodia through the respective activation of Cdc42 and Rac. The Cdc42 activation is necessary, but not sufficient, for the Rac-induced formation of lamellipodia, whereas the Rac activation is not necessary for the Cdc42-induced formation of filopodia. These effects of nectins require their cytoplasmic tail but not their association with afadin. We propose here the functional relationship between nectins and the small G proteins in the organization of AJs.     

Roles and modes of action of nectins in cell-cell adhesion

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell-cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell-cell junctions and cell-cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell-cell adhesion and recruit E-cadherin to the nectin-based cell-cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.     

Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectin and E-cadherin, through afadin and alpha-actinin in epithelial cells

Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules that are involved in formation of cadherin-based adherens junctions (AJs). The nectin-based cell-cell adhesion induces activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of AJs through reorganization of the actin cytoskeleton. Although evidence has accumulated that nectins recruit cadherins to the nectin-based cell-cell adhesion sites through their cytoplasm-associated proteins, afadin and catenins, it is not fully understood how nectins are physically associated with cadherins. Here we identified a rat counterpart of the human LIM domain only 7 (LMO7) as an afadin- and alpha-actinin-binding protein. Rat LMO7 has two splice variants, LMO7a and LMO7b, consisting of 1,729 and 1,395 amino acids, respectively. LMO7 has calponin homology, PDZ, and LIM domains. Western blotting revealed that LMO7 was expressed ubiquitously in various rat tissues. Immunofluorescence and immunoelectron microscopy revealed that LMO7 localized at cell-cell AJs, where afadin localized, in epithelial cells of rat gallbladder. In addition, LMO7 localized at the cytoplasmic faces of apical membranes in the same epithelial cells. We furthermore revealed that LMO7 bound alpha-actinin, an actin filament-bundling protein, which bound to alpha-catenin. Immunoprecipitation analysis revealed that LMO7 was associated with both the nectin-afadin and E-cadherin-catenin systems. LMO7 was assembled at the cell-cell adhesion sites after both the nectin-afadin and E-cadherin-catenin systems had been assembled. These results indicate that LMO7 is an afadin- and alpha-actinin-binding protein that connects the nectin-afadin and E-cadherin-catenin systems through alpha-actinin.     

Involvement of nectin-activated Cdc42 small G protein in organization of adherens and tight junctions in Madin-Darby canine kidney cells

Nectins, Ca2+-independent immunoglobulin-like cell-cell adhesion molecules, trans-interact and form cell-cell adhesion, which increases the velocities of the formation of the E-cadherin-based adherens junctions (AJs) and the claudin-based tight junctions (TJs) in Madin-Darby canine kidney (MDCK) cells. The trans-interactions of nectins furthermore induce activation of Cdc42 and Rac small G proteins, but the roles of these small G proteins activated in this way remain unknown. We examined here the role and the mode of action of Cdc42 in the organization of AJs and TJs in MDCK cells. We first made the NWASP-Cdc42 and Rac interactive binding (CRIB) domain, an inhibitor of activated Cdc42, fused to the Ki-Ras CAAX motif (NWASP-CRIB-CAAX; where A is aliphatic amino acid), which was targeted to the cell-cell adhesion sites. We then found that overexpression of NWASP-CRIB-CAAX reduced the velocities of the formation of AJs and TJs. Conversely, overexpression of a constitutively active mutant of Cdc42 (V12Cdc42) increased their velocities, and the inhibitory effect of NWASP-CRIB-CAAX was suppressed by co-expression with V12Cdc42. The inhibitory effect of NWASP-CRIB-CAAX on the formation of AJs and TJs was suppressed by co-expression of nectin-1 of which trans-interaction activated endogenous Cdc42. Moreover, the formation of the claudin-based TJs required a greater amount of activated Cdc42 than that of the E-cadherin-based AJs. These results indicate that the Cdc42 activated by the trans-interactions of nectins is involved in the organization of AJs and TJs in different mechanisms in MDCK cells.     

Roles and modes of action of nectins in cell–cell adhesion

Nectins are Ca²⁺-independent immunoglobulin (Ig)-like cell–cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell–cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell–cell junctions and cell–cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell–cell adhesion and recruit E-cadherin to the nectin-based cell–cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.     

Interaction of integrin alpha(v)beta3 with nectin. Implication in cross-talk between cell-matrix and cell-cell junctions

Cell-matrix and cell-cell junctions cross-talk together, and these two junctions cooperatively regulate cell movement, proliferation, adhesion, and polarization. However, the mechanism of this cross-talk remains unknown. An immunoglobulin-like cell-cell adhesion molecule nectin first trans-interacts with each other to form cell-cell adhesion and induces activation of Rap1, Cdc42, and Rac small G proteins through c-Src. Trans-interacting nectin then recruits another cell-cell adhesion molecule cadherin to the nectin-based cell-cell adhesion sites and forms adherens junctions (AJs). Here, we show that integrin alpha(v)beta3 functionally and physically associates with nectin. Integrin alpha(v)beta3 colocalized with nectin at the nectin-based cell-cell adhesion sites. The association of integrin alpha(v)beta3 with nectin was direct and was mediated through their extracellular regions. This interaction was necessary for the nectin-induced signaling. Focal adhesion kinase, which relays the integrin-initiated outside-in signals to the intracellular signaling molecules, was also involved in the nectin-induced signaling. During the formation of AJs, the high affinity form of integrin alpha(v)beta3 co-localized with nectin at the primordial cell-cell contact sites, and then after the establishment of AJs, this high affinity form of integrin alpha(v)beta3 was converted to the low affinity form, which continued to co-localize with nectin. Thus, integrin alpha(v)beta3 and nectin play pivotal roles in the cross-talk between cell-matrix and cell-cell junctions and the formation of cadherin-based AJs.     

Role of multiple bonds between the single cell adhesion molecules, nectin and cadherin, revealed by high sensitive force measurements

Nectins and cadherins, members of cell adhesion molecules (CAMs), are the primary mediators for various types of cell-cell junctions. Here, intermolecular force microscopy (IFM) with force sensitivity at sub-picoNewtons is used to characterize the extracellular trans-interactions between paired nectins and paired cadherins at the single molecule level. Three and four different bound states between paired nectins and paired cadherins are, respectively, identified and characterized based on bond strength distributions where each bound state has a unique lifetime and bond length. The results indicate that multiple domains of nectins act uncooperatively, as a zipper-like multiply bonded system whereas those of cadherins act cooperatively, as a parallel-like multiply bonded system, consistent with a "fork initiation and zipper" hypothesis for the formation of cell-cell adhesion. The observed dynamic properties among multiple bonds are expected to be advantageous such that nectins search adaptively in the cell-cell exploratory recognition process while cadherins slowly stabilize in the cell-cell zippering process.     

The Cool-2/alpha-Pix protein mediates a Cdc42-Rac signaling cascade

BACKGROUND: Cloned-out of library-2 (Cool-2)/PAK-interactive exchange factor (alpha-Pix) was identified through its ability to bind the Cdc42/Rac target p21-activated kinase (PAK) and has been implicated in certain forms of X-linked mental retardation as well as in growth factor- and chemoattractant-coupled signaling pathways. We recently found that the dimeric form of Cool-2 is a specific guanine nucleotide exchange factor (GEF) for Rac, whereas monomeric Cool-2 is a GEF for Cdc42 as well as Rac. However, unlike many GEFs, Cool-2 binds to activated forms of Cdc42 and Rac. Thus, we have investigated the functional consequences of these interactions. RESULTS: We show that the binding of activated Cdc42 to the Cool-2 dimer markedly enhances its ability to associate with GDP bound Rac1, resulting in a significant activation of Rac-GEF activity. While the Rac-specific GEF activity of Cool-2 is mediated through the Dbl homology (DH) domain from one monomer and the Pleckstrin homology domain from the other, activated Cdc42 interacts with the DH domain, most likely opposite the DH domain binding site for GDP bound Rac. Activated Rac also binds to Cool-2; however, it strongly inhibits the GEF activity of dimeric Cool-2. CONCLUSIONS: We provide evidence for novel mechanisms of allosteric regulation of the Rac-GEF activity of the Cool-2 dimer, involving stimulatory effects by Cdc42 and feedback inhibition by Rac. These findings demonstrate that by serving as a target for GTP bound Cdc42 and a GEF for Rac, Cool-2 mediates a GTPase cascade where the activation of Cdc42 is translated into the activation of Rac.     

Roles of nectins in cell adhesion, migration and polarization

Nectins are Ca2+-independent immunoglobulin (Ig)-like cell-cell adhesion molecules, which comprise a family consisting of four members. Nectins have five activities: (1) they show Ca2+-independent cell-cell adhesion activity by homo- and hetero-trans-interactions through their extracellular regions; (2) they bind afadin, an actin filament (F-actin)-binding protein, through their cytoplasmic tails and are connected to the actin cytoskeleton; (3) they induce activation of Cdc42 and Rac small G proteins through their cytoplasmic tails; (4) they bind Par-3, a cell polarity protein, through their cytoplasmic tails; and (5) they heterophilically trans-interact with Necls, nectin-like molecules, through their extracellular regions. Through these activities, nectins regulate a variety of cellular functions, including adhesion, migration, and polarization. Here we describe these activities and functions of nectins.     

The roles of cadherins and nectins in interneuronal synapse formation

Cadherins are Ca(2+)-dependent intercellular adhesion molecules (CAMs) and they play key roles in the intercellular junctions of a wide variety of cells, including interneuronal synapses. Nectins are Ca(2+)-independent immunoglobulin-like CAMs and they are also involved in the organization of various types of intercellular junctions, including interneuronal synapses, either in cooperation with or independently of cadherins. Intercellular adhesion through nectins induces activation of Cdc42 and Rac small G proteins, leading to a reorganization of the actin cytoskeleton, gene expression, and cell polarization.     

Induction of rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) following phosphorylation by the nonreceptor tyrosine kinase Src

Ras-GRF1/CDC25(Mm) has been implicated as a Ras-guanine nucleotide exchange factor (GEF) expressed in brain. Ras-GEF activity of Ras-GRF1 is augmented in response to Ca(2+) influx and G protein betagamma subunit (Gbetagamma) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by Gbetagamma-mediated signals. Tyrosine phosphorylation of Ras-GRF1 is critical for the induction of Rac-GEF activity as evidenced by inhibition by tyrosine kinase inhibitors. Herein, we show that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity. Ras-GRF1 transiently expressed with v-Src was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by Gbetagamma. In contrast, Ras-GEF activity remained unchanged. The recombinant c-Src protein phosphorylated affinity-purified glutathione S-transferase-tagged Ras-GRF1 in vitro and thereby elicited Rac-GEF activity. Taken together, tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of Gbetagamma to regulate Ras-GRF1.     

The roles of nectins in cell adhesions: cooperation with other cell adhesion molecules and growth factor receptors

Nectins are Ca(2+)-independent Ig-like cell adhesion molecules (CAMs) which homophilically and heterophilically interact in trans with nectins and form cell-cell adhesion. This cell-cell adhesion is involved in the formation of many types of cell-cell junctions such as adherens junctions, tight junctions, and synaptic junctions, cooperatively with other CAMs such as cadherins and claudins. Nectins transduce signals cooperatively with integrin alpha(v)beta(3), and regulate formation of cell-cell junctions. In addition, nectin interacts in cis with PDGF receptor and regulates its signaling for anti-apoptosis. Furthermore, nectin interacts in trans with nectin-like molecule-5 (Necl-5) and regulate cell movement and proliferation. We describe cooperative roles of nectins with other CAMs and growth factor receptors.     

Cooperative role of nectin-nectin and nectin-afadin interactions in formation of nectin-based cell-cell adhesion

The nectin cell adhesion molecules interact in trans with each other through their extracellular regions and with afadin through their cytoplasmic tails, forming adherens junctions in cooperation with cadherins. In a single cell, Necl-5 (nectin-like molecule-5) localizes at the leading edge and regulates directional cell movement in response to a chemoattractant. In such a single cell, afadin also localizes at the leading edge without interacting with nectins or Necl-5. It remains unknown how the nectin-nectin and nectin-afadin interactions are initiated when moving cells contact each other to initiate the formation of adherens junctions. We show here that the Necl-5-nectin interaction induced by cell-cell contact enhances the nectin-afadin interaction. This interaction then enhances the nectin-nectin interaction, which further enhances the nectin-afadin interaction in a positive feedback manner. Thus, the Necl-5-nectin, nectin-nectin, and nectin-afadin interactions cooperatively increase the clustering of the nectin-afadin complex at the cell-cell contact sites, promoting the formation of the nectin-based cell-cell adhesion.     

Vav2 is an activator of Cdc42, Rac1, and RhoA

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.     

Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells

Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes.     

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120(ctn), beta-catenin, and alpha-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells.     

Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.