We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin–mediated cell–cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin–mediated cell–cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin–mediated cell– cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase– PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin–mediated cell– cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.

No abstract

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin-Darby canine kidney (MDCK) cells, Tiam1 localized to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibited hepatocyte growth factor-induced scattering by increasing E-cadherin-mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restored E-cadherin-mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells.

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.

Proteins of the Rho family control signalling pathways that regulate the actin cytoskeleton and gene transcription. In vitro studies have implicated Rho-like GTP-hydrolysing enzymes (GTPases) in cell migration, cell-cycle progression, and Ras-induced focus formation, suggesting a role for these GTPases in the formation and progression of tumours in vivo. To study this, we have generated mice lacking the Rac-specific activator Tiam1, a T-lymphoma invasion and metastasis inducing protein. Here we show that such Tiam1(-/-) mice are resistant to the development of Ras-induced skin tumours initiated with 7,12-dimethylbenzanthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate. Moreover, the few tumours produced in Tiam1(-/-) mice grew much slower than did tumours in wild-type mice. Tiam1-deficient primary embryonic fibroblasts were also resistant to Ras(V12)-induced focus formation. Analysis of Tiam1 heterozygotes indicated that both tumour initiation and promotion were dependent on the Tiam1 gene dose. Tiam1 deficiency was associated with increased apoptosis during initiation, and with impeded proliferation during promotion. Although the number of tumours in Tiam1(-/-) mice was small, a greater proportion progressed to malignancy, suggesting that Tiam1 deficiency promotes malignant conversion. Our studies identify the Rac activator Tiam1 as a critical regulator of different aspects of Ras-induced tumour formation.

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

Rho-like GTPases, including Cdc42, Rac, and Rho, regulate signaling pathways that control actin cytoskeletal structures and transcriptional activation. The Tiam1 gene encodes an activator of Rac1, and similarly to constitutively activated (V12)Rac1, overexpression of Tiam1 in fibroblasts induces the formation of membrane ruffles. Tiam1 contains a Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, hallmarks for activators of Rho-like GTPases. Unique for Tiam1 are an additional PH domain and a Discs-large homology region in the NH2-terminal part of the protein. Here we show that both in fibroblasts and COS cells, membrane localization of Tiam1 is required for the induction of membrane ruffling. A detailed mutational analysis, in combination with confocal laser scanning microscopy and immunoelectron microscopy, demonstrates that the NH2-terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH2-terminal PH domain of Tiam1 can be functionally replaced by the myristoylated membrane localization domain of c-Src, indicating that the primary function of this PH domain is to localize the protein at the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor stimulation induces membrane translocation of Tiam1. Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK). This Rac-dependent stimulation of JNK also requires membrane association of Tiam1. We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.

Proteins of the Rho family regulate cytoskeletal rearrangements in response to receptor stimulation and are involved in the establishment and maintenance of epithelial cell morphology. We recently showed that Rac is able to downregulate Rho activity and that the reciprocal balance between Rac and Rho activity is a major determinant of cellular morphology and motility in NIH3T3 fibroblasts. Using biochemical pull-down assays, we analyzed the effect of transient and sustained oncogenic Ras signaling on the activation state of Rac and Rho in epithelial MDCK cells. In contrast to the activation of Rac by growth factor-induced Ras signaling, we found that sustained signaling by oncogenic RasV12 permanently downregulates Rac activity, which leads to upregulation of Rho activity and epithelial–mesenchymal transition. Oncogenic Ras decreases Rac activity through sustained Raf/MAP kinase signaling, which causes transcriptional downregulation of Tiam1, an activator of Rac in epithelial cells. Reconstitution of Rac activity by expression of Tiam1 or RacV12 leads to downregulation of Rho activity and restores an epithelial phenotype in mesenchymal RasV12- or RafCAAX-transformed cells. The present data reveal a novel mechanism by which oncogenic Ras is able to interfere with the balance between Rac and Rho activity to achieve morphological transformation of epithelial cells.