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Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation.

Michiels F, Stam JC, Hordijk PL, van der Kammen RA, Ruuls-Van Stalle L, Feltkamp CA, Collard JG - J. Cell Biol. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, Division of Cell Biology, Amsterdam.

ABSTRACT
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.

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Related in: MedlinePlus

Confocal images of COS-7 cells expressing mutated C1199 Tiam1 proteins. In this figure, the first column depicts the mutant  Tiam1 proteins that were used. The second column shows the FITC-staining for Tiam1 expression. The third column shows the TRITClabeled phalloidin staining for F-actin, while the merged images of Tiam1 (green) and F-actin (red) are shown in the fourth column.  Colocalization appears in yellow. Deletions within the conserved domains are indicated by arrowheads. See Materials and Methods section for a description of the mutations. For abbreviations, see legend to Fig. 1. Magnification is 200×. Bar, 40 μm.
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Figure 3: Confocal images of COS-7 cells expressing mutated C1199 Tiam1 proteins. In this figure, the first column depicts the mutant Tiam1 proteins that were used. The second column shows the FITC-staining for Tiam1 expression. The third column shows the TRITClabeled phalloidin staining for F-actin, while the merged images of Tiam1 (green) and F-actin (red) are shown in the fourth column. Colocalization appears in yellow. Deletions within the conserved domains are indicated by arrowheads. See Materials and Methods section for a description of the mutations. For abbreviations, see legend to Fig. 1. Magnification is 200×. Bar, 40 μm.

Mentions: To investigate which of the conserved domains in Tiam1 determines the intracellular localization, small deletions were made in each of these domains within the C1199 construct (see Fig. 3 and Materials and Methods). These mutant proteins were transiently expressed in COS-7 cells to analyze their intracellular localization and ability to induce membrane ruffling. Transfection of these mutant Tiam1 constructs in NIH3T3 cells resulted in the same phenotypic changes (not shown). Although Tiam1 is endogenously expressed in COS-7 cells and NIH3T3 cells, the levels are too low to be visualized by immunocytochemistry or Western blotting. If phenotypic changes were induced by the transfection of Tiam1 mutants, they were found in >80% of the transfected cells.


Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation.

Michiels F, Stam JC, Hordijk PL, van der Kammen RA, Ruuls-Van Stalle L, Feltkamp CA, Collard JG - J. Cell Biol. (1997)

Confocal images of COS-7 cells expressing mutated C1199 Tiam1 proteins. In this figure, the first column depicts the mutant  Tiam1 proteins that were used. The second column shows the FITC-staining for Tiam1 expression. The third column shows the TRITClabeled phalloidin staining for F-actin, while the merged images of Tiam1 (green) and F-actin (red) are shown in the fourth column.  Colocalization appears in yellow. Deletions within the conserved domains are indicated by arrowheads. See Materials and Methods section for a description of the mutations. For abbreviations, see legend to Fig. 1. Magnification is 200×. Bar, 40 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139766&req=5

Figure 3: Confocal images of COS-7 cells expressing mutated C1199 Tiam1 proteins. In this figure, the first column depicts the mutant Tiam1 proteins that were used. The second column shows the FITC-staining for Tiam1 expression. The third column shows the TRITClabeled phalloidin staining for F-actin, while the merged images of Tiam1 (green) and F-actin (red) are shown in the fourth column. Colocalization appears in yellow. Deletions within the conserved domains are indicated by arrowheads. See Materials and Methods section for a description of the mutations. For abbreviations, see legend to Fig. 1. Magnification is 200×. Bar, 40 μm.
Mentions: To investigate which of the conserved domains in Tiam1 determines the intracellular localization, small deletions were made in each of these domains within the C1199 construct (see Fig. 3 and Materials and Methods). These mutant proteins were transiently expressed in COS-7 cells to analyze their intracellular localization and ability to induce membrane ruffling. Transfection of these mutant Tiam1 constructs in NIH3T3 cells resulted in the same phenotypic changes (not shown). Although Tiam1 is endogenously expressed in COS-7 cells and NIH3T3 cells, the levels are too low to be visualized by immunocytochemistry or Western blotting. If phenotypic changes were induced by the transfection of Tiam1 mutants, they were found in >80% of the transfected cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, Division of Cell Biology, Amsterdam.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus