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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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Confocal images of NIH3T3 cells expressing truncated  Tiam1 proteins. A depicts the Tiam1 constructs encoding the  COOH-terminal 1,199 amino acids (C1199) or 682 amino acids  (C682) that were used for transfection of NIH3T3 cells, in comparison to the full-length Tiam1 protein (FL1591). Established  cell lines that express the C1199 Tiam1 protein (B) or the C682  Tiam1 protein (C) were fixed and stained with anti-Tiam1 antibodies (Habets et al., 1994), followed by FITC-coupled secondary antibodies (left) and by TRITC-labeled phalloidin for F-actin  (right). Note the prominent membrane ruffling in cells that express the C1199 Tiam1 protein. Stress fibers are hardly visible in  these cells, since the optical sections were made through the upper half of the cells to reveal the presence or absence of membrane ruffling. No significant differences were found in the  amount or appearance of stress fibers between Tiam1-expressing  cells and controls. M, myristoylation signal; P, PEST regions;  PHn and PHc, NH2-terminal and COOH-terminal pleckstrin homology domains. Magnification is 250×. Bar, 40 μm.
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Figure 1: Confocal images of NIH3T3 cells expressing truncated Tiam1 proteins. A depicts the Tiam1 constructs encoding the COOH-terminal 1,199 amino acids (C1199) or 682 amino acids (C682) that were used for transfection of NIH3T3 cells, in comparison to the full-length Tiam1 protein (FL1591). Established cell lines that express the C1199 Tiam1 protein (B) or the C682 Tiam1 protein (C) were fixed and stained with anti-Tiam1 antibodies (Habets et al., 1994), followed by FITC-coupled secondary antibodies (left) and by TRITC-labeled phalloidin for F-actin (right). Note the prominent membrane ruffling in cells that express the C1199 Tiam1 protein. Stress fibers are hardly visible in these cells, since the optical sections were made through the upper half of the cells to reveal the presence or absence of membrane ruffling. No significant differences were found in the amount or appearance of stress fibers between Tiam1-expressing cells and controls. M, myristoylation signal; P, PEST regions; PHn and PHc, NH2-terminal and COOH-terminal pleckstrin homology domains. Magnification is 250×. Bar, 40 μm.

Mentions: Apart from the DH domain, Tiam1 contains several other conserved motifs, including a consensus myristoylation sequence at the NH2 terminus, a Discs-large homology region (DHR), and two pleckstrin homology (PH) domains (Habets et al., 1994; Collard, 1996; see Fig. 1 A). One of the PH domains is present COOH-terminally adjacent to the DH domain, as found in all other GDS proteins for Rho-like GTPases (Collard, 1996), while a second PH domain is located in the NH2-terminal part of Tiam1. DHR domains have been implicated as protein–protein interaction motifs (Ponting and Phillips, 1995). PH domains, primarily found in signaling molecules, are thought to determine the cellular localization and/or activity of these proteins by interacting with proteins or phospholipids (for review see Lemmon et al., 1996).


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 NIH3T3 cells expressing truncated  Tiam1 proteins. A depicts the Tiam1 constructs encoding the  COOH-terminal 1,199 amino acids (C1199) or 682 amino acids  (C682) that were used for transfection of NIH3T3 cells, in comparison to the full-length Tiam1 protein (FL1591). Established  cell lines that express the C1199 Tiam1 protein (B) or the C682  Tiam1 protein (C) were fixed and stained with anti-Tiam1 antibodies (Habets et al., 1994), followed by FITC-coupled secondary antibodies (left) and by TRITC-labeled phalloidin for F-actin  (right). Note the prominent membrane ruffling in cells that express the C1199 Tiam1 protein. Stress fibers are hardly visible in  these cells, since the optical sections were made through the upper half of the cells to reveal the presence or absence of membrane ruffling. No significant differences were found in the  amount or appearance of stress fibers between Tiam1-expressing  cells and controls. M, myristoylation signal; P, PEST regions;  PHn and PHc, NH2-terminal and COOH-terminal pleckstrin homology domains. Magnification is 250×. Bar, 40 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139766&req=5

Figure 1: Confocal images of NIH3T3 cells expressing truncated Tiam1 proteins. A depicts the Tiam1 constructs encoding the COOH-terminal 1,199 amino acids (C1199) or 682 amino acids (C682) that were used for transfection of NIH3T3 cells, in comparison to the full-length Tiam1 protein (FL1591). Established cell lines that express the C1199 Tiam1 protein (B) or the C682 Tiam1 protein (C) were fixed and stained with anti-Tiam1 antibodies (Habets et al., 1994), followed by FITC-coupled secondary antibodies (left) and by TRITC-labeled phalloidin for F-actin (right). Note the prominent membrane ruffling in cells that express the C1199 Tiam1 protein. Stress fibers are hardly visible in these cells, since the optical sections were made through the upper half of the cells to reveal the presence or absence of membrane ruffling. No significant differences were found in the amount or appearance of stress fibers between Tiam1-expressing cells and controls. M, myristoylation signal; P, PEST regions; PHn and PHc, NH2-terminal and COOH-terminal pleckstrin homology domains. Magnification is 250×. Bar, 40 μm.
Mentions: Apart from the DH domain, Tiam1 contains several other conserved motifs, including a consensus myristoylation sequence at the NH2 terminus, a Discs-large homology region (DHR), and two pleckstrin homology (PH) domains (Habets et al., 1994; Collard, 1996; see Fig. 1 A). One of the PH domains is present COOH-terminally adjacent to the DH domain, as found in all other GDS proteins for Rho-like GTPases (Collard, 1996), while a second PH domain is located in the NH2-terminal part of Tiam1. DHR domains have been implicated as protein–protein interaction motifs (Ponting and Phillips, 1995). PH domains, primarily found in signaling molecules, are thought to determine the cellular localization and/or activity of these proteins by interacting with proteins or phospholipids (for review see Lemmon et al., 1996).

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