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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 COS-7 cells expressing mutant Tiam1 proteins. Confocal laser scanning microscopic images of COS-7 cells  expressing C580 Tiam1 (A), MS-C580 Tiam1 (B), full-length Tiam1 (FL1591) (C), full-length Tiam1 with a deletion in the NH2-terminal PH  domain (FL-ΔPHn) (D), and FL-ΔPHn containing a Src myristoylation signal at the NH2 terminus (MS-FL-ΔPHn) (E). The insert in E is  a higher magnification of the framed region showing the vesicle-like structures that are surrounded by the MS-FL-ΔPHn Tiam1 protein.  Indicated in blue is the myristoylation signal from c-Src (MS), containing the NH2-terminal 20 amino acids. In the merged images, Tiam1  is shown in green and F-actin in red. Magnification is 200×. Bar, 40 μm.
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Figure 6: Confocal images of COS-7 cells expressing mutant Tiam1 proteins. Confocal laser scanning microscopic images of COS-7 cells expressing C580 Tiam1 (A), MS-C580 Tiam1 (B), full-length Tiam1 (FL1591) (C), full-length Tiam1 with a deletion in the NH2-terminal PH domain (FL-ΔPHn) (D), and FL-ΔPHn containing a Src myristoylation signal at the NH2 terminus (MS-FL-ΔPHn) (E). The insert in E is a higher magnification of the framed region showing the vesicle-like structures that are surrounded by the MS-FL-ΔPHn Tiam1 protein. Indicated in blue is the myristoylation signal from c-Src (MS), containing the NH2-terminal 20 amino acids. In the merged images, Tiam1 is shown in green and F-actin in red. Magnification is 200×. Bar, 40 μm.

Mentions: To determine whether membrane localization is the only function of the NH2-terminal PH domain, we fused the NH2terminal 20 amino acids of c-Src, containing the myristoylation site and a basic region, in front of a C580 Tiam1 protein, resulting in MS-C580 Tiam1 (see Materials and Methods). C580 Tiam1 encompasses the COOH-terminal 580 amino acids and encodes the DH domain and adjacent PH domain (Fig. 6 A). Similar to the C682 Tiam1 protein (see Fig. 2 B), the C580 Tiam1 protein did not induce membrane ruffling and was not localized at the plasma membrane (Figs. 6 A and 7 A). In contrast, the MS-C580 Tiam1 protein induced ruffling in both NIH3T3 cells and COS-7 cells (Fig. 6 B), albeit less extensively than C1199 Tiam1 (Fig. 3 A). No differences were observed in the expression levels of these proteins (Fig. 4, lanes 10–12). Immuno-EM showed that MS-C580 Tiam1 was able to associate with the plasma membrane (Fig. 7 B), although this protein seemed to be more interiorly located than the mutant C1199 Tiam1 proteins (compare Figs. 5 and 7 B). Furthermore, part of the expressed MS-C580 Tiam1 protein was present around vesicle-like structures in the cytoplasm, similar to other MS-containing Tiam1 fusion proteins (see for example Fig. 6 E, inset). A fusion protein containing the NH2-terminal Src domain in front of the C1199-ΔPHn Tiam1 protein was also able to localize at the plasma membrane and to cause ruffling (data not shown). The Src membrane localization domain thus enables C580 Tiam1 and C1199-ΔPHn to induce membrane ruffling by tethering these proteins to the plasma membrane.


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 mutant Tiam1 proteins. Confocal laser scanning microscopic images of COS-7 cells  expressing C580 Tiam1 (A), MS-C580 Tiam1 (B), full-length Tiam1 (FL1591) (C), full-length Tiam1 with a deletion in the NH2-terminal PH  domain (FL-ΔPHn) (D), and FL-ΔPHn containing a Src myristoylation signal at the NH2 terminus (MS-FL-ΔPHn) (E). The insert in E is  a higher magnification of the framed region showing the vesicle-like structures that are surrounded by the MS-FL-ΔPHn Tiam1 protein.  Indicated in blue is the myristoylation signal from c-Src (MS), containing the NH2-terminal 20 amino acids. In the merged images, Tiam1  is shown in green and F-actin in red. Magnification is 200×. Bar, 40 μm.
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Figure 6: Confocal images of COS-7 cells expressing mutant Tiam1 proteins. Confocal laser scanning microscopic images of COS-7 cells expressing C580 Tiam1 (A), MS-C580 Tiam1 (B), full-length Tiam1 (FL1591) (C), full-length Tiam1 with a deletion in the NH2-terminal PH domain (FL-ΔPHn) (D), and FL-ΔPHn containing a Src myristoylation signal at the NH2 terminus (MS-FL-ΔPHn) (E). The insert in E is a higher magnification of the framed region showing the vesicle-like structures that are surrounded by the MS-FL-ΔPHn Tiam1 protein. Indicated in blue is the myristoylation signal from c-Src (MS), containing the NH2-terminal 20 amino acids. In the merged images, Tiam1 is shown in green and F-actin in red. Magnification is 200×. Bar, 40 μm.
Mentions: To determine whether membrane localization is the only function of the NH2-terminal PH domain, we fused the NH2terminal 20 amino acids of c-Src, containing the myristoylation site and a basic region, in front of a C580 Tiam1 protein, resulting in MS-C580 Tiam1 (see Materials and Methods). C580 Tiam1 encompasses the COOH-terminal 580 amino acids and encodes the DH domain and adjacent PH domain (Fig. 6 A). Similar to the C682 Tiam1 protein (see Fig. 2 B), the C580 Tiam1 protein did not induce membrane ruffling and was not localized at the plasma membrane (Figs. 6 A and 7 A). In contrast, the MS-C580 Tiam1 protein induced ruffling in both NIH3T3 cells and COS-7 cells (Fig. 6 B), albeit less extensively than C1199 Tiam1 (Fig. 3 A). No differences were observed in the expression levels of these proteins (Fig. 4, lanes 10–12). Immuno-EM showed that MS-C580 Tiam1 was able to associate with the plasma membrane (Fig. 7 B), although this protein seemed to be more interiorly located than the mutant C1199 Tiam1 proteins (compare Figs. 5 and 7 B). Furthermore, part of the expressed MS-C580 Tiam1 protein was present around vesicle-like structures in the cytoplasm, similar to other MS-containing Tiam1 fusion proteins (see for example Fig. 6 E, inset). A fusion protein containing the NH2-terminal Src domain in front of the C1199-ΔPHn Tiam1 protein was also able to localize at the plasma membrane and to cause ruffling (data not shown). The Src membrane localization domain thus enables C580 Tiam1 and C1199-ΔPHn to induce membrane ruffling by tethering these proteins to the plasma membrane.

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