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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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Immunoelectron microscopic localization of truncated  Tiam1 proteins. Frozen sections of established NIH3T3 cell lines  expressing either C1199 Tiam1 (A) or C682 Tiam1 (B) were stained  with anti-Tiam1 antibodies followed by gold-conjugated secondary antibodies. The C682 Tiam1 protein is almost exclusively located in the cytoplasm, whereas the C1199 Tiam1 protein is also  present at the plasma membrane and in the membrane ruffles.  Bar, 400 nm.
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Figure 2: Immunoelectron microscopic localization of truncated Tiam1 proteins. Frozen sections of established NIH3T3 cell lines expressing either C1199 Tiam1 (A) or C682 Tiam1 (B) were stained with anti-Tiam1 antibodies followed by gold-conjugated secondary antibodies. The C682 Tiam1 protein is almost exclusively located in the cytoplasm, whereas the C1199 Tiam1 protein is also present at the plasma membrane and in the membrane ruffles. Bar, 400 nm.

Mentions: We have shown previously that full-length Tiam1 (FL1591) or a large COOH-terminal fragment of the Tiam1 protein (C1199) causes Rac1-dependent induction of membrane ruffling in NIH3T3 fibroblast cells (Michiels et al., 1995). Established NIH3T3 cell lines expressing either of these proteins are flat and epithelial-like and contain many membrane ruffles (Fig. 1 B), a phenotype that is also induced by transfection or microinjection of constitutively activated (V12)Rac1 (Ridley et al., 1992; Michiels et al., 1995; Van Leeuwen et al., 1995). In contrast, a smaller COOH-terminal part of the Tiam1 protein (C682), which contains only the DH domain and the adjacent PH domain, did not induce this phenotype (Fig. 1 C). As shown by confocal laser scanning immunofluorescence microscopy, the truncated large C1199 Tiam1 protein was present in the cytoplasm and colocalized with F-actin in membrane ruffles (Fig. 1 B). In contrast, the short C682 Tiam1 protein seemed to be restricted to the cytoplasm (Fig. 1 C). Western blot analyses (see Fig. 4, lanes 1–3) indicated that both proteins were intact and equally expressed. This suggested that the difference in phenotypes induced by these truncated proteins is probably caused by a different intracellular localization, and not by differences in stability. Immunoelectron microscopy (immuno-EM) indeed confirmed that the C1199 Tiam1 protein is present in the cytoplasm as well as at the cell membrane and particularly in the membrane ruffles, whereas the C682 Tiam1 protein is almost exclusively located in the cytoplasm (Fig. 2). We hypothesized, therefore, that membrane localization of Tiam1 is required for morphological transformation of NIH3T3 cells, including the formation of membrane ruffles.


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)

Immunoelectron microscopic localization of truncated  Tiam1 proteins. Frozen sections of established NIH3T3 cell lines  expressing either C1199 Tiam1 (A) or C682 Tiam1 (B) were stained  with anti-Tiam1 antibodies followed by gold-conjugated secondary antibodies. The C682 Tiam1 protein is almost exclusively located in the cytoplasm, whereas the C1199 Tiam1 protein is also  present at the plasma membrane and in the membrane ruffles.  Bar, 400 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139766&req=5

Figure 2: Immunoelectron microscopic localization of truncated Tiam1 proteins. Frozen sections of established NIH3T3 cell lines expressing either C1199 Tiam1 (A) or C682 Tiam1 (B) were stained with anti-Tiam1 antibodies followed by gold-conjugated secondary antibodies. The C682 Tiam1 protein is almost exclusively located in the cytoplasm, whereas the C1199 Tiam1 protein is also present at the plasma membrane and in the membrane ruffles. Bar, 400 nm.
Mentions: We have shown previously that full-length Tiam1 (FL1591) or a large COOH-terminal fragment of the Tiam1 protein (C1199) causes Rac1-dependent induction of membrane ruffling in NIH3T3 fibroblast cells (Michiels et al., 1995). Established NIH3T3 cell lines expressing either of these proteins are flat and epithelial-like and contain many membrane ruffles (Fig. 1 B), a phenotype that is also induced by transfection or microinjection of constitutively activated (V12)Rac1 (Ridley et al., 1992; Michiels et al., 1995; Van Leeuwen et al., 1995). In contrast, a smaller COOH-terminal part of the Tiam1 protein (C682), which contains only the DH domain and the adjacent PH domain, did not induce this phenotype (Fig. 1 C). As shown by confocal laser scanning immunofluorescence microscopy, the truncated large C1199 Tiam1 protein was present in the cytoplasm and colocalized with F-actin in membrane ruffles (Fig. 1 B). In contrast, the short C682 Tiam1 protein seemed to be restricted to the cytoplasm (Fig. 1 C). Western blot analyses (see Fig. 4, lanes 1–3) indicated that both proteins were intact and equally expressed. This suggested that the difference in phenotypes induced by these truncated proteins is probably caused by a different intracellular localization, and not by differences in stability. Immunoelectron microscopy (immuno-EM) indeed confirmed that the C1199 Tiam1 protein is present in the cytoplasm as well as at the cell membrane and particularly in the membrane ruffles, whereas the C682 Tiam1 protein is almost exclusively located in the cytoplasm (Fig. 2). We hypothesized, therefore, that membrane localization of Tiam1 is required for morphological transformation of NIH3T3 cells, including the formation of membrane ruffles.

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