Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunoblotting of mutant Tiam1 proteins. Cell lysates  were separated on a 7.5% SDS-polyacrylamide gel and blotted to  nitrocellulose. All mutant C1199 and C682 Tiam1 proteins were  detected with a polyclonal anti-Tiam1 antibody (Habets et al.,  1994). Mutant C580 Tiam1 proteins were detected with a monoclonal antibody against the HA-tag (12CA5). Proteins were visualized using enhanced chemiluminescence (Amersham Corp.). Molecular mass markers (in kD) are shown. The arrow indicates the  position of the mutant C1199 Tiam1 proteins.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139766&req=5

Figure 4: Immunoblotting of mutant Tiam1 proteins. Cell lysates were separated on a 7.5% SDS-polyacrylamide gel and blotted to nitrocellulose. All mutant C1199 and C682 Tiam1 proteins were detected with a polyclonal anti-Tiam1 antibody (Habets et al., 1994). Mutant C580 Tiam1 proteins were detected with a monoclonal antibody against the HA-tag (12CA5). Proteins were visualized using enhanced chemiluminescence (Amersham Corp.). Molecular mass markers (in kD) are shown. The arrow indicates the position of the mutant C1199 Tiam1 proteins.

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)

Immunoblotting of mutant Tiam1 proteins. Cell lysates  were separated on a 7.5% SDS-polyacrylamide gel and blotted to  nitrocellulose. All mutant C1199 and C682 Tiam1 proteins were  detected with a polyclonal anti-Tiam1 antibody (Habets et al.,  1994). Mutant C580 Tiam1 proteins were detected with a monoclonal antibody against the HA-tag (12CA5). Proteins were visualized using enhanced chemiluminescence (Amersham Corp.). Molecular mass markers (in kD) are shown. The arrow indicates the  position of the mutant C1199 Tiam1 proteins.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Immunoblotting of mutant Tiam1 proteins. Cell lysates were separated on a 7.5% SDS-polyacrylamide gel and blotted to nitrocellulose. All mutant C1199 and C682 Tiam1 proteins were detected with a polyclonal anti-Tiam1 antibody (Habets et al., 1994). Mutant C580 Tiam1 proteins were detected with a monoclonal antibody against the HA-tag (12CA5). Proteins were visualized using enhanced chemiluminescence (Amersham Corp.). Molecular mass markers (in kD) are shown. The arrow indicates the position of the mutant C1199 Tiam1 proteins.
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