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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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Model for activation of Rac by Tiam1. Activation of  Rac requires membrane translocation of Tiam1 and might be  analogous to activation of Ras by receptor-induced translocation  of SOS. Membrane association of Tiam1 could be mediated by  interactions between the NH2-terminal PH domain and putative  effectors of activated G-protein coupled receptors (GPCR), tyrosine kinase receptors (TKR), or Ras, which may include Gβγ  subunits, PIP2, PIP3, IP3, and perhaps Gα subunits. Activation of  Rac by membrane-associated Tiam1 leads to membrane ruffling  and JNK activation. It is unlikely that JNK is involved in the induction of an oncogenic phenotype in NIH3T3 cells, since mutant  C682 Tiam1 induces an oncogenic phenotype without activation  of JNK. Which Tiam1/Rac-mediated signaling pathways are involved in the induction of an invasive phenotype in T-lymphoma  cells remains to be established.
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Figure 10: Model for activation of Rac by Tiam1. Activation of Rac requires membrane translocation of Tiam1 and might be analogous to activation of Ras by receptor-induced translocation of SOS. Membrane association of Tiam1 could be mediated by interactions between the NH2-terminal PH domain and putative effectors of activated G-protein coupled receptors (GPCR), tyrosine kinase receptors (TKR), or Ras, which may include Gβγ subunits, PIP2, PIP3, IP3, and perhaps Gα subunits. Activation of Rac by membrane-associated Tiam1 leads to membrane ruffling and JNK activation. It is unlikely that JNK is involved in the induction of an oncogenic phenotype in NIH3T3 cells, since mutant C682 Tiam1 induces an oncogenic phenotype without activation of JNK. Which Tiam1/Rac-mediated signaling pathways are involved in the induction of an invasive phenotype in T-lymphoma cells remains to be established.

Mentions: Several reports have demonstrated that isolated PH domains can bind in vitro to Gβγ subunits and phospholipids like phosphatidylinositol (4,5)bisphosphate (PIP2) and inositol (3,4,5)trisphosphate (IP3) (Harlan et al., 1994; Ferguson et al., 1995; Mahadevan et al., 1995; Touhara et al., 1995). Phosphoinositide-3-kinase, which stimulates the synthesis of phosphatidylinositol (3,4,5)trisphosphate (PIP3) from PIP2 (Hiles et al., 1992), has been implicated in the activation of Rac by PDGF and insulin (Wennstrom et al., 1994; Hawkins et al., 1995) and might be required for the activation of Rac by Ras (Rodriguez-Viciana et al., 1994). Although we have shown that the association of Tiam1 with the plasma membrane is induced by serum, neither PDGF nor insulin could substitute for serum in NIH3T3 cells. Gβγ subunits and phospholipids represent potential downstream effectors of G-protein–coupled receptors and/or activated tyrosine kinase receptors. Based on the in vitro binding data of PH domains, it is tempting to speculate that Gβγ subunits and/or specific phospholipids regulate the association of Tiam1 with the plasma membrane, as depicted in Fig. 10. The fact that we were not able to functionally exchange the NH2-terminal PH domain of Tiam1 for other PH domains argues for a specific targeting function for this PH domain, and similar results have been reported for PH domains in other signaling proteins (Buchsbaum et al., 1996; Kolanus et al., 1996). However, at the moment we cannot determine whether PH domains can function as independent modules or whether additional sequences are required for their activity.


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)

Model for activation of Rac by Tiam1. Activation of  Rac requires membrane translocation of Tiam1 and might be  analogous to activation of Ras by receptor-induced translocation  of SOS. Membrane association of Tiam1 could be mediated by  interactions between the NH2-terminal PH domain and putative  effectors of activated G-protein coupled receptors (GPCR), tyrosine kinase receptors (TKR), or Ras, which may include Gβγ  subunits, PIP2, PIP3, IP3, and perhaps Gα subunits. Activation of  Rac by membrane-associated Tiam1 leads to membrane ruffling  and JNK activation. It is unlikely that JNK is involved in the induction of an oncogenic phenotype in NIH3T3 cells, since mutant  C682 Tiam1 induces an oncogenic phenotype without activation  of JNK. Which Tiam1/Rac-mediated signaling pathways are involved in the induction of an invasive phenotype in T-lymphoma  cells remains to be established.
© Copyright Policy
Related In: Results  -  Collection

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Figure 10: Model for activation of Rac by Tiam1. Activation of Rac requires membrane translocation of Tiam1 and might be analogous to activation of Ras by receptor-induced translocation of SOS. Membrane association of Tiam1 could be mediated by interactions between the NH2-terminal PH domain and putative effectors of activated G-protein coupled receptors (GPCR), tyrosine kinase receptors (TKR), or Ras, which may include Gβγ subunits, PIP2, PIP3, IP3, and perhaps Gα subunits. Activation of Rac by membrane-associated Tiam1 leads to membrane ruffling and JNK activation. It is unlikely that JNK is involved in the induction of an oncogenic phenotype in NIH3T3 cells, since mutant C682 Tiam1 induces an oncogenic phenotype without activation of JNK. Which Tiam1/Rac-mediated signaling pathways are involved in the induction of an invasive phenotype in T-lymphoma cells remains to be established.
Mentions: Several reports have demonstrated that isolated PH domains can bind in vitro to Gβγ subunits and phospholipids like phosphatidylinositol (4,5)bisphosphate (PIP2) and inositol (3,4,5)trisphosphate (IP3) (Harlan et al., 1994; Ferguson et al., 1995; Mahadevan et al., 1995; Touhara et al., 1995). Phosphoinositide-3-kinase, which stimulates the synthesis of phosphatidylinositol (3,4,5)trisphosphate (PIP3) from PIP2 (Hiles et al., 1992), has been implicated in the activation of Rac by PDGF and insulin (Wennstrom et al., 1994; Hawkins et al., 1995) and might be required for the activation of Rac by Ras (Rodriguez-Viciana et al., 1994). Although we have shown that the association of Tiam1 with the plasma membrane is induced by serum, neither PDGF nor insulin could substitute for serum in NIH3T3 cells. Gβγ subunits and phospholipids represent potential downstream effectors of G-protein–coupled receptors and/or activated tyrosine kinase receptors. Based on the in vitro binding data of PH domains, it is tempting to speculate that Gβγ subunits and/or specific phospholipids regulate the association of Tiam1 with the plasma membrane, as depicted in Fig. 10. The fact that we were not able to functionally exchange the NH2-terminal PH domain of Tiam1 for other PH domains argues for a specific targeting function for this PH domain, and similar results have been reported for PH domains in other signaling proteins (Buchsbaum et al., 1996; Kolanus et al., 1996). However, at the moment we cannot determine whether PH domains can function as independent modules or whether additional sequences are required for their activity.

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