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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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Stimulation of JNK activity by Tiam1. (A) COS-7 cells  were transfected with pCMV-Flag-JNK1 and empty vector  (pcDNA-neo), full-length Tiam1 (FL1591), or constitutively  activated (V12)Rac1. Cotransfection with dominant-negative  (N17)Rac1 was performed as indicated. The activity of immunoprecipitated Flag-JNK was determined 48 h after transfection by  an in vitro kinase assay using bacterially expressed GST-Jun as a  substrate. The expression of Tiam1, Myc-tagged (N17)Rac, and  Flag-tagged JNK was analyzed by Western blotting and is shown  underneath the graph. (B) NIH3T3 cells were transfected with  pCMV-Flag-JNK1 and mutant Tiam1 constructs. JNK activity  was determined as described. All data are presented as fold stimulation of empty vector controls. Equal amounts of expressed  Flag-JNK1 and Tiam1 proteins, as determined by Western blotting, were used. Data represent mean ± standard deviation of at  least two independent experiments.
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Figure 9: Stimulation of JNK activity by Tiam1. (A) COS-7 cells were transfected with pCMV-Flag-JNK1 and empty vector (pcDNA-neo), full-length Tiam1 (FL1591), or constitutively activated (V12)Rac1. Cotransfection with dominant-negative (N17)Rac1 was performed as indicated. The activity of immunoprecipitated Flag-JNK was determined 48 h after transfection by an in vitro kinase assay using bacterially expressed GST-Jun as a substrate. The expression of Tiam1, Myc-tagged (N17)Rac, and Flag-tagged JNK was analyzed by Western blotting and is shown underneath the graph. (B) NIH3T3 cells were transfected with pCMV-Flag-JNK1 and mutant Tiam1 constructs. JNK activity was determined as described. All data are presented as fold stimulation of empty vector controls. Equal amounts of expressed Flag-JNK1 and Tiam1 proteins, as determined by Western blotting, were used. Data represent mean ± standard deviation of at least two independent experiments.

Mentions: To investigate whether membrane association of Tiam1 is also required for the induction of other Rac-mediated signaling pathways besides membrane ruffling, the activity of JNK was determined after cotransfection with different Tiam1 mutants (see Materials and Methods). Both in COS-7 cells and NIH3T3 cells, cotransfection of fulllength Tiam1 led to an almost sevenfold stimulation of JNK activity, similar to constitutively activated (V12)Rac1 (Fig. 9 A). Cotransfection of tagged mitogen-activated protein kinase (MAPK) with full-length or mutant Tiam1 or (V12)Rac did not result in activation of MAPK (not shown). The stimulation of JNK by Tiam1 was dependent on the activation of Rac, since cotransfection of dominantnegative (N17)Rac1 partly blocked the activation of JNK by Tiam1 (Fig. 9 A). Higher amounts of (N17)Rac interfered with the expression of JNK. Furthermore, a deletion in the catalytic DH domain of Tiam1 prevented JNK activation (not shown). Cotransfection of dominant-negative (N17)Cdc42 did not interfere with the activation of JNK by Tiam1 (data not shown). Transfection of C1199 Tiam1 stimulated JNK activity to a similar extent as FL1591 (Fig. 9 B). However, shorter COOH-terminal Tiam1 fragments such as C682 and C580 Tiam1, which did not localize to the plasma membrane, were not able to activate JNK above background levels. Also, mutant C1199-ΔPHn Tiam1, which did not localize to the plasma membrane because of the deletion in the NH2-terminal PH domain, was unable to activate JNK (Fig. 9 B). MS-C580 Tiam1 caused no stimulation of JNK activity (Fig. 9 B), and neither did MS-C1199-ΔPHn or MS-FL1591-ΔPHn Tiam1 proteins (data not shown). These proteins, however, induced membrane ruffling and were present at the plasma membrane (Fig. 6). A possible explanation for this somewhat unexpected result is that activation of endogenous Rac by MS-containing Tiam1 proteins is sufficient for induction of membrane ruffling but not for stimulation of JNK. Alternatively, the lack of induction of JNK activity is caused by the different ultrastructural localization of the MS-C580 Tiam1 protein (compare Figs. 5 and 7) or may reflect the need for an intact NH2-terminal PH domain. Interestingly, similar results were obtained with membrane-targeted p110 phosphoinositide-3-kinase which was able to stimulate membrane ruffling but not gene transcription from the Jun-inducible Fos promoter (Reif et al., 1996). Whatever the mechanism, these results indicate that the localization of Tiam1 at the plasma membrane is also required for Rac-mediated stimulation of JNK 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)

Stimulation of JNK activity by Tiam1. (A) COS-7 cells  were transfected with pCMV-Flag-JNK1 and empty vector  (pcDNA-neo), full-length Tiam1 (FL1591), or constitutively  activated (V12)Rac1. Cotransfection with dominant-negative  (N17)Rac1 was performed as indicated. The activity of immunoprecipitated Flag-JNK was determined 48 h after transfection by  an in vitro kinase assay using bacterially expressed GST-Jun as a  substrate. The expression of Tiam1, Myc-tagged (N17)Rac, and  Flag-tagged JNK was analyzed by Western blotting and is shown  underneath the graph. (B) NIH3T3 cells were transfected with  pCMV-Flag-JNK1 and mutant Tiam1 constructs. JNK activity  was determined as described. All data are presented as fold stimulation of empty vector controls. Equal amounts of expressed  Flag-JNK1 and Tiam1 proteins, as determined by Western blotting, were used. Data represent mean ± standard deviation of at  least two independent experiments.
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Figure 9: Stimulation of JNK activity by Tiam1. (A) COS-7 cells were transfected with pCMV-Flag-JNK1 and empty vector (pcDNA-neo), full-length Tiam1 (FL1591), or constitutively activated (V12)Rac1. Cotransfection with dominant-negative (N17)Rac1 was performed as indicated. The activity of immunoprecipitated Flag-JNK was determined 48 h after transfection by an in vitro kinase assay using bacterially expressed GST-Jun as a substrate. The expression of Tiam1, Myc-tagged (N17)Rac, and Flag-tagged JNK was analyzed by Western blotting and is shown underneath the graph. (B) NIH3T3 cells were transfected with pCMV-Flag-JNK1 and mutant Tiam1 constructs. JNK activity was determined as described. All data are presented as fold stimulation of empty vector controls. Equal amounts of expressed Flag-JNK1 and Tiam1 proteins, as determined by Western blotting, were used. Data represent mean ± standard deviation of at least two independent experiments.
Mentions: To investigate whether membrane association of Tiam1 is also required for the induction of other Rac-mediated signaling pathways besides membrane ruffling, the activity of JNK was determined after cotransfection with different Tiam1 mutants (see Materials and Methods). Both in COS-7 cells and NIH3T3 cells, cotransfection of fulllength Tiam1 led to an almost sevenfold stimulation of JNK activity, similar to constitutively activated (V12)Rac1 (Fig. 9 A). Cotransfection of tagged mitogen-activated protein kinase (MAPK) with full-length or mutant Tiam1 or (V12)Rac did not result in activation of MAPK (not shown). The stimulation of JNK by Tiam1 was dependent on the activation of Rac, since cotransfection of dominantnegative (N17)Rac1 partly blocked the activation of JNK by Tiam1 (Fig. 9 A). Higher amounts of (N17)Rac interfered with the expression of JNK. Furthermore, a deletion in the catalytic DH domain of Tiam1 prevented JNK activation (not shown). Cotransfection of dominant-negative (N17)Cdc42 did not interfere with the activation of JNK by Tiam1 (data not shown). Transfection of C1199 Tiam1 stimulated JNK activity to a similar extent as FL1591 (Fig. 9 B). However, shorter COOH-terminal Tiam1 fragments such as C682 and C580 Tiam1, which did not localize to the plasma membrane, were not able to activate JNK above background levels. Also, mutant C1199-ΔPHn Tiam1, which did not localize to the plasma membrane because of the deletion in the NH2-terminal PH domain, was unable to activate JNK (Fig. 9 B). MS-C580 Tiam1 caused no stimulation of JNK activity (Fig. 9 B), and neither did MS-C1199-ΔPHn or MS-FL1591-ΔPHn Tiam1 proteins (data not shown). These proteins, however, induced membrane ruffling and were present at the plasma membrane (Fig. 6). A possible explanation for this somewhat unexpected result is that activation of endogenous Rac by MS-containing Tiam1 proteins is sufficient for induction of membrane ruffling but not for stimulation of JNK. Alternatively, the lack of induction of JNK activity is caused by the different ultrastructural localization of the MS-C580 Tiam1 protein (compare Figs. 5 and 7) or may reflect the need for an intact NH2-terminal PH domain. Interestingly, similar results were obtained with membrane-targeted p110 phosphoinositide-3-kinase which was able to stimulate membrane ruffling but not gene transcription from the Jun-inducible Fos promoter (Reif et al., 1996). Whatever the mechanism, these results indicate that the localization of Tiam1 at the plasma membrane is also required for Rac-mediated stimulation of JNK 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