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Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.

Sander EE, ten Klooster JP, van Delft S, van der Kammen RA, Collard JG - J. Cell Biol. (1999)

Bottom Line: We found that Cdc42 also downregulates Rho activity.Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho.We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: The Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

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Activation of endogenous Rac by Tiam1 requires membrane localization of the GEF and inhibits migration of NIH3T3 fibroblasts. (A) Activation of endogenous Rac in control NIH3T3 cells or cells expressing C1199- or C580Tiam1. Lysates of the indicated cell lines (from 3 × 106 cells) were incubated with GST-PAK-CD and the bound Rac molecules were detected by Western blot. Aliquots of the respective lysates serve as controls for analyzing total amounts of Rac protein. (B) Tiam1 is a specific activator of Rac and not Cdc42. Lysates of control or Tiam1-expressing NIH3T3 cells were incubated with GST-PAK-CD and the precipitated molecules probed with antibodies against Cdc42. (C) Migration rates of 7 × 104 control or C1199Tiam1-expressing NIH3T3 cells per well coated with fibronectin were determined towards 20 ng/ml PDGF contained in the lower chamber of the transwell. Each bar represents the mean ± SD of triplicate migration assays. Shown is one representative example of three independent experiments.
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Figure 2: Activation of endogenous Rac by Tiam1 requires membrane localization of the GEF and inhibits migration of NIH3T3 fibroblasts. (A) Activation of endogenous Rac in control NIH3T3 cells or cells expressing C1199- or C580Tiam1. Lysates of the indicated cell lines (from 3 × 106 cells) were incubated with GST-PAK-CD and the bound Rac molecules were detected by Western blot. Aliquots of the respective lysates serve as controls for analyzing total amounts of Rac protein. (B) Tiam1 is a specific activator of Rac and not Cdc42. Lysates of control or Tiam1-expressing NIH3T3 cells were incubated with GST-PAK-CD and the precipitated molecules probed with antibodies against Cdc42. (C) Migration rates of 7 × 104 control or C1199Tiam1-expressing NIH3T3 cells per well coated with fibronectin were determined towards 20 ng/ml PDGF contained in the lower chamber of the transwell. Each bar represents the mean ± SD of triplicate migration assays. Shown is one representative example of three independent experiments.

Mentions: C580Tiam1 still contains the catalytic DH domain, but does not localize at the cell membrane and does not induce an epithelial-like morphology, including membrane ruffling. To study whether C580Tiam1 is able to activate Rac, we compared the activation of endogenous Rac in cells expressing C1199- or C580Tiam1 (Fig. 2 A). Cell lysates expressing either one of the Tiam1 proteins were incubated with a fusion protein of glutathione-S-transferase (GST) and the CRIB domain of the Rac/Cdc42 effector molecule Pak (GST-PAK-CD). Activated, i.e., GTP-loaded Rac bound to GST-PAK-CD, was analyzed by Western blot with an antibody directed against Rac1. C1199Tiam1 strongly activated endogenous Rac, compared with control NIH3T3 cells (Fig. 2 A, lanes 1 and 2). Expression of the cytoplasmic C580Tiam1 containing the catalytic DH domain only weakly stimulated Rac activity (Fig. 2 A, lane 3). This indicates that efficient activation of endogenous Rac by Tiam1 requires membrane localization of the GEF.


Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.

Sander EE, ten Klooster JP, van Delft S, van der Kammen RA, Collard JG - J. Cell Biol. (1999)

Activation of endogenous Rac by Tiam1 requires membrane localization of the GEF and inhibits migration of NIH3T3 fibroblasts. (A) Activation of endogenous Rac in control NIH3T3 cells or cells expressing C1199- or C580Tiam1. Lysates of the indicated cell lines (from 3 × 106 cells) were incubated with GST-PAK-CD and the bound Rac molecules were detected by Western blot. Aliquots of the respective lysates serve as controls for analyzing total amounts of Rac protein. (B) Tiam1 is a specific activator of Rac and not Cdc42. Lysates of control or Tiam1-expressing NIH3T3 cells were incubated with GST-PAK-CD and the precipitated molecules probed with antibodies against Cdc42. (C) Migration rates of 7 × 104 control or C1199Tiam1-expressing NIH3T3 cells per well coated with fibronectin were determined towards 20 ng/ml PDGF contained in the lower chamber of the transwell. Each bar represents the mean ± SD of triplicate migration assays. Shown is one representative example of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169355&req=5

Figure 2: Activation of endogenous Rac by Tiam1 requires membrane localization of the GEF and inhibits migration of NIH3T3 fibroblasts. (A) Activation of endogenous Rac in control NIH3T3 cells or cells expressing C1199- or C580Tiam1. Lysates of the indicated cell lines (from 3 × 106 cells) were incubated with GST-PAK-CD and the bound Rac molecules were detected by Western blot. Aliquots of the respective lysates serve as controls for analyzing total amounts of Rac protein. (B) Tiam1 is a specific activator of Rac and not Cdc42. Lysates of control or Tiam1-expressing NIH3T3 cells were incubated with GST-PAK-CD and the precipitated molecules probed with antibodies against Cdc42. (C) Migration rates of 7 × 104 control or C1199Tiam1-expressing NIH3T3 cells per well coated with fibronectin were determined towards 20 ng/ml PDGF contained in the lower chamber of the transwell. Each bar represents the mean ± SD of triplicate migration assays. Shown is one representative example of three independent experiments.
Mentions: C580Tiam1 still contains the catalytic DH domain, but does not localize at the cell membrane and does not induce an epithelial-like morphology, including membrane ruffling. To study whether C580Tiam1 is able to activate Rac, we compared the activation of endogenous Rac in cells expressing C1199- or C580Tiam1 (Fig. 2 A). Cell lysates expressing either one of the Tiam1 proteins were incubated with a fusion protein of glutathione-S-transferase (GST) and the CRIB domain of the Rac/Cdc42 effector molecule Pak (GST-PAK-CD). Activated, i.e., GTP-loaded Rac bound to GST-PAK-CD, was analyzed by Western blot with an antibody directed against Rac1. C1199Tiam1 strongly activated endogenous Rac, compared with control NIH3T3 cells (Fig. 2 A, lanes 1 and 2). Expression of the cytoplasmic C580Tiam1 containing the catalytic DH domain only weakly stimulated Rac activity (Fig. 2 A, lane 3). This indicates that efficient activation of endogenous Rac by Tiam1 requires membrane localization of the GEF.

Bottom Line: We found that Cdc42 also downregulates Rho activity.Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho.We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: The Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

Show MeSH
Related in: MedlinePlus