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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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PDGF-mediated Rac activation simultaneously results in inactivation of Rho. 4 × 106 NIH3T3 cells were deprived of serum for 24 h, and then stimulated with 20 ng/ml PDGF for 0, 3, 7, or 60 min. Cell lysates were assayed for Rac and Rho activities and incubated either with GST-PAK-CD or GST-C21. Bound GTPases were analyzed by Western blot using specific Rac and Rho antibodies.
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Figure 9: PDGF-mediated Rac activation simultaneously results in inactivation of Rho. 4 × 106 NIH3T3 cells were deprived of serum for 24 h, and then stimulated with 20 ng/ml PDGF for 0, 3, 7, or 60 min. Cell lysates were assayed for Rac and Rho activities and incubated either with GST-PAK-CD or GST-C21. Bound GTPases were analyzed by Western blot using specific Rac and Rho antibodies.

Mentions: To address whether inactivation of Rho is a phenomenon restricted to sustained activation of Rac in established cell lines or whether growth factor–induced transient activation of Rac leads to downregulation of Rho, we stimulated serum-starved control NIH3T3 cells with PDGF. In agreement with the established link between PDGF-mediated receptor stimulation and Rac activation, PDGF activated Rac (Fig. 9) and not Cdc42 (data not shown) after 3 and 7 min of stimulation. PDGF-mediated activation of Rac was found to be a transient effect and was no longer detected 1 h after PDGF stimulation (Fig. 9). Simultaneously with activation of Rac, we found a time-dependent inactivation of Rho in response to PDGF stimulation (Fig. 9). Similarly to the activation of Rac, Rho activity returned to basal levels 1 h after PDGF stimulation. These data suggest that PDGF-mediated Rac activation also results in downregulation of Rho activity, as the kinetics of Rac activation correspond with inactivation of Rho.


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)

PDGF-mediated Rac activation simultaneously results in inactivation of Rho. 4 × 106 NIH3T3 cells were deprived of serum for 24 h, and then stimulated with 20 ng/ml PDGF for 0, 3, 7, or 60 min. Cell lysates were assayed for Rac and Rho activities and incubated either with GST-PAK-CD or GST-C21. Bound GTPases were analyzed by Western blot using specific Rac and Rho antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: PDGF-mediated Rac activation simultaneously results in inactivation of Rho. 4 × 106 NIH3T3 cells were deprived of serum for 24 h, and then stimulated with 20 ng/ml PDGF for 0, 3, 7, or 60 min. Cell lysates were assayed for Rac and Rho activities and incubated either with GST-PAK-CD or GST-C21. Bound GTPases were analyzed by Western blot using specific Rac and Rho antibodies.
Mentions: To address whether inactivation of Rho is a phenomenon restricted to sustained activation of Rac in established cell lines or whether growth factor–induced transient activation of Rac leads to downregulation of Rho, we stimulated serum-starved control NIH3T3 cells with PDGF. In agreement with the established link between PDGF-mediated receptor stimulation and Rac activation, PDGF activated Rac (Fig. 9) and not Cdc42 (data not shown) after 3 and 7 min of stimulation. PDGF-mediated activation of Rac was found to be a transient effect and was no longer detected 1 h after PDGF stimulation (Fig. 9). Simultaneously with activation of Rac, we found a time-dependent inactivation of Rho in response to PDGF stimulation (Fig. 9). Similarly to the activation of Rac, Rho activity returned to basal levels 1 h after PDGF stimulation. These data suggest that PDGF-mediated Rac activation also results in downregulation of Rho activity, as the kinetics of Rac activation correspond with inactivation of Rho.

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