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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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Rac activation downregulates Rho activity. (A) Cell lysates (from 3 × 106 cells) of control, C1199Tiam1- or C580Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho activities. Lysates were incubated with GST-C21 and bound Rho was analyzed with antibodies against RhoA. (B) Cell lysates of control, C1199Tiam1- or V12Rac-expressing NIH3T3 fibroblasts were assayed for Rho activities, similar as in A. Aliquots of total lysates were probed with anti–Rho antibodies to control for total amounts of Rho protein or with anti–Myc (9E10) antibodies to control expression of V12Rac.
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Figure 4: Rac activation downregulates Rho activity. (A) Cell lysates (from 3 × 106 cells) of control, C1199Tiam1- or C580Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho activities. Lysates were incubated with GST-C21 and bound Rho was analyzed with antibodies against RhoA. (B) Cell lysates of control, C1199Tiam1- or V12Rac-expressing NIH3T3 fibroblasts were assayed for Rho activities, similar as in A. Aliquots of total lysates were probed with anti–Rho antibodies to control for total amounts of Rho protein or with anti–Myc (9E10) antibodies to control expression of V12Rac.

Mentions: However, comparing control and Tiam1-expressing fibroblasts, we found that in serum-starved C1199Tiam1-expressing cells almost no Rho activity was detectable (compare lanes 5 and 2), whereas C1199Tiam1-expressing cells grown in the presence of serum still showed some Rho activity (data not shown and see Fig. 4). Similarly, Rho activation after stimulation with LPA was much lower in C1199Tiam1-expressing cells than the activation of Rho by LPA in control cells (compare Fig. 3, lanes 4 and 3). Thus, expression of C1199Tiam1 resulted in decreased Rho activity and a desensitized stimulation by LPA.


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)

Rac activation downregulates Rho activity. (A) Cell lysates (from 3 × 106 cells) of control, C1199Tiam1- or C580Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho activities. Lysates were incubated with GST-C21 and bound Rho was analyzed with antibodies against RhoA. (B) Cell lysates of control, C1199Tiam1- or V12Rac-expressing NIH3T3 fibroblasts were assayed for Rho activities, similar as in A. Aliquots of total lysates were probed with anti–Rho antibodies to control for total amounts of Rho protein or with anti–Myc (9E10) antibodies to control expression of V12Rac.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Rac activation downregulates Rho activity. (A) Cell lysates (from 3 × 106 cells) of control, C1199Tiam1- or C580Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho activities. Lysates were incubated with GST-C21 and bound Rho was analyzed with antibodies against RhoA. (B) Cell lysates of control, C1199Tiam1- or V12Rac-expressing NIH3T3 fibroblasts were assayed for Rho activities, similar as in A. Aliquots of total lysates were probed with anti–Rho antibodies to control for total amounts of Rho protein or with anti–Myc (9E10) antibodies to control expression of V12Rac.
Mentions: However, comparing control and Tiam1-expressing fibroblasts, we found that in serum-starved C1199Tiam1-expressing cells almost no Rho activity was detectable (compare lanes 5 and 2), whereas C1199Tiam1-expressing cells grown in the presence of serum still showed some Rho activity (data not shown and see Fig. 4). Similarly, Rho activation after stimulation with LPA was much lower in C1199Tiam1-expressing cells than the activation of Rho by LPA in control cells (compare Fig. 3, lanes 4 and 3). Thus, expression of C1199Tiam1 resulted in decreased Rho activity and a desensitized stimulation by LPA.

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