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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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Model showing the crosstalk of Rho-like GTPases regulating cell spreading and contractility. Constitutively active V12Rac or PDGF receptor-mediated Rac activation results in inactivation of Rho activity. Cdc42 similarly mediates inactivation of Rho, either by activation of Rac or by signaling directly to Rho. LPA stimulation activates Rho, but does not affect Rac activity. Moreover, in cells exhibiting high Rac activity (Tiam1- or V12Rac-expressing cells), LPA-mediated activation of Rho is desensitized. The Rac-mediated regulation of Rho activity might involve GAP, GEF or GDI proteins, as Rho-GTP levels are reduced by activation of Rac.
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Figure 11: Model showing the crosstalk of Rho-like GTPases regulating cell spreading and contractility. Constitutively active V12Rac or PDGF receptor-mediated Rac activation results in inactivation of Rho activity. Cdc42 similarly mediates inactivation of Rho, either by activation of Rac or by signaling directly to Rho. LPA stimulation activates Rho, but does not affect Rac activity. Moreover, in cells exhibiting high Rac activity (Tiam1- or V12Rac-expressing cells), LPA-mediated activation of Rho is desensitized. The Rac-mediated regulation of Rho activity might involve GAP, GEF or GDI proteins, as Rho-GTP levels are reduced by activation of Rac.

Mentions: Rac-mediated downregulation of Rho occurred downstream of Rac. This signal regulates Rho activity upstream of Rho since low levels of GTP-bound Rho were found. The biochemical analysis of the actual GTPase activities in NIH3T3 fibroblasts supports a model (Fig. 11) that Rac and Rho signaling antagonize each other to control the cellular phenotype and migratory behavior. Activation of Rac induces cell spreading, resulting in an epithelioid phenotype. Activation of Rho promotes a more fibroblastoid, motile phenotype because of enhanced contractility by the formation of stress fibers. Note that for an epithelioid as well as for a migratory phenotype, the activities of both Rac and Rho GTPases are required, but the balance of their reciprocal activities determines the cellular morphology and the migratory behavior. At the molecular level, this is controlled by the negative regulation of Rho activity involving Cdc42 and Rac signaling. Activity of the distinct GTPases might be regulated locally in cells in response to extracellular stimuli, allowing cellular migration by coordinated activation/inactivation of Rho-like proteins. Our current research is focusing on the signaling pathways downstream of Cdc42 and Rac involved in downregulation of Rho. Complex formation and/or activity of regulators of Rho, including GAP, GEF, and GDI proteins, might be altered. p190RhoGAP may be a good candidate involved in Rac-mediated downregulation of Rho, as p190 becomes phosphorylated on tyrosine after EGF stimulation (Chang et al. 1995) and β1 integrin signaling (Nakahara et al. 1998). A PKC-mediated pathway could also play a role, since in Swiss 3T3 fibroblast, LPA-induced stress fiber formation is inhibited by short pretreatment with PDGF or the phorbol ester PMA. Stress fibers induced by microinjected V14Rho are not sensitive to PMA treatment (Ridley and Hall 1994). In light of the present data, Rac might activate a PMA-sensitive isoform of PKC, thereby negatively regulating Rho activity. As phosphorylation of the myosin light chain by Rho contributes to increased contractility (Amano et al. 1996; Kimura et al. 1996), a consequence of Rac-induced downregulation of Rho might be the inhibition of myosin light chain phosphorylation leading to loss of cellular contraction and allowing cell spreading. Taken together, we conclude that the molecular crosstalk of different Rho family GTPases, specifically Rac-mediated downregulation of Rho activity, determines cellular morphology and migratory behavior of NIH3T3 fibroblasts.


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)

Model showing the crosstalk of Rho-like GTPases regulating cell spreading and contractility. Constitutively active V12Rac or PDGF receptor-mediated Rac activation results in inactivation of Rho activity. Cdc42 similarly mediates inactivation of Rho, either by activation of Rac or by signaling directly to Rho. LPA stimulation activates Rho, but does not affect Rac activity. Moreover, in cells exhibiting high Rac activity (Tiam1- or V12Rac-expressing cells), LPA-mediated activation of Rho is desensitized. The Rac-mediated regulation of Rho activity might involve GAP, GEF or GDI proteins, as Rho-GTP levels are reduced by activation of Rac.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169355&req=5

Figure 11: Model showing the crosstalk of Rho-like GTPases regulating cell spreading and contractility. Constitutively active V12Rac or PDGF receptor-mediated Rac activation results in inactivation of Rho activity. Cdc42 similarly mediates inactivation of Rho, either by activation of Rac or by signaling directly to Rho. LPA stimulation activates Rho, but does not affect Rac activity. Moreover, in cells exhibiting high Rac activity (Tiam1- or V12Rac-expressing cells), LPA-mediated activation of Rho is desensitized. The Rac-mediated regulation of Rho activity might involve GAP, GEF or GDI proteins, as Rho-GTP levels are reduced by activation of Rac.
Mentions: Rac-mediated downregulation of Rho occurred downstream of Rac. This signal regulates Rho activity upstream of Rho since low levels of GTP-bound Rho were found. The biochemical analysis of the actual GTPase activities in NIH3T3 fibroblasts supports a model (Fig. 11) that Rac and Rho signaling antagonize each other to control the cellular phenotype and migratory behavior. Activation of Rac induces cell spreading, resulting in an epithelioid phenotype. Activation of Rho promotes a more fibroblastoid, motile phenotype because of enhanced contractility by the formation of stress fibers. Note that for an epithelioid as well as for a migratory phenotype, the activities of both Rac and Rho GTPases are required, but the balance of their reciprocal activities determines the cellular morphology and the migratory behavior. At the molecular level, this is controlled by the negative regulation of Rho activity involving Cdc42 and Rac signaling. Activity of the distinct GTPases might be regulated locally in cells in response to extracellular stimuli, allowing cellular migration by coordinated activation/inactivation of Rho-like proteins. Our current research is focusing on the signaling pathways downstream of Cdc42 and Rac involved in downregulation of Rho. Complex formation and/or activity of regulators of Rho, including GAP, GEF, and GDI proteins, might be altered. p190RhoGAP may be a good candidate involved in Rac-mediated downregulation of Rho, as p190 becomes phosphorylated on tyrosine after EGF stimulation (Chang et al. 1995) and β1 integrin signaling (Nakahara et al. 1998). A PKC-mediated pathway could also play a role, since in Swiss 3T3 fibroblast, LPA-induced stress fiber formation is inhibited by short pretreatment with PDGF or the phorbol ester PMA. Stress fibers induced by microinjected V14Rho are not sensitive to PMA treatment (Ridley and Hall 1994). In light of the present data, Rac might activate a PMA-sensitive isoform of PKC, thereby negatively regulating Rho activity. As phosphorylation of the myosin light chain by Rho contributes to increased contractility (Amano et al. 1996; Kimura et al. 1996), a consequence of Rac-induced downregulation of Rho might be the inhibition of myosin light chain phosphorylation leading to loss of cellular contraction and allowing cell spreading. Taken together, we conclude that the molecular crosstalk of different Rho family GTPases, specifically Rac-mediated downregulation of Rho activity, determines cellular morphology and migratory behavior of NIH3T3 fibroblasts.

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