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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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Interplay of Cdc42, Rac, and Rho activities. (A) Expression of Myc-tagged V14Rho and F28LCdc42 in established NIH3T3 cell lines. Western blot of cell lysates from control and V14Rho- and F28LCdc42-expressing cells using a mixture of anti–Rho and anti–Myc antibodies. (B) V14Rho does not affect endogenous Rac activity. Cell lysates (from 4 × 106 cells) of control and V14Rho-expressing NIH3T3 fibroblasts were assayed for Rac activity. Lysates were incubated with GST-PAK-CD and bound Rac was analyzed by Western blot with antibodies against Rac. Aliquots of total lysates were probed with antibodies against Rac to control for total amounts of Rac proteins. (C) Cdc42 inactivates Rho. Cell lysates (from 4 × 106 cells) of control, F28LCdc42- and V12Rac-expressing NIH3T3 fibroblasts were assayed for Rho activity. Lysates were incubated with GST-C21, and bound Rho was analyzed by Western blot with antibodies against Rho. Aliquots of total lysates were probed with antibodies against Rho to control for total amounts of Rho proteins. (D) F28LCdc42 is an activated Cdc42 mutant and activates Rac. Cell lysates (from 4 × 106 cells) of control and F28LCdc42 NIH3T3 fibroblasts were assayed for Cdc42 activity. After incubation with GST-PAK-CD, bound F28LCdc42 was analyzed by Western blot with antibodies recognizing the Myc-epitope tag and bound Rac with anti–Rac antibodies (Upstate Biotechnology Inc.). (E) Dominant negative N19Rho does not affect endogenous Rac activity. Cell lysates (from 4 × 106 cells) of control and N19Rho-expressing NIH3T3 fibroblasts were assayed for Rac activity and analyzed as in B.
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Figure 8: Interplay of Cdc42, Rac, and Rho activities. (A) Expression of Myc-tagged V14Rho and F28LCdc42 in established NIH3T3 cell lines. Western blot of cell lysates from control and V14Rho- and F28LCdc42-expressing cells using a mixture of anti–Rho and anti–Myc antibodies. (B) V14Rho does not affect endogenous Rac activity. Cell lysates (from 4 × 106 cells) of control and V14Rho-expressing NIH3T3 fibroblasts were assayed for Rac activity. Lysates were incubated with GST-PAK-CD and bound Rac was analyzed by Western blot with antibodies against Rac. Aliquots of total lysates were probed with antibodies against Rac to control for total amounts of Rac proteins. (C) Cdc42 inactivates Rho. Cell lysates (from 4 × 106 cells) of control, F28LCdc42- and V12Rac-expressing NIH3T3 fibroblasts were assayed for Rho activity. Lysates were incubated with GST-C21, and bound Rho was analyzed by Western blot with antibodies against Rho. Aliquots of total lysates were probed with antibodies against Rho to control for total amounts of Rho proteins. (D) F28LCdc42 is an activated Cdc42 mutant and activates Rac. Cell lysates (from 4 × 106 cells) of control and F28LCdc42 NIH3T3 fibroblasts were assayed for Cdc42 activity. After incubation with GST-PAK-CD, bound F28LCdc42 was analyzed by Western blot with antibodies recognizing the Myc-epitope tag and bound Rac with anti–Rac antibodies (Upstate Biotechnology Inc.). (E) Dominant negative N19Rho does not affect endogenous Rac activity. Cell lysates (from 4 × 106 cells) of control and N19Rho-expressing NIH3T3 fibroblasts were assayed for Rac activity and analyzed as in B.

Mentions: To address whether Rac and Rho regulate their activities in a reciprocal manner or whether downregulation of Rho activity is specifically mediated by Rac, we established an NIH3T3 cell line stably expressing Myc-tagged V14Rho (Fig. 8 A, lane 2). Rac activity in V14Rho-expressing cells was not significantly changed compared with control cells (Fig. 8 B), consistent with the equal Rac activities found in serum-starved Tiam1-expressing cells and cells stimulated with LPA (Fig. 3 C). Similarly, blocking Rho activity by dominant negative N19Rho (Fig. 8 E) or treatment of cells with the Rho-kinase inhibitor Y27632 (not shown) did not affect endogenous Rac activity. From these data, we conclude that V14Rho or LPA-mediated Rho activation does not result in downmodulation of Rac activity, and neither does dominant negative N19Rho result in upregulation of Rac activity. Apparently, modulation of Rho activity itself does not affect cellular Rac activity in NIH3T3 cells.


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)

Interplay of Cdc42, Rac, and Rho activities. (A) Expression of Myc-tagged V14Rho and F28LCdc42 in established NIH3T3 cell lines. Western blot of cell lysates from control and V14Rho- and F28LCdc42-expressing cells using a mixture of anti–Rho and anti–Myc antibodies. (B) V14Rho does not affect endogenous Rac activity. Cell lysates (from 4 × 106 cells) of control and V14Rho-expressing NIH3T3 fibroblasts were assayed for Rac activity. Lysates were incubated with GST-PAK-CD and bound Rac was analyzed by Western blot with antibodies against Rac. Aliquots of total lysates were probed with antibodies against Rac to control for total amounts of Rac proteins. (C) Cdc42 inactivates Rho. Cell lysates (from 4 × 106 cells) of control, F28LCdc42- and V12Rac-expressing NIH3T3 fibroblasts were assayed for Rho activity. Lysates were incubated with GST-C21, and bound Rho was analyzed by Western blot with antibodies against Rho. Aliquots of total lysates were probed with antibodies against Rho to control for total amounts of Rho proteins. (D) F28LCdc42 is an activated Cdc42 mutant and activates Rac. Cell lysates (from 4 × 106 cells) of control and F28LCdc42 NIH3T3 fibroblasts were assayed for Cdc42 activity. After incubation with GST-PAK-CD, bound F28LCdc42 was analyzed by Western blot with antibodies recognizing the Myc-epitope tag and bound Rac with anti–Rac antibodies (Upstate Biotechnology Inc.). (E) Dominant negative N19Rho does not affect endogenous Rac activity. Cell lysates (from 4 × 106 cells) of control and N19Rho-expressing NIH3T3 fibroblasts were assayed for Rac activity and analyzed as in B.
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Figure 8: Interplay of Cdc42, Rac, and Rho activities. (A) Expression of Myc-tagged V14Rho and F28LCdc42 in established NIH3T3 cell lines. Western blot of cell lysates from control and V14Rho- and F28LCdc42-expressing cells using a mixture of anti–Rho and anti–Myc antibodies. (B) V14Rho does not affect endogenous Rac activity. Cell lysates (from 4 × 106 cells) of control and V14Rho-expressing NIH3T3 fibroblasts were assayed for Rac activity. Lysates were incubated with GST-PAK-CD and bound Rac was analyzed by Western blot with antibodies against Rac. Aliquots of total lysates were probed with antibodies against Rac to control for total amounts of Rac proteins. (C) Cdc42 inactivates Rho. Cell lysates (from 4 × 106 cells) of control, F28LCdc42- and V12Rac-expressing NIH3T3 fibroblasts were assayed for Rho activity. Lysates were incubated with GST-C21, and bound Rho was analyzed by Western blot with antibodies against Rho. Aliquots of total lysates were probed with antibodies against Rho to control for total amounts of Rho proteins. (D) F28LCdc42 is an activated Cdc42 mutant and activates Rac. Cell lysates (from 4 × 106 cells) of control and F28LCdc42 NIH3T3 fibroblasts were assayed for Cdc42 activity. After incubation with GST-PAK-CD, bound F28LCdc42 was analyzed by Western blot with antibodies recognizing the Myc-epitope tag and bound Rac with anti–Rac antibodies (Upstate Biotechnology Inc.). (E) Dominant negative N19Rho does not affect endogenous Rac activity. Cell lysates (from 4 × 106 cells) of control and N19Rho-expressing NIH3T3 fibroblasts were assayed for Rac activity and analyzed as in B.
Mentions: To address whether Rac and Rho regulate their activities in a reciprocal manner or whether downregulation of Rho activity is specifically mediated by Rac, we established an NIH3T3 cell line stably expressing Myc-tagged V14Rho (Fig. 8 A, lane 2). Rac activity in V14Rho-expressing cells was not significantly changed compared with control cells (Fig. 8 B), consistent with the equal Rac activities found in serum-starved Tiam1-expressing cells and cells stimulated with LPA (Fig. 3 C). Similarly, blocking Rho activity by dominant negative N19Rho (Fig. 8 E) or treatment of cells with the Rho-kinase inhibitor Y27632 (not shown) did not affect endogenous Rac activity. From these data, we conclude that V14Rho or LPA-mediated Rho activation does not result in downmodulation of Rac activity, and neither does dominant negative N19Rho result in upregulation of Rac activity. Apparently, modulation of Rho activity itself does not affect cellular Rac activity in NIH3T3 cells.

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