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TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase.

Goncharova E, Goncharov D, Noonan D, Krymskaya VP - J. Cell Biol. (2004)

Bottom Line: Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM).The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of stress fibers.Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM). However, TSC1 also activates Rho and regulates cell adhesion. We found that TSC2 modulates actin dynamics and cell adhesion and the TSC1-binding domain (TSC2-HBD) is essential for this function of TSC2. Expression of TSC2 or TSC2-HBD in TSC2-/- cells promoted Rac1 activation, inhibition of Rho, stress fiber disassembly, and focal adhesion remodeling. The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of stress fibers. Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1. Because TSC1 and TSC2 regulate Rho and Rac1, whose activities are interconnected in a reciprocal fashion, loss of either TSC1 or TSC2 function may result in the deregulation of cell motility and adhesion, which are associated with the pathobiology of TSC and LAM.

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TSC2 and TSC2-HBD inhibit Rho activity in TSC2−/− cells. (A) TSC2−/− cells were transfected with pEBG-V12Rho or GFP-TSC2; GFP-TSC2 was cotransfected with pEBG-V12Rho plasmids; or pEGFP-TSC2-HBD was cotransfected with pEBG-V12Rho plasmid; and then cells were stained with anti-GFP to detect GFP, GFP-TSC2, or GFP-TSC2-HBD (green), anti-GST to detect GST, or GST-V12Rho (blue) and rhodamine phalloidin to detect F-actin (red). Images are representative of three independent experiments. Bar, 20 μm. (B) Cells were transfected with pEGFP-TSC2, pEGFP-TSC2-HBD, and control pEGFP plasmids expressing GFP-tagged TSC2, GFP-TSC2-HBD, and control GFP, respectively, and then Rho activity assay was performed. Immunoblot analysis of Rho-GTP pull-down with Rhotekin-RBD agarose (top) and whole cell lysates (bottom) was performed with anti-Rho antibodies. White lines indicate that intervening lanes have been spliced out. Quantitative analysis of three independent experiments was performed using Gel-Pro Analyzer Software. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP by ANOVA (Bonferroni-Dunn test).
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fig7: TSC2 and TSC2-HBD inhibit Rho activity in TSC2−/− cells. (A) TSC2−/− cells were transfected with pEBG-V12Rho or GFP-TSC2; GFP-TSC2 was cotransfected with pEBG-V12Rho plasmids; or pEGFP-TSC2-HBD was cotransfected with pEBG-V12Rho plasmid; and then cells were stained with anti-GFP to detect GFP, GFP-TSC2, or GFP-TSC2-HBD (green), anti-GST to detect GST, or GST-V12Rho (blue) and rhodamine phalloidin to detect F-actin (red). Images are representative of three independent experiments. Bar, 20 μm. (B) Cells were transfected with pEGFP-TSC2, pEGFP-TSC2-HBD, and control pEGFP plasmids expressing GFP-tagged TSC2, GFP-TSC2-HBD, and control GFP, respectively, and then Rho activity assay was performed. Immunoblot analysis of Rho-GTP pull-down with Rhotekin-RBD agarose (top) and whole cell lysates (bottom) was performed with anti-Rho antibodies. White lines indicate that intervening lanes have been spliced out. Quantitative analysis of three independent experiments was performed using Gel-Pro Analyzer Software. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP by ANOVA (Bonferroni-Dunn test).

Mentions: The small GTPase RhoA is necessary for stress fiber and focal adhesion formation (Etienne-Manneville and Hall, 2002) and TSC1 activates Rho (Lamb et al., 2000). To determine whether or not TSC2-induced stress fiber disassembly was by impaired signaling downstream of Rho, we cotransfected TSC2 or TSC2-HBD with constitutively active Rho, V14Rho. As seen in Fig. 7 A (panels III and IV, respectively), in cells cotransfected with V14Rho, stress fibers were maintained compared with cells expressing TSC2 alone (Fig. 7 A, panel II). These data suggest that stress fiber disassembly induced by TSC2 and TSC2-HBD is not due to failure in pathways downstream of Rho that regulate assembly and maintenance of stress fibers.


TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase.

Goncharova E, Goncharov D, Noonan D, Krymskaya VP - J. Cell Biol. (2004)

TSC2 and TSC2-HBD inhibit Rho activity in TSC2−/− cells. (A) TSC2−/− cells were transfected with pEBG-V12Rho or GFP-TSC2; GFP-TSC2 was cotransfected with pEBG-V12Rho plasmids; or pEGFP-TSC2-HBD was cotransfected with pEBG-V12Rho plasmid; and then cells were stained with anti-GFP to detect GFP, GFP-TSC2, or GFP-TSC2-HBD (green), anti-GST to detect GST, or GST-V12Rho (blue) and rhodamine phalloidin to detect F-actin (red). Images are representative of three independent experiments. Bar, 20 μm. (B) Cells were transfected with pEGFP-TSC2, pEGFP-TSC2-HBD, and control pEGFP plasmids expressing GFP-tagged TSC2, GFP-TSC2-HBD, and control GFP, respectively, and then Rho activity assay was performed. Immunoblot analysis of Rho-GTP pull-down with Rhotekin-RBD agarose (top) and whole cell lysates (bottom) was performed with anti-Rho antibodies. White lines indicate that intervening lanes have been spliced out. Quantitative analysis of three independent experiments was performed using Gel-Pro Analyzer Software. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP by ANOVA (Bonferroni-Dunn test).
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fig7: TSC2 and TSC2-HBD inhibit Rho activity in TSC2−/− cells. (A) TSC2−/− cells were transfected with pEBG-V12Rho or GFP-TSC2; GFP-TSC2 was cotransfected with pEBG-V12Rho plasmids; or pEGFP-TSC2-HBD was cotransfected with pEBG-V12Rho plasmid; and then cells were stained with anti-GFP to detect GFP, GFP-TSC2, or GFP-TSC2-HBD (green), anti-GST to detect GST, or GST-V12Rho (blue) and rhodamine phalloidin to detect F-actin (red). Images are representative of three independent experiments. Bar, 20 μm. (B) Cells were transfected with pEGFP-TSC2, pEGFP-TSC2-HBD, and control pEGFP plasmids expressing GFP-tagged TSC2, GFP-TSC2-HBD, and control GFP, respectively, and then Rho activity assay was performed. Immunoblot analysis of Rho-GTP pull-down with Rhotekin-RBD agarose (top) and whole cell lysates (bottom) was performed with anti-Rho antibodies. White lines indicate that intervening lanes have been spliced out. Quantitative analysis of three independent experiments was performed using Gel-Pro Analyzer Software. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP by ANOVA (Bonferroni-Dunn test).
Mentions: The small GTPase RhoA is necessary for stress fiber and focal adhesion formation (Etienne-Manneville and Hall, 2002) and TSC1 activates Rho (Lamb et al., 2000). To determine whether or not TSC2-induced stress fiber disassembly was by impaired signaling downstream of Rho, we cotransfected TSC2 or TSC2-HBD with constitutively active Rho, V14Rho. As seen in Fig. 7 A (panels III and IV, respectively), in cells cotransfected with V14Rho, stress fibers were maintained compared with cells expressing TSC2 alone (Fig. 7 A, panel II). These data suggest that stress fiber disassembly induced by TSC2 and TSC2-HBD is not due to failure in pathways downstream of Rho that regulate assembly and maintenance of stress fibers.

Bottom Line: Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM).The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of stress fibers.Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM). However, TSC1 also activates Rho and regulates cell adhesion. We found that TSC2 modulates actin dynamics and cell adhesion and the TSC1-binding domain (TSC2-HBD) is essential for this function of TSC2. Expression of TSC2 or TSC2-HBD in TSC2-/- cells promoted Rac1 activation, inhibition of Rho, stress fiber disassembly, and focal adhesion remodeling. The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of stress fibers. Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1. Because TSC1 and TSC2 regulate Rho and Rac1, whose activities are interconnected in a reciprocal fashion, loss of either TSC1 or TSC2 function may result in the deregulation of cell motility and adhesion, which are associated with the pathobiology of TSC and LAM.

Show MeSH
Related in: MedlinePlus