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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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Dominant-negative Rac1 abrogates TSC2- and siRNA TSC1-induced stress fiber disassembly. (A) Serum-deprived cells were transfected with pEBG-V12Rac1 expressing activated Rac1 or the cells were cotransfected with pEGFP-TSC2 and pEBG-N17Rac1 plasmids, expressing GFP-TSC2 and GST-N17Rac1, respectively, and were stained with anti-GFP to detect GFP-TSC2 (green), anti-GST to detect GST, GST-V12Rac1, GST-N17Rac1 (blue), and phalloidin rhodamine to detect F-actin (red). Images are representative of three independent experiments. Bar, 20 μm. (B) Cells, microinjected with GST-N17Rac1, comicroinjected with siRNA TSC1 and GST to identify microinjected cells, or comicroinjected with siRNA TSC1 and N17Rac1, were stained with anti-GST antibody to detect GST and GST-N1Rac1 (green) and phalloidin rhodamine to detect F-actin (red). Images are representative of three independent experiments. (C) Quantitative analysis of F-actin staining. Data represent the percentage of cells with stress fibers per total number of cells transfected with plasmids expressing GST, GST-V12Rac1, GST-N17Rac1, GFP-TSC2, or coexpressed GFP-TSC2 and GST-N17Rac1, siRNA TSC1 and GST-N17Rac1, or siRNA TSC1 and control GST taken as 100%. Data represent the mean ± SE from three independent experiments. *, P < 0.001 for GST-V12Rac1 versus GST, siRNA TSC1 + GST versus siRNA TSC1 + GST-N17Rac1, and GFP-TSC2 versus GFP-TSC2 + GST-N17Rac1 by ANOVA (Bonferroni-Dunn test).
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fig8: Dominant-negative Rac1 abrogates TSC2- and siRNA TSC1-induced stress fiber disassembly. (A) Serum-deprived cells were transfected with pEBG-V12Rac1 expressing activated Rac1 or the cells were cotransfected with pEGFP-TSC2 and pEBG-N17Rac1 plasmids, expressing GFP-TSC2 and GST-N17Rac1, respectively, and were stained with anti-GFP to detect GFP-TSC2 (green), anti-GST to detect GST, GST-V12Rac1, GST-N17Rac1 (blue), and phalloidin rhodamine to detect F-actin (red). Images are representative of three independent experiments. Bar, 20 μm. (B) Cells, microinjected with GST-N17Rac1, comicroinjected with siRNA TSC1 and GST to identify microinjected cells, or comicroinjected with siRNA TSC1 and N17Rac1, were stained with anti-GST antibody to detect GST and GST-N1Rac1 (green) and phalloidin rhodamine to detect F-actin (red). Images are representative of three independent experiments. (C) Quantitative analysis of F-actin staining. Data represent the percentage of cells with stress fibers per total number of cells transfected with plasmids expressing GST, GST-V12Rac1, GST-N17Rac1, GFP-TSC2, or coexpressed GFP-TSC2 and GST-N17Rac1, siRNA TSC1 and GST-N17Rac1, or siRNA TSC1 and control GST taken as 100%. Data represent the mean ± SE from three independent experiments. *, P < 0.001 for GST-V12Rac1 versus GST, siRNA TSC1 + GST versus siRNA TSC1 + GST-N17Rac1, and GFP-TSC2 versus GFP-TSC2 + GST-N17Rac1 by ANOVA (Bonferroni-Dunn test).

Mentions: Rho and Rac1 both regulate stress fiber formation and focal adhesion remodeling in a reciprocal manner, as such activation of Rac1 results in the inhibition of Rho and vice versa (Horwitz and Parsons, 1999). To clarify the hierarchy of Rho inhibition and Rac1 activation in TSC2-dependent stress fiber disassembly, we analyzed whether or not the activated form of Rac1, V12Rac1, could promote actin rearrangements in TSC2−/− cells; and then we performed cotransfection experiments of dominant-negative GST-tagged Rac1 (N17Rac1) with TSC2. Expression of V12Rac1 promoted stress fiber disassembly in the cell center and formation of cortical actin at the cell periphery similar to the effect of TSC2 (Fig. 8 A, top and middle, respectively). In contrast, in cells coexpressing N17Rac1 and TSC2, stress fibers were maintained (Fig. 8 A, bottom), suggesting that TSC2-induced stress fiber disassembly requires Rac1 activation. Furthermore, when N17Rac1 was comicroinjected with siRNA TSC1, stress fibers also remained (Fig. 8 B, bottom), indicating that TSC1-dependent stress fiber formation requires the negative regulation of Rac1 activity. Quantitative analysis of these experiments is presented in Fig. 8 C. Because stress fibers are maintained by active Rho, and inactive Rac1 coexpressed with TSC2 or siRNA TSC1 does not promote stress fiber disassembly, this data indicates that TSC2 requires activation of Rac1, followed by inhibition of Rho, in regulating actin remodeling.


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)

Dominant-negative Rac1 abrogates TSC2- and siRNA TSC1-induced stress fiber disassembly. (A) Serum-deprived cells were transfected with pEBG-V12Rac1 expressing activated Rac1 or the cells were cotransfected with pEGFP-TSC2 and pEBG-N17Rac1 plasmids, expressing GFP-TSC2 and GST-N17Rac1, respectively, and were stained with anti-GFP to detect GFP-TSC2 (green), anti-GST to detect GST, GST-V12Rac1, GST-N17Rac1 (blue), and phalloidin rhodamine to detect F-actin (red). Images are representative of three independent experiments. Bar, 20 μm. (B) Cells, microinjected with GST-N17Rac1, comicroinjected with siRNA TSC1 and GST to identify microinjected cells, or comicroinjected with siRNA TSC1 and N17Rac1, were stained with anti-GST antibody to detect GST and GST-N1Rac1 (green) and phalloidin rhodamine to detect F-actin (red). Images are representative of three independent experiments. (C) Quantitative analysis of F-actin staining. Data represent the percentage of cells with stress fibers per total number of cells transfected with plasmids expressing GST, GST-V12Rac1, GST-N17Rac1, GFP-TSC2, or coexpressed GFP-TSC2 and GST-N17Rac1, siRNA TSC1 and GST-N17Rac1, or siRNA TSC1 and control GST taken as 100%. Data represent the mean ± SE from three independent experiments. *, P < 0.001 for GST-V12Rac1 versus GST, siRNA TSC1 + GST versus siRNA TSC1 + GST-N17Rac1, and GFP-TSC2 versus GFP-TSC2 + GST-N17Rac1 by ANOVA (Bonferroni-Dunn test).
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fig8: Dominant-negative Rac1 abrogates TSC2- and siRNA TSC1-induced stress fiber disassembly. (A) Serum-deprived cells were transfected with pEBG-V12Rac1 expressing activated Rac1 or the cells were cotransfected with pEGFP-TSC2 and pEBG-N17Rac1 plasmids, expressing GFP-TSC2 and GST-N17Rac1, respectively, and were stained with anti-GFP to detect GFP-TSC2 (green), anti-GST to detect GST, GST-V12Rac1, GST-N17Rac1 (blue), and phalloidin rhodamine to detect F-actin (red). Images are representative of three independent experiments. Bar, 20 μm. (B) Cells, microinjected with GST-N17Rac1, comicroinjected with siRNA TSC1 and GST to identify microinjected cells, or comicroinjected with siRNA TSC1 and N17Rac1, were stained with anti-GST antibody to detect GST and GST-N1Rac1 (green) and phalloidin rhodamine to detect F-actin (red). Images are representative of three independent experiments. (C) Quantitative analysis of F-actin staining. Data represent the percentage of cells with stress fibers per total number of cells transfected with plasmids expressing GST, GST-V12Rac1, GST-N17Rac1, GFP-TSC2, or coexpressed GFP-TSC2 and GST-N17Rac1, siRNA TSC1 and GST-N17Rac1, or siRNA TSC1 and control GST taken as 100%. Data represent the mean ± SE from three independent experiments. *, P < 0.001 for GST-V12Rac1 versus GST, siRNA TSC1 + GST versus siRNA TSC1 + GST-N17Rac1, and GFP-TSC2 versus GFP-TSC2 + GST-N17Rac1 by ANOVA (Bonferroni-Dunn test).
Mentions: Rho and Rac1 both regulate stress fiber formation and focal adhesion remodeling in a reciprocal manner, as such activation of Rac1 results in the inhibition of Rho and vice versa (Horwitz and Parsons, 1999). To clarify the hierarchy of Rho inhibition and Rac1 activation in TSC2-dependent stress fiber disassembly, we analyzed whether or not the activated form of Rac1, V12Rac1, could promote actin rearrangements in TSC2−/− cells; and then we performed cotransfection experiments of dominant-negative GST-tagged Rac1 (N17Rac1) with TSC2. Expression of V12Rac1 promoted stress fiber disassembly in the cell center and formation of cortical actin at the cell periphery similar to the effect of TSC2 (Fig. 8 A, top and middle, respectively). In contrast, in cells coexpressing N17Rac1 and TSC2, stress fibers were maintained (Fig. 8 A, bottom), suggesting that TSC2-induced stress fiber disassembly requires Rac1 activation. Furthermore, when N17Rac1 was comicroinjected with siRNA TSC1, stress fibers also remained (Fig. 8 B, bottom), indicating that TSC1-dependent stress fiber formation requires the negative regulation of Rac1 activity. Quantitative analysis of these experiments is presented in Fig. 8 C. Because stress fibers are maintained by active Rho, and inactive Rac1 coexpressed with TSC2 or siRNA TSC1 does not promote stress fiber disassembly, this data indicates that TSC2 requires activation of Rac1, followed by inhibition of Rho, in regulating actin remodeling.

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