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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, TSC2-HBD, and siRNA TSC1 activate Rac1. (A) TSC2−/− cells were stimulated with 10 ng/ml of PDGF or diluent for 10 min or transfected with plasmids expressing GFP-tagged TSC2 or TSC2-HBD, or GFP as a control, or microinjected with siRNA TSC1 or control siRNA, and then subjected to the Rac1 activity assay. (top) Immunoblot analysis with anti-Rac1 antibody to detect Rac1 in pull-down assay with PAK-1 PBD agarose (top images) and in whole cell lysates (bottom images). Images are representative of three separate experiments. (bottom) Quantitative analysis of Rac1 activity assays using Gel-Pro Analyzer Software. Rac1 activity in cells transfected with control pEGFP plasmid was taken as a onefold. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP; ***, P < 0.001 for siRNA TSC1 versus control siRNA. Data represent the mean ± SE from three independent experiments. (B) LAMD cells were transfected with GFP-TSC2, GFP-TSC2-HBD, or GFP as a control, and then Rac1 activity assay was performed. (top) Immunoblot analysis with anti-Rac1 antibody to detect Rac1 in pull-down assay with PAK-1 PBD agarose (top images) and in whole cell lysates (bottom images). Images are representative of two separate experiments. (bottom) Quantitative analysis of Rac1 activity. Data represent the mean ± SE from two independent experiments. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP by ANOVA (Bonferroni-Dunn test). White lines indicate that intervening lanes have been spliced out.
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fig6: TSC2, TSC2-HBD, and siRNA TSC1 activate Rac1. (A) TSC2−/− cells were stimulated with 10 ng/ml of PDGF or diluent for 10 min or transfected with plasmids expressing GFP-tagged TSC2 or TSC2-HBD, or GFP as a control, or microinjected with siRNA TSC1 or control siRNA, and then subjected to the Rac1 activity assay. (top) Immunoblot analysis with anti-Rac1 antibody to detect Rac1 in pull-down assay with PAK-1 PBD agarose (top images) and in whole cell lysates (bottom images). Images are representative of three separate experiments. (bottom) Quantitative analysis of Rac1 activity assays using Gel-Pro Analyzer Software. Rac1 activity in cells transfected with control pEGFP plasmid was taken as a onefold. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP; ***, P < 0.001 for siRNA TSC1 versus control siRNA. Data represent the mean ± SE from three independent experiments. (B) LAMD cells were transfected with GFP-TSC2, GFP-TSC2-HBD, or GFP as a control, and then Rac1 activity assay was performed. (top) Immunoblot analysis with anti-Rac1 antibody to detect Rac1 in pull-down assay with PAK-1 PBD agarose (top images) and in whole cell lysates (bottom images). Images are representative of two separate experiments. (bottom) Quantitative analysis of Rac1 activity. Data represent the mean ± SE from two independent experiments. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP by ANOVA (Bonferroni-Dunn test). White lines indicate that intervening lanes have been spliced out.

Mentions: To clarify the function of Rac1 in TSC2-induced stress fiber disassembly and focal adhesion remodeling, we next examined Rac1 activation in TSC2−/− cells. Stimulation of TSC2−/− cells with PDGF had little effect on the basal Rac1 activity (Fig. 6 A), whereas PDGF-stimulated Rac1 activity in 3T3 cells (not depicted), which were used as a model cell line. By expressing TSC2 in TSC2−/− cells, we found that TSC2 alone was sufficient to markedly increase Rac1 activity compared with cells transfected with control GFP (Fig. 6 A). Similarly, Rac1 activity was increased in LAMD cells transfected with TSC2, indicating that TSC2 may elicit activation of Rac1 (Fig. 6 B). Importantly, down-regulation of TSC1 with TSC1 siRNA or expression of TSC2-HBD was also sufficient for stimulating Rac1 activity (Fig. 6 A), suggesting that TCS2 interaction with TSC1 may be involved in the regulation of Rac1 activity. We conclude that TSC2 acts upstream of Rac1 in pathways regulating actin and focal adhesion 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)

TSC2, TSC2-HBD, and siRNA TSC1 activate Rac1. (A) TSC2−/− cells were stimulated with 10 ng/ml of PDGF or diluent for 10 min or transfected with plasmids expressing GFP-tagged TSC2 or TSC2-HBD, or GFP as a control, or microinjected with siRNA TSC1 or control siRNA, and then subjected to the Rac1 activity assay. (top) Immunoblot analysis with anti-Rac1 antibody to detect Rac1 in pull-down assay with PAK-1 PBD agarose (top images) and in whole cell lysates (bottom images). Images are representative of three separate experiments. (bottom) Quantitative analysis of Rac1 activity assays using Gel-Pro Analyzer Software. Rac1 activity in cells transfected with control pEGFP plasmid was taken as a onefold. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP; ***, P < 0.001 for siRNA TSC1 versus control siRNA. Data represent the mean ± SE from three independent experiments. (B) LAMD cells were transfected with GFP-TSC2, GFP-TSC2-HBD, or GFP as a control, and then Rac1 activity assay was performed. (top) Immunoblot analysis with anti-Rac1 antibody to detect Rac1 in pull-down assay with PAK-1 PBD agarose (top images) and in whole cell lysates (bottom images). Images are representative of two separate experiments. (bottom) Quantitative analysis of Rac1 activity. Data represent the mean ± SE from two independent experiments. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP by ANOVA (Bonferroni-Dunn test). White lines indicate that intervening lanes have been spliced out.
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fig6: TSC2, TSC2-HBD, and siRNA TSC1 activate Rac1. (A) TSC2−/− cells were stimulated with 10 ng/ml of PDGF or diluent for 10 min or transfected with plasmids expressing GFP-tagged TSC2 or TSC2-HBD, or GFP as a control, or microinjected with siRNA TSC1 or control siRNA, and then subjected to the Rac1 activity assay. (top) Immunoblot analysis with anti-Rac1 antibody to detect Rac1 in pull-down assay with PAK-1 PBD agarose (top images) and in whole cell lysates (bottom images). Images are representative of three separate experiments. (bottom) Quantitative analysis of Rac1 activity assays using Gel-Pro Analyzer Software. Rac1 activity in cells transfected with control pEGFP plasmid was taken as a onefold. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP; ***, P < 0.001 for siRNA TSC1 versus control siRNA. Data represent the mean ± SE from three independent experiments. (B) LAMD cells were transfected with GFP-TSC2, GFP-TSC2-HBD, or GFP as a control, and then Rac1 activity assay was performed. (top) Immunoblot analysis with anti-Rac1 antibody to detect Rac1 in pull-down assay with PAK-1 PBD agarose (top images) and in whole cell lysates (bottom images). Images are representative of two separate experiments. (bottom) Quantitative analysis of Rac1 activity. Data represent the mean ± SE from two independent experiments. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-HBD versus GFP by ANOVA (Bonferroni-Dunn test). White lines indicate that intervening lanes have been spliced out.
Mentions: To clarify the function of Rac1 in TSC2-induced stress fiber disassembly and focal adhesion remodeling, we next examined Rac1 activation in TSC2−/− cells. Stimulation of TSC2−/− cells with PDGF had little effect on the basal Rac1 activity (Fig. 6 A), whereas PDGF-stimulated Rac1 activity in 3T3 cells (not depicted), which were used as a model cell line. By expressing TSC2 in TSC2−/− cells, we found that TSC2 alone was sufficient to markedly increase Rac1 activity compared with cells transfected with control GFP (Fig. 6 A). Similarly, Rac1 activity was increased in LAMD cells transfected with TSC2, indicating that TSC2 may elicit activation of Rac1 (Fig. 6 B). Importantly, down-regulation of TSC1 with TSC1 siRNA or expression of TSC2-HBD was also sufficient for stimulating Rac1 activity (Fig. 6 A), suggesting that TCS2 interaction with TSC1 may be involved in the regulation of Rac1 activity. We conclude that TSC2 acts upstream of Rac1 in pathways regulating actin and focal adhesion 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