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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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Expression of TSC2, but not TSC2-HBD, inhibits ribosomal protein S6 phosphorylation and DNA synthesis in TSC2−/− cells. TSC2−/− cells were transfected with plasmids expressing GFP-tagged TSC2, TSC2-ΔHBD, or TSC2-HBD, or comicroinjected with pEGFP-TSC2-HBD and pEGFP-TSC2-ΔHBD plasmids, serum-deprived for 24 h, and immunostained with anti–phospho-S6 (red) antibody and anti-GFP antiserum (green); or BrdU incorporation analysis was performed. (A) Representative images of three separate experiments were taken. Arrows indicate transfected cells. (B) Quantitative analysis of P-S6 immunostaining. Data represent the percentage of P-S6–positive transfected cells per total number of transfected cells. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-ΔHBD versus GFP; ***, P < 0.001 for GFP-TSC2-HBD + GFP-TSC2-ΔHBD versus GFP. (C) BrdU incorporation analysis of transfected cells. Mitotic index represents the percentage of BrdU-positive transfected cells compared with the total number of transfected cells. Data are mean ± SE of three separate experiments. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-ΔHBD versus GFP; ***, P < 0.001 for GFP-TSC2-HBD + GFP-TSC2-ΔHBD versus GFP.
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fig9: Expression of TSC2, but not TSC2-HBD, inhibits ribosomal protein S6 phosphorylation and DNA synthesis in TSC2−/− cells. TSC2−/− cells were transfected with plasmids expressing GFP-tagged TSC2, TSC2-ΔHBD, or TSC2-HBD, or comicroinjected with pEGFP-TSC2-HBD and pEGFP-TSC2-ΔHBD plasmids, serum-deprived for 24 h, and immunostained with anti–phospho-S6 (red) antibody and anti-GFP antiserum (green); or BrdU incorporation analysis was performed. (A) Representative images of three separate experiments were taken. Arrows indicate transfected cells. (B) Quantitative analysis of P-S6 immunostaining. Data represent the percentage of P-S6–positive transfected cells per total number of transfected cells. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-ΔHBD versus GFP; ***, P < 0.001 for GFP-TSC2-HBD + GFP-TSC2-ΔHBD versus GFP. (C) BrdU incorporation analysis of transfected cells. Mitotic index represents the percentage of BrdU-positive transfected cells compared with the total number of transfected cells. Data are mean ± SE of three separate experiments. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-ΔHBD versus GFP; ***, P < 0.001 for GFP-TSC2-HBD + GFP-TSC2-ΔHBD versus GFP.

Mentions: Because TSC2 negatively regulates the activity of ribosomal protein S6 by inhibition of its phosphorylation (Goncharova et al., 2002), we investigated whether TSC2-HBD or TSC2-ΔHBD affects ribosomal protein S6 hyperphosphorylation in TSC2−/− cells. Consistent with previously published results (Goncharova et al., 2002), full-length TSC2 inhibited S6 phosphorylation by 56.2 ± 3.9% compared with GFP-transfected cells (Fig. 9, A and B). TSC2-ΔHBD also significantly, however to a lesser extent than full-length TSC2, inhibited S6 phosphorylation by 22.7 ± 2.8%. Importantly, comicroinjection of TSC2-HBD and TSC2-ΔHBD inhibited phospho-S6 by 47.4 ± 3.3%, which was comparable to the inhibitory effect of full-length TSC2 (Fig. 9 B). In contrast, expression of TSC2-HBD had little effect on S6 phosphorylation, suggesting that this domain of TSC2 is not required for the regulation of ribosomal protein S6 activation (Fig. 9, A and B).


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)

Expression of TSC2, but not TSC2-HBD, inhibits ribosomal protein S6 phosphorylation and DNA synthesis in TSC2−/− cells. TSC2−/− cells were transfected with plasmids expressing GFP-tagged TSC2, TSC2-ΔHBD, or TSC2-HBD, or comicroinjected with pEGFP-TSC2-HBD and pEGFP-TSC2-ΔHBD plasmids, serum-deprived for 24 h, and immunostained with anti–phospho-S6 (red) antibody and anti-GFP antiserum (green); or BrdU incorporation analysis was performed. (A) Representative images of three separate experiments were taken. Arrows indicate transfected cells. (B) Quantitative analysis of P-S6 immunostaining. Data represent the percentage of P-S6–positive transfected cells per total number of transfected cells. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-ΔHBD versus GFP; ***, P < 0.001 for GFP-TSC2-HBD + GFP-TSC2-ΔHBD versus GFP. (C) BrdU incorporation analysis of transfected cells. Mitotic index represents the percentage of BrdU-positive transfected cells compared with the total number of transfected cells. Data are mean ± SE of three separate experiments. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-ΔHBD versus GFP; ***, P < 0.001 for GFP-TSC2-HBD + GFP-TSC2-ΔHBD versus GFP.
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fig9: Expression of TSC2, but not TSC2-HBD, inhibits ribosomal protein S6 phosphorylation and DNA synthesis in TSC2−/− cells. TSC2−/− cells were transfected with plasmids expressing GFP-tagged TSC2, TSC2-ΔHBD, or TSC2-HBD, or comicroinjected with pEGFP-TSC2-HBD and pEGFP-TSC2-ΔHBD plasmids, serum-deprived for 24 h, and immunostained with anti–phospho-S6 (red) antibody and anti-GFP antiserum (green); or BrdU incorporation analysis was performed. (A) Representative images of three separate experiments were taken. Arrows indicate transfected cells. (B) Quantitative analysis of P-S6 immunostaining. Data represent the percentage of P-S6–positive transfected cells per total number of transfected cells. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-ΔHBD versus GFP; ***, P < 0.001 for GFP-TSC2-HBD + GFP-TSC2-ΔHBD versus GFP. (C) BrdU incorporation analysis of transfected cells. Mitotic index represents the percentage of BrdU-positive transfected cells compared with the total number of transfected cells. Data are mean ± SE of three separate experiments. *, P < 0.001 for GFP-TSC2 versus GFP; **, P < 0.001 for GFP-TSC2-ΔHBD versus GFP; ***, P < 0.001 for GFP-TSC2-HBD + GFP-TSC2-ΔHBD versus GFP.
Mentions: Because TSC2 negatively regulates the activity of ribosomal protein S6 by inhibition of its phosphorylation (Goncharova et al., 2002), we investigated whether TSC2-HBD or TSC2-ΔHBD affects ribosomal protein S6 hyperphosphorylation in TSC2−/− cells. Consistent with previously published results (Goncharova et al., 2002), full-length TSC2 inhibited S6 phosphorylation by 56.2 ± 3.9% compared with GFP-transfected cells (Fig. 9, A and B). TSC2-ΔHBD also significantly, however to a lesser extent than full-length TSC2, inhibited S6 phosphorylation by 22.7 ± 2.8%. Importantly, comicroinjection of TSC2-HBD and TSC2-ΔHBD inhibited phospho-S6 by 47.4 ± 3.3%, which was comparable to the inhibitory effect of full-length TSC2 (Fig. 9 B). In contrast, expression of TSC2-HBD had little effect on S6 phosphorylation, suggesting that this domain of TSC2 is not required for the regulation of ribosomal protein S6 activation (Fig. 9, A and B).

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