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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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PDGF has little effect on stress fiber disassembly in TSC2−/− cells. (A) Rhodamine phalloidin staining of F-actin of serum-deprived TSC2−/− and 3T3 cells, which were either stimulated with 10 ng/ml of PDGF (+) or diluent (−) for 10 min. PDGFR activation in TSC2−/− cells: serum-deprived cells, transfected with either GFP or GFP-TSC2, were stimulated with PDGF. Equalized in protein content whole cell lysates were subjected to immunoprecipitation with either anti-PDGFRβ (B) or PDGFRα (C) antibodies; and then immunoblot analysis was performed with either anti-PDGFRβ, PDGFRα, or anti-phosphotyrosine (PY) antibodies.
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fig2: PDGF has little effect on stress fiber disassembly in TSC2−/− cells. (A) Rhodamine phalloidin staining of F-actin of serum-deprived TSC2−/− and 3T3 cells, which were either stimulated with 10 ng/ml of PDGF (+) or diluent (−) for 10 min. PDGFR activation in TSC2−/− cells: serum-deprived cells, transfected with either GFP or GFP-TSC2, were stimulated with PDGF. Equalized in protein content whole cell lysates were subjected to immunoprecipitation with either anti-PDGFRβ (B) or PDGFRα (C) antibodies; and then immunoblot analysis was performed with either anti-PDGFRβ, PDGFRα, or anti-phosphotyrosine (PY) antibodies.

Mentions: Because cell motility is regulated by actin remodeling, we examined actin rearrangements in TSC2−/− cells. F-actin staining revealed abundant stress fiber formation (Fig. 2 A, top). Surprisingly, PDGF, which promotes stress fiber disassembly and lamellipodia formation in most cell types (Zigmond, 1996) and promoted stress fiber disassembly in 3T3 cells (Fig. 2 A, bottom), had little effect on actin rearrangements and lamellipodia formation in TSC2−/− cells (Fig. 2 A, top). This finding was more surprising because we previously demonstrated that PDGF stimulates migration of TSC2−/− ELT3 cells (smooth muscle cells derived from Eker rat uterine leiomyomas; Irani et al., 2002). Because Zhang et al. (2003) demonstrated that PDGF receptor (PDGFR) α and PDGFRβ levels were reduced in TSC2−/−TP53−/− murine embryonic fibroblasts, we examined their expression in TSC2−/− rat ELT3 cells. Immunoblot analysis revealed that PDGFRα and PDGFRβ are expressed in these cells, and TSC2 expression had little effect on both receptor levels (Fig. 2, B and C). Stimulation of cells with PDGF-BB induced activation of PDGFRβ, but has little effect on PDGFRα (Fig. 2, B and C, respectively). These data suggest that PDGF-induced signaling is not defective in rat TSC2−/− cells at the receptor levels.


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)

PDGF has little effect on stress fiber disassembly in TSC2−/− cells. (A) Rhodamine phalloidin staining of F-actin of serum-deprived TSC2−/− and 3T3 cells, which were either stimulated with 10 ng/ml of PDGF (+) or diluent (−) for 10 min. PDGFR activation in TSC2−/− cells: serum-deprived cells, transfected with either GFP or GFP-TSC2, were stimulated with PDGF. Equalized in protein content whole cell lysates were subjected to immunoprecipitation with either anti-PDGFRβ (B) or PDGFRα (C) antibodies; and then immunoblot analysis was performed with either anti-PDGFRβ, PDGFRα, or anti-phosphotyrosine (PY) antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172598&req=5

fig2: PDGF has little effect on stress fiber disassembly in TSC2−/− cells. (A) Rhodamine phalloidin staining of F-actin of serum-deprived TSC2−/− and 3T3 cells, which were either stimulated with 10 ng/ml of PDGF (+) or diluent (−) for 10 min. PDGFR activation in TSC2−/− cells: serum-deprived cells, transfected with either GFP or GFP-TSC2, were stimulated with PDGF. Equalized in protein content whole cell lysates were subjected to immunoprecipitation with either anti-PDGFRβ (B) or PDGFRα (C) antibodies; and then immunoblot analysis was performed with either anti-PDGFRβ, PDGFRα, or anti-phosphotyrosine (PY) antibodies.
Mentions: Because cell motility is regulated by actin remodeling, we examined actin rearrangements in TSC2−/− cells. F-actin staining revealed abundant stress fiber formation (Fig. 2 A, top). Surprisingly, PDGF, which promotes stress fiber disassembly and lamellipodia formation in most cell types (Zigmond, 1996) and promoted stress fiber disassembly in 3T3 cells (Fig. 2 A, bottom), had little effect on actin rearrangements and lamellipodia formation in TSC2−/− cells (Fig. 2 A, top). This finding was more surprising because we previously demonstrated that PDGF stimulates migration of TSC2−/− ELT3 cells (smooth muscle cells derived from Eker rat uterine leiomyomas; Irani et al., 2002). Because Zhang et al. (2003) demonstrated that PDGF receptor (PDGFR) α and PDGFRβ levels were reduced in TSC2−/−TP53−/− murine embryonic fibroblasts, we examined their expression in TSC2−/− rat ELT3 cells. Immunoblot analysis revealed that PDGFRα and PDGFRβ are expressed in these cells, and TSC2 expression had little effect on both receptor levels (Fig. 2, B and C). Stimulation of cells with PDGF-BB induced activation of PDGFRβ, but has little effect on PDGFRα (Fig. 2, B and C, respectively). These data suggest that PDGF-induced signaling is not defective in rat TSC2−/− cells at the receptor levels.

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