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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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Schematic representation and expression of GFP-tagged TSC2 constructs in the TSC2−/− cells. (A) TSC2 includes leucine zipper (LZ), two coiled-coiled (CC), two transcription-activating domains (TAD), GAP homology (GAP), and calmodulin (CaM)-binding domain. (B) To identify expression of TSC2 mutants, cells were transfected with pEGFP vectors expressing GFP-tagged TSC2, N-TSC2, C-TSC2, TSC2-HBD, TSC2-ΔHBD, or control GFP. After 24 h of transient transfection, cells were lysed, and whole cell lysates were subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-GFP antiserum. TSC2-positive rat TRKE cells, used as a positive control, were lysed and subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-TSC2 antibody.
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fig3: Schematic representation and expression of GFP-tagged TSC2 constructs in the TSC2−/− cells. (A) TSC2 includes leucine zipper (LZ), two coiled-coiled (CC), two transcription-activating domains (TAD), GAP homology (GAP), and calmodulin (CaM)-binding domain. (B) To identify expression of TSC2 mutants, cells were transfected with pEGFP vectors expressing GFP-tagged TSC2, N-TSC2, C-TSC2, TSC2-HBD, TSC2-ΔHBD, or control GFP. After 24 h of transient transfection, cells were lysed, and whole cell lysates were subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-GFP antiserum. TSC2-positive rat TRKE cells, used as a positive control, were lysed and subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-TSC2 antibody.

Mentions: To examine whether TSC2 is required for actin remodeling, and to identify which domain of TSC2 is important for stress fiber disassembly, we tested a panel of GFP-tagged deletion constructs of TSC2 (Fig. 3). The reexpression of full-length TSC2 in TSC2−/− cells markedly promoted stress fiber disassembly and the formation of cortical actin compared with cells transfected with control GFP (Fig. 4 A). Interestingly, the expression of TSC2-ΔHBD in TSC2−/− cells containing the Rheb GAP domain (Fig. 4 A) or the expression of C-TSC2 (not depicted) had little effect on stress fiber disassembly. In contrast, expression of N-TSC2 (not depicted) or TSC2-HBD was sufficient to induce stress fiber disassembly (Fig. 4 A).


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)

Schematic representation and expression of GFP-tagged TSC2 constructs in the TSC2−/− cells. (A) TSC2 includes leucine zipper (LZ), two coiled-coiled (CC), two transcription-activating domains (TAD), GAP homology (GAP), and calmodulin (CaM)-binding domain. (B) To identify expression of TSC2 mutants, cells were transfected with pEGFP vectors expressing GFP-tagged TSC2, N-TSC2, C-TSC2, TSC2-HBD, TSC2-ΔHBD, or control GFP. After 24 h of transient transfection, cells were lysed, and whole cell lysates were subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-GFP antiserum. TSC2-positive rat TRKE cells, used as a positive control, were lysed and subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-TSC2 antibody.
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Related In: Results  -  Collection

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fig3: Schematic representation and expression of GFP-tagged TSC2 constructs in the TSC2−/− cells. (A) TSC2 includes leucine zipper (LZ), two coiled-coiled (CC), two transcription-activating domains (TAD), GAP homology (GAP), and calmodulin (CaM)-binding domain. (B) To identify expression of TSC2 mutants, cells were transfected with pEGFP vectors expressing GFP-tagged TSC2, N-TSC2, C-TSC2, TSC2-HBD, TSC2-ΔHBD, or control GFP. After 24 h of transient transfection, cells were lysed, and whole cell lysates were subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-GFP antiserum. TSC2-positive rat TRKE cells, used as a positive control, were lysed and subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-TSC2 antibody.
Mentions: To examine whether TSC2 is required for actin remodeling, and to identify which domain of TSC2 is important for stress fiber disassembly, we tested a panel of GFP-tagged deletion constructs of TSC2 (Fig. 3). The reexpression of full-length TSC2 in TSC2−/− cells markedly promoted stress fiber disassembly and the formation of cortical actin compared with cells transfected with control GFP (Fig. 4 A). Interestingly, the expression of TSC2-ΔHBD in TSC2−/− cells containing the Rheb GAP domain (Fig. 4 A) or the expression of C-TSC2 (not depicted) had little effect on stress fiber disassembly. In contrast, expression of N-TSC2 (not depicted) or TSC2-HBD was sufficient to induce stress fiber disassembly (Fig. 4 A).

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