Limits...
The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions.

Garvalov BK, Higgins TE, Sutherland JD, Zettl M, Scaplehorn N, Köcher T, Piddini E, Griffiths G, Way M - J. Cell Biol. (2003)

Bottom Line: The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts.These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule.Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.

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Related in: MedlinePlus

Tes is recruited to focal adhesions. (A) Schematic representation of Tes and the GFP-tagged expression clones including the location of point mutations inactivating each LIM domain used in this study. The in vivo localization of the GFP-tagged proteins and their interacting partners based on Western blot analysis of pull-down assays are indicated. Only the interactions between LIM1-zyxin and the two halves of Tes have been shown to be direct. N.D, not determined. (B) Immunofluorescence analysis of HeLa cells reveals that GFP-Tes or endogenous Tes colocalize with paxillin at focal adhesions. Bar, 20 μm. (C) Western blot analysis with Tes antibody detects a single band of the correct predicted size in HeLa cell extracts. Molecular mass markers are indicated in kDa.
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fig1: Tes is recruited to focal adhesions. (A) Schematic representation of Tes and the GFP-tagged expression clones including the location of point mutations inactivating each LIM domain used in this study. The in vivo localization of the GFP-tagged proteins and their interacting partners based on Western blot analysis of pull-down assays are indicated. Only the interactions between LIM1-zyxin and the two halves of Tes have been shown to be direct. N.D, not determined. (B) Immunofluorescence analysis of HeLa cells reveals that GFP-Tes or endogenous Tes colocalize with paxillin at focal adhesions. Bar, 20 μm. (C) Western blot analysis with Tes antibody detects a single band of the correct predicted size in HeLa cell extracts. Molecular mass markers are indicated in kDa.

Mentions: Given its sequence homology to a number of cytoskeletal proteins, as well as its possible role as a tumor suppressor, we set out to investigate whether Tes is a cytoskeleton-associated protein. We found that GFP-tagged human Tes was recruited to focal adhesions in HeLa cells (Fig. 1, A and B) . In contrast to other focal adhesion proteins such as α-actinin, Mena, vasodilator-stimulated phosphoprotein (VASP),* and zyxin, GFP-Tes was not observed along stress fibers (Fig. S1, available at www.jcb.org/cgi/content/full/jcb.200211015/DC1). Immunofluorescence analysis with an anti-Tes antibody confirmed that the endogenous protein is also found at focal adhesions, whereas Western blot analysis identified a single band of the correct predicted size (Fig. 1, B and C).


The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions.

Garvalov BK, Higgins TE, Sutherland JD, Zettl M, Scaplehorn N, Köcher T, Piddini E, Griffiths G, Way M - J. Cell Biol. (2003)

Tes is recruited to focal adhesions. (A) Schematic representation of Tes and the GFP-tagged expression clones including the location of point mutations inactivating each LIM domain used in this study. The in vivo localization of the GFP-tagged proteins and their interacting partners based on Western blot analysis of pull-down assays are indicated. Only the interactions between LIM1-zyxin and the two halves of Tes have been shown to be direct. N.D, not determined. (B) Immunofluorescence analysis of HeLa cells reveals that GFP-Tes or endogenous Tes colocalize with paxillin at focal adhesions. Bar, 20 μm. (C) Western blot analysis with Tes antibody detects a single band of the correct predicted size in HeLa cell extracts. Molecular mass markers are indicated in kDa.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172870&req=5

fig1: Tes is recruited to focal adhesions. (A) Schematic representation of Tes and the GFP-tagged expression clones including the location of point mutations inactivating each LIM domain used in this study. The in vivo localization of the GFP-tagged proteins and their interacting partners based on Western blot analysis of pull-down assays are indicated. Only the interactions between LIM1-zyxin and the two halves of Tes have been shown to be direct. N.D, not determined. (B) Immunofluorescence analysis of HeLa cells reveals that GFP-Tes or endogenous Tes colocalize with paxillin at focal adhesions. Bar, 20 μm. (C) Western blot analysis with Tes antibody detects a single band of the correct predicted size in HeLa cell extracts. Molecular mass markers are indicated in kDa.
Mentions: Given its sequence homology to a number of cytoskeletal proteins, as well as its possible role as a tumor suppressor, we set out to investigate whether Tes is a cytoskeleton-associated protein. We found that GFP-tagged human Tes was recruited to focal adhesions in HeLa cells (Fig. 1, A and B) . In contrast to other focal adhesion proteins such as α-actinin, Mena, vasodilator-stimulated phosphoprotein (VASP),* and zyxin, GFP-Tes was not observed along stress fibers (Fig. S1, available at www.jcb.org/cgi/content/full/jcb.200211015/DC1). Immunofluorescence analysis with an anti-Tes antibody confirmed that the endogenous protein is also found at focal adhesions, whereas Western blot analysis identified a single band of the correct predicted size (Fig. 1, B and C).

Bottom Line: The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts.These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule.Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.

Show MeSH
Related in: MedlinePlus