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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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Tes negatively regulates growth of T47D cells. (A) Phase–contrast images of T47D cells, wild-type or expressing GFP or GFP-Tes, taken from movie sequences at the times indicated. Video available at www.jcb.org/cgi/content/full/jcb.200211015/DC1. (B) Graph showing average fold increase in cell number over times indicated. Error bars represent standard deviation of three independent experiments. (C) T47D colonies in soft-agar stained with nitroblue tetrazolium after 14-d growth. (D) Graph showing number of colonies (gray) formed by T47D cells (wild-type or expressing GFP or GFP-Tes) and the number of colonies with diameter greater than 100 μm (black). Data represent mean ± standard deviation between three independent experiments. Expression of GFP-Tes in T47D cells reduces the number and size of colonies formed when compared with cells expressing GFP.
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fig5: Tes negatively regulates growth of T47D cells. (A) Phase–contrast images of T47D cells, wild-type or expressing GFP or GFP-Tes, taken from movie sequences at the times indicated. Video available at www.jcb.org/cgi/content/full/jcb.200211015/DC1. (B) Graph showing average fold increase in cell number over times indicated. Error bars represent standard deviation of three independent experiments. (C) T47D colonies in soft-agar stained with nitroblue tetrazolium after 14-d growth. (D) Graph showing number of colonies (gray) formed by T47D cells (wild-type or expressing GFP or GFP-Tes) and the number of colonies with diameter greater than 100 μm (black). Data represent mean ± standard deviation between three independent experiments. Expression of GFP-Tes in T47D cells reduces the number and size of colonies formed when compared with cells expressing GFP.

Mentions: What is the cellular role of Tes? Previous observations have demonstrated that forced expression of Tes inhibited stable colony formation during antibiotic selection (Tobias et al., 2001). These experiments, however, do not rule out the possibility that reduced colony formation was due to cell death rather than suppression of cell growth. Notwithstanding this possibility, the expression profiles of Tes are consistent with a possible role as a tumor suppressor (Tatarelli et al., 2000; Tobias et al., 2001). To investigate whether Tes is able to suppress cell growth, we performed long-term video analysis of T47D cells and those stably expressing GFP or GFP-Tes (Fig. 5 A). T47D cells are human invasive ductal breast carcinoma cells, which do not express Tes based on Northern blot and RT-PCR analysis (Tatarelli et al., 2000), as well as immunofluorescence and Western blot analysis with anti-Tes antibody (not shown). GFP-Tes is detected at focal adhesions when stably expressed in T47D cells (not shown). We found that although expression of Tes had no appreciable effect on the motility of T47D cells, it severely reduced their overall growth rate (Fig. 5, A and B; and Videos 1–3, available at www.jcb.org/cgi/content/full/jcb.200211015/DC1). We also found that expression of GFP-Tes had an inhibitory effect on anchorage-independent growth of T47D cells in soft agarose (Fig. 5, C and D). Fewer colonies were formed by GFP-Tes expressing cells compared with those with GFP. Furthermore, those colonies that did form were significantly smaller (Fig. 5, C and D). The role of focal adhesions in mediating integrin-dependent modulation of cell growth through the action of MAP kinases is well established (Schwartz and Assoian, 2001). Given its numerous interactions, it is likely that Tes is also involved in this complex signaling cascade. Our future studies will aim to understand the regulation of the conformational state of Tes and how this influences cell growth.


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 negatively regulates growth of T47D cells. (A) Phase–contrast images of T47D cells, wild-type or expressing GFP or GFP-Tes, taken from movie sequences at the times indicated. Video available at www.jcb.org/cgi/content/full/jcb.200211015/DC1. (B) Graph showing average fold increase in cell number over times indicated. Error bars represent standard deviation of three independent experiments. (C) T47D colonies in soft-agar stained with nitroblue tetrazolium after 14-d growth. (D) Graph showing number of colonies (gray) formed by T47D cells (wild-type or expressing GFP or GFP-Tes) and the number of colonies with diameter greater than 100 μm (black). Data represent mean ± standard deviation between three independent experiments. Expression of GFP-Tes in T47D cells reduces the number and size of colonies formed when compared with cells expressing GFP.
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Related In: Results  -  Collection

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fig5: Tes negatively regulates growth of T47D cells. (A) Phase–contrast images of T47D cells, wild-type or expressing GFP or GFP-Tes, taken from movie sequences at the times indicated. Video available at www.jcb.org/cgi/content/full/jcb.200211015/DC1. (B) Graph showing average fold increase in cell number over times indicated. Error bars represent standard deviation of three independent experiments. (C) T47D colonies in soft-agar stained with nitroblue tetrazolium after 14-d growth. (D) Graph showing number of colonies (gray) formed by T47D cells (wild-type or expressing GFP or GFP-Tes) and the number of colonies with diameter greater than 100 μm (black). Data represent mean ± standard deviation between three independent experiments. Expression of GFP-Tes in T47D cells reduces the number and size of colonies formed when compared with cells expressing GFP.
Mentions: What is the cellular role of Tes? Previous observations have demonstrated that forced expression of Tes inhibited stable colony formation during antibiotic selection (Tobias et al., 2001). These experiments, however, do not rule out the possibility that reduced colony formation was due to cell death rather than suppression of cell growth. Notwithstanding this possibility, the expression profiles of Tes are consistent with a possible role as a tumor suppressor (Tatarelli et al., 2000; Tobias et al., 2001). To investigate whether Tes is able to suppress cell growth, we performed long-term video analysis of T47D cells and those stably expressing GFP or GFP-Tes (Fig. 5 A). T47D cells are human invasive ductal breast carcinoma cells, which do not express Tes based on Northern blot and RT-PCR analysis (Tatarelli et al., 2000), as well as immunofluorescence and Western blot analysis with anti-Tes antibody (not shown). GFP-Tes is detected at focal adhesions when stably expressed in T47D cells (not shown). We found that although expression of Tes had no appreciable effect on the motility of T47D cells, it severely reduced their overall growth rate (Fig. 5, A and B; and Videos 1–3, available at www.jcb.org/cgi/content/full/jcb.200211015/DC1). We also found that expression of GFP-Tes had an inhibitory effect on anchorage-independent growth of T47D cells in soft agarose (Fig. 5, C and D). Fewer colonies were formed by GFP-Tes expressing cells compared with those with GFP. Furthermore, those colonies that did form were significantly smaller (Fig. 5, C and D). The role of focal adhesions in mediating integrin-dependent modulation of cell growth through the action of MAP kinases is well established (Schwartz and Assoian, 2001). Given its numerous interactions, it is likely that Tes is also involved in this complex signaling cascade. Our future studies will aim to understand the regulation of the conformational state of Tes and how this influences cell growth.

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