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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Recruitment of Tes and VASP to focal adhesions is dependent on zyxin. (A) Immunofluorescence analysis demonstrates that endogenous paxillin, zyxin, and Tes (red) are still recruited to focal adhesions in the absence of Ena/VASP proteins in MVD7 cells. The actin cytoskeleton is visualized with phalloidin (green). Bar, 20 μm. (B) Immunofluorescence analysis of mixed populations of HeLa cells transfected with zyxin and control siRNA oligos. The left column corresponds to the zyxin signal, whereas the right column shows the indicated protein. In cells lacking zyxin (white arrowheads), there is a corresponding absence of Tes, VASP, and Mena (not depicted) but not paxillin or vinculin at focal adhesions. Bar, 20 μm. (C) Schematic representation of the conformation changes in Tes and the proteins with which it associates. Double-headed arrows indicate associations based on pull-downs or immunoprecipitations, which may not represent direct interactions. In the cytoplasm, the molecule is in a “closed” conformation but can still associate with actin and VASP/Mena. Upon “activation” by an unknown mechanism, the molecule adopts a more “open” conformation and is recruited to and/or stabilized at focal adhesions. In this “open” conformation, Tes is able to associate with α-actinin, actin, paxillin, Mena, VASP, and bind directly to zyxin.
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fig4: Recruitment of Tes and VASP to focal adhesions is dependent on zyxin. (A) Immunofluorescence analysis demonstrates that endogenous paxillin, zyxin, and Tes (red) are still recruited to focal adhesions in the absence of Ena/VASP proteins in MVD7 cells. The actin cytoskeleton is visualized with phalloidin (green). Bar, 20 μm. (B) Immunofluorescence analysis of mixed populations of HeLa cells transfected with zyxin and control siRNA oligos. The left column corresponds to the zyxin signal, whereas the right column shows the indicated protein. In cells lacking zyxin (white arrowheads), there is a corresponding absence of Tes, VASP, and Mena (not depicted) but not paxillin or vinculin at focal adhesions. Bar, 20 μm. (C) Schematic representation of the conformation changes in Tes and the proteins with which it associates. Double-headed arrows indicate associations based on pull-downs or immunoprecipitations, which may not represent direct interactions. In the cytoplasm, the molecule is in a “closed” conformation but can still associate with actin and VASP/Mena. Upon “activation” by an unknown mechanism, the molecule adopts a more “open” conformation and is recruited to and/or stabilized at focal adhesions. In this “open” conformation, Tes is able to associate with α-actinin, actin, paxillin, Mena, VASP, and bind directly to zyxin.

Mentions: Our localization studies suggest that in the context of the full molecule, the LIM3 domain is required for recruitment of the protein to focal adhesions. This could imply that Ena/VASP proteins are involved in recruiting Tes to focal adhesions. However, immunofluorescence analysis of MVD7 cells deficient in Mena and VASP (Bear et al., 2000) revealed that Tes is still present at focal adhesions (Fig. 4 A). That Tes recruitment to focal adhesions is independent of Ena/VASP proteins is consistent with the observation that mutation of the LIM3 domain in the isolated COOH-terminal half of the molecule does not inhibit its recruitment to focal adhesions (Fig. 1 A). Furthermore, LIM3 alone is not recruited to focal adhesions (Figs. 1 A and 2). We suggest that disruption of the LIM3 domain abolishes recruitment of Tes to focal adhesions because it is involved in regulating the conformational state of the full-length protein and thus its ability to be recruited to focal adhesions. One can imagine that upon transition to an “open” conformational state, Tes is recruited and/or stabilized at focal adhesions through a direct interaction of its LIM1 domain with zyxin (Fig. 4 C). The recruitment of Tes to focal adhesions by zyxin would also be stabilized by the ability of the NH2-terminal domain to associate with α-actinin and paxillin (Fig. 3 A). Consistent with this hypothesis, disruption of LIM1 but not LIM2 or LIM3 in the isolated COOH-terminal half of the molecule results in the elimination of its recruitment to focal adhesions (Fig. 1 A).


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)

Recruitment of Tes and VASP to focal adhesions is dependent on zyxin. (A) Immunofluorescence analysis demonstrates that endogenous paxillin, zyxin, and Tes (red) are still recruited to focal adhesions in the absence of Ena/VASP proteins in MVD7 cells. The actin cytoskeleton is visualized with phalloidin (green). Bar, 20 μm. (B) Immunofluorescence analysis of mixed populations of HeLa cells transfected with zyxin and control siRNA oligos. The left column corresponds to the zyxin signal, whereas the right column shows the indicated protein. In cells lacking zyxin (white arrowheads), there is a corresponding absence of Tes, VASP, and Mena (not depicted) but not paxillin or vinculin at focal adhesions. Bar, 20 μm. (C) Schematic representation of the conformation changes in Tes and the proteins with which it associates. Double-headed arrows indicate associations based on pull-downs or immunoprecipitations, which may not represent direct interactions. In the cytoplasm, the molecule is in a “closed” conformation but can still associate with actin and VASP/Mena. Upon “activation” by an unknown mechanism, the molecule adopts a more “open” conformation and is recruited to and/or stabilized at focal adhesions. In this “open” conformation, Tes is able to associate with α-actinin, actin, paxillin, Mena, VASP, and bind directly to zyxin.
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Related In: Results  -  Collection

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fig4: Recruitment of Tes and VASP to focal adhesions is dependent on zyxin. (A) Immunofluorescence analysis demonstrates that endogenous paxillin, zyxin, and Tes (red) are still recruited to focal adhesions in the absence of Ena/VASP proteins in MVD7 cells. The actin cytoskeleton is visualized with phalloidin (green). Bar, 20 μm. (B) Immunofluorescence analysis of mixed populations of HeLa cells transfected with zyxin and control siRNA oligos. The left column corresponds to the zyxin signal, whereas the right column shows the indicated protein. In cells lacking zyxin (white arrowheads), there is a corresponding absence of Tes, VASP, and Mena (not depicted) but not paxillin or vinculin at focal adhesions. Bar, 20 μm. (C) Schematic representation of the conformation changes in Tes and the proteins with which it associates. Double-headed arrows indicate associations based on pull-downs or immunoprecipitations, which may not represent direct interactions. In the cytoplasm, the molecule is in a “closed” conformation but can still associate with actin and VASP/Mena. Upon “activation” by an unknown mechanism, the molecule adopts a more “open” conformation and is recruited to and/or stabilized at focal adhesions. In this “open” conformation, Tes is able to associate with α-actinin, actin, paxillin, Mena, VASP, and bind directly to zyxin.
Mentions: Our localization studies suggest that in the context of the full molecule, the LIM3 domain is required for recruitment of the protein to focal adhesions. This could imply that Ena/VASP proteins are involved in recruiting Tes to focal adhesions. However, immunofluorescence analysis of MVD7 cells deficient in Mena and VASP (Bear et al., 2000) revealed that Tes is still present at focal adhesions (Fig. 4 A). That Tes recruitment to focal adhesions is independent of Ena/VASP proteins is consistent with the observation that mutation of the LIM3 domain in the isolated COOH-terminal half of the molecule does not inhibit its recruitment to focal adhesions (Fig. 1 A). Furthermore, LIM3 alone is not recruited to focal adhesions (Figs. 1 A and 2). We suggest that disruption of the LIM3 domain abolishes recruitment of Tes to focal adhesions because it is involved in regulating the conformational state of the full-length protein and thus its ability to be recruited to focal adhesions. One can imagine that upon transition to an “open” conformational state, Tes is recruited and/or stabilized at focal adhesions through a direct interaction of its LIM1 domain with zyxin (Fig. 4 C). The recruitment of Tes to focal adhesions by zyxin would also be stabilized by the ability of the NH2-terminal domain to associate with α-actinin and paxillin (Fig. 3 A). Consistent with this hypothesis, disruption of LIM1 but not LIM2 or LIM3 in the isolated COOH-terminal half of the molecule results in the elimination of its recruitment to focal adhesions (Fig. 1 A).

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