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.

Show MeSH

Related in: MedlinePlus

Tes interacts directly with zyxin; the two halves of Tes interact with each other. (A) Western blot analysis with the indicated antibodies (left) of pull-down assays performed on HeLa cell extracts using the Ni affinity resins indicated (top). The His-Gem affinity resin (Gem) represents a negative control. The input extract (Extract) as well as the bound fraction (Bound) and supernatant (Sup) for each resin are indicated. (B) Western blot analysis with the indicated antibodies (left) of Tes immunoprecipitation assays. The input extract (Extract), anti-Tes antiserum (anti-Tes) or the preimmune serum (Pre-imm) are indicated. (C) Western blot analysis of pull-down assays performed on HeLa cell extracts using the His-LIM resins indicated (top) shows that the LIM1 domain interacts with zyxin, whereas LIM3 binds Mena and VASP. (D) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing GST-zyxin with the His-LIM resins shows that only the LIM1 domain is able to interact directly with zyxin. (E) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing the GST fusion protein (bottom) with purified His protein resins (top) demonstrates that the NH2-terminal half of Tes can interact directly with the COOH-terminal half of the molecule in vitro. (F) Western blot analysis of Ni resin pull-downs from extracts of HeLa cells cotransfected with His-Tes-C-term and GFP-Tes-N-term. The anti-GFP blot demonstrates that GFP-Tes-N-term copurifies with His-Tes-C-term but not His-Gem, indicating the halves of the molecule interact with each other in vivo.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172870&req=5

fig3: Tes interacts directly with zyxin; the two halves of Tes interact with each other. (A) Western blot analysis with the indicated antibodies (left) of pull-down assays performed on HeLa cell extracts using the Ni affinity resins indicated (top). The His-Gem affinity resin (Gem) represents a negative control. The input extract (Extract) as well as the bound fraction (Bound) and supernatant (Sup) for each resin are indicated. (B) Western blot analysis with the indicated antibodies (left) of Tes immunoprecipitation assays. The input extract (Extract), anti-Tes antiserum (anti-Tes) or the preimmune serum (Pre-imm) are indicated. (C) Western blot analysis of pull-down assays performed on HeLa cell extracts using the His-LIM resins indicated (top) shows that the LIM1 domain interacts with zyxin, whereas LIM3 binds Mena and VASP. (D) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing GST-zyxin with the His-LIM resins shows that only the LIM1 domain is able to interact directly with zyxin. (E) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing the GST fusion protein (bottom) with purified His protein resins (top) demonstrates that the NH2-terminal half of Tes can interact directly with the COOH-terminal half of the molecule in vitro. (F) Western blot analysis of Ni resin pull-downs from extracts of HeLa cells cotransfected with His-Tes-C-term and GFP-Tes-N-term. The anti-GFP blot demonstrates that GFP-Tes-N-term copurifies with His-Tes-C-term but not His-Gem, indicating the halves of the molecule interact with each other in vivo.

Mentions: To examine which protein(s) might be responsible for recruiting Tes to focal adhesions, we performed pull-down assays on HeLa cell extracts using Tes produced in E. coli. Western blot analysis of pull-downs with a panel of antibodies against known focal adhesion proteins reveals that the Tes affinity resin retains actin, Mena, and VASP, but not α-actinin, Nck, FAK, paxillin, talin, vinculin, or zyxin, from cell extracts (Fig. 3 A). Coimmunoprecipitation experiments using anti-Tes antibody confirmed that Tes forms complexes with actin, Mena, and VASP, but not α-actinin, paxillin, or zyxin, in vivo (Fig. 3 B). Given our observations on the different in vivo localizations of the GFP-tagged Tes domains, we also performed pull-downs with the NH2- and COOH-terminal halves of the molecule. We found that an affinity column of the NH2-terminal half of Tes produced in E. coli can interact with actin, whereas the COOH-terminal half of the molecule associates with Mena and VASP (Fig. 3 A). Interestingly, the isolated halves of the protein also engage in additional interactions that are not observed with full-length Tes. The NH2-terminal half is able to associate with α-actinin and paxillin, whereas the COOH-terminal half interacts with zyxin (Fig. 3 A). To further define which region in the COOH-terminal half of Tes is responsible for interacting with Mena, VASP, and zyxin, we performed pull-down assays using the individual LIM domains (Fig. 3 C). Western blot analysis of the pull-downs reveals that LIM1 binds zyxin, whereas LIM3 associates with Mena and VASP (Fig. 3 C). These interactions were abolished when a disrupting point mutation was introduced into the respective LIM domain (data not shown). To examine if the binding of Tes to VASP or zyxin is direct, we performed pull-down assays using protein produced in E. coli. We could find no evidence for a direct interaction between LIM3 and VASP (not shown). In contrast, we found that LIM1 was able to bind zyxin directly (Fig. 3 D).


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 interacts directly with zyxin; the two halves of Tes interact with each other. (A) Western blot analysis with the indicated antibodies (left) of pull-down assays performed on HeLa cell extracts using the Ni affinity resins indicated (top). The His-Gem affinity resin (Gem) represents a negative control. The input extract (Extract) as well as the bound fraction (Bound) and supernatant (Sup) for each resin are indicated. (B) Western blot analysis with the indicated antibodies (left) of Tes immunoprecipitation assays. The input extract (Extract), anti-Tes antiserum (anti-Tes) or the preimmune serum (Pre-imm) are indicated. (C) Western blot analysis of pull-down assays performed on HeLa cell extracts using the His-LIM resins indicated (top) shows that the LIM1 domain interacts with zyxin, whereas LIM3 binds Mena and VASP. (D) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing GST-zyxin with the His-LIM resins shows that only the LIM1 domain is able to interact directly with zyxin. (E) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing the GST fusion protein (bottom) with purified His protein resins (top) demonstrates that the NH2-terminal half of Tes can interact directly with the COOH-terminal half of the molecule in vitro. (F) Western blot analysis of Ni resin pull-downs from extracts of HeLa cells cotransfected with His-Tes-C-term and GFP-Tes-N-term. The anti-GFP blot demonstrates that GFP-Tes-N-term copurifies with His-Tes-C-term but not His-Gem, indicating the halves of the molecule interact with each other in vivo.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Tes interacts directly with zyxin; the two halves of Tes interact with each other. (A) Western blot analysis with the indicated antibodies (left) of pull-down assays performed on HeLa cell extracts using the Ni affinity resins indicated (top). The His-Gem affinity resin (Gem) represents a negative control. The input extract (Extract) as well as the bound fraction (Bound) and supernatant (Sup) for each resin are indicated. (B) Western blot analysis with the indicated antibodies (left) of Tes immunoprecipitation assays. The input extract (Extract), anti-Tes antiserum (anti-Tes) or the preimmune serum (Pre-imm) are indicated. (C) Western blot analysis of pull-down assays performed on HeLa cell extracts using the His-LIM resins indicated (top) shows that the LIM1 domain interacts with zyxin, whereas LIM3 binds Mena and VASP. (D) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing GST-zyxin with the His-LIM resins shows that only the LIM1 domain is able to interact directly with zyxin. (E) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing the GST fusion protein (bottom) with purified His protein resins (top) demonstrates that the NH2-terminal half of Tes can interact directly with the COOH-terminal half of the molecule in vitro. (F) Western blot analysis of Ni resin pull-downs from extracts of HeLa cells cotransfected with His-Tes-C-term and GFP-Tes-N-term. The anti-GFP blot demonstrates that GFP-Tes-N-term copurifies with His-Tes-C-term but not His-Gem, indicating the halves of the molecule interact with each other in vivo.
Mentions: To examine which protein(s) might be responsible for recruiting Tes to focal adhesions, we performed pull-down assays on HeLa cell extracts using Tes produced in E. coli. Western blot analysis of pull-downs with a panel of antibodies against known focal adhesion proteins reveals that the Tes affinity resin retains actin, Mena, and VASP, but not α-actinin, Nck, FAK, paxillin, talin, vinculin, or zyxin, from cell extracts (Fig. 3 A). Coimmunoprecipitation experiments using anti-Tes antibody confirmed that Tes forms complexes with actin, Mena, and VASP, but not α-actinin, paxillin, or zyxin, in vivo (Fig. 3 B). Given our observations on the different in vivo localizations of the GFP-tagged Tes domains, we also performed pull-downs with the NH2- and COOH-terminal halves of the molecule. We found that an affinity column of the NH2-terminal half of Tes produced in E. coli can interact with actin, whereas the COOH-terminal half of the molecule associates with Mena and VASP (Fig. 3 A). Interestingly, the isolated halves of the protein also engage in additional interactions that are not observed with full-length Tes. The NH2-terminal half is able to associate with α-actinin and paxillin, whereas the COOH-terminal half interacts with zyxin (Fig. 3 A). To further define which region in the COOH-terminal half of Tes is responsible for interacting with Mena, VASP, and zyxin, we performed pull-down assays using the individual LIM domains (Fig. 3 C). Western blot analysis of the pull-downs reveals that LIM1 binds zyxin, whereas LIM3 associates with Mena and VASP (Fig. 3 C). These interactions were abolished when a disrupting point mutation was introduced into the respective LIM domain (data not shown). To examine if the binding of Tes to VASP or zyxin is direct, we performed pull-down assays using protein produced in E. coli. We could find no evidence for a direct interaction between LIM3 and VASP (not shown). In contrast, we found that LIM1 was able to bind zyxin directly (Fig. 3 D).

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