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Tetratricopeptide repeat domain 9A is an interacting protein for tropomyosin Tm5NM-1.

Cao S, Ho GH, Lin VC - BMC Cancer (2008)

Bottom Line: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins.The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role.In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore. ashleycsl@gmail.com

ABSTRACT

Background: Tetratricopeptide repeat domain 9A (TTC9A) protein is a recently identified protein which contains three tetratricopeptide repeats (TPRs) on its C-terminus. In our previous studies, we have shown that TTC9A was a hormonally-regulated gene in breast cancer cells. In this study, we found that TTC9A was over-expressed in breast cancer tissues compared with the adjacent controls (P < 0.00001), suggesting it might be involved in the breast cancer development process. The aim of the current study was to further elucidate the function of TTC9A.

Methods: Breast samples from 25 patients including the malignant breast tissues and the adjacent normal tissues were processed for Southern blot analysis. Yeast-two-hybrid assay, GST pull-down assay and co-immunoprecipitation were used to identify and verify the interaction between TTC9A and other proteins.

Results: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins. The interaction between TTC9A and Tm5NM-1 was further confirmed by GST pull-down assay and co-immunoprecipitation in mammalian cells. TTC9A domains required for the interaction were also characterized in this study. The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role.

Conclusion: Since the primary function of tropomyosin is to stabilize actin filament, its interaction with TTC9A may play a role in cell shape and motility. In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading. We speculate that TTC9A acts as a chaperone protein to facilitate the function of tropomyosins in stabilizing microfilament and it may play a role in cancer cell invasion and metastasis.

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Related in: MedlinePlus

TTC9A binds to Tm5NM-1. (A) GST-TTC9A binds to Tm5NM-1-(His)6. COS-7 cells were transfected with Tm5NM-1-(His)6 expression vector or control vector and total cell lysates were collected at 48 h post-transfection. 60 μg GST-TTC9A protein was immobilized onto Glutathione Sepharose 4B gel (Amersham Biosciences) and 300 μg total cell lysates were used for Tm5NM-1-(His)6 pull-down. The proteins bound to the beads were eluted with 2 × SDS-PAGE sample buffer and were separated on an SDS-PAGE gel. Tm5NM-1-(His)6 was detected using anti-His antibody (Amersham Biosciences). GST protein expressed by empty pGEX-5X-3 vector was included as a negative control. 15 μg total cell lysates (5% of input) were loaded in the first lane to indicate the position of Tm5NM-1-(His)6 band. (B) Tm5NM-1-(His)6 pull-down by GST-TTC9A is concentration-dependent. GST-pull down assay was carried out with 2 μg or 20 μg GST-TTC9A as bait protein. The amount of Tm5NM-1-(His)6 pulled down was proportional to the amount of bait protein used. (C) TTC9A-flag interacted with Tm5NM-1-(His)6. Expression vectors for TTC9A-flag and Tm5NM-1-(His)6 were co-transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-flag agarose beads (Sigma-Aldrich) and Tm5NM-1 was detected by anti-His antibody (Amersham Biosciences). Upper panel: Tm5NM-1-(His)6 was expressed at similar level in control vector and TTC9A-flag transfected COS-7 cells; lower panel: Tm5NM-1-(His)6 was pulled down by TTC9A-flag. (D) TTC9A-flag interacted with endogenous Tm5NM-1-(His)6. Expression vector for TTC9A-flag was transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-Tm5NM-1/2 (Chemicon) antibody and TTC9A was detected by anti-flag antibody (Sigma-Aldrich). Co-immunoprecipitation with goat pre-immune serum was included as a negative control.
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Figure 2: TTC9A binds to Tm5NM-1. (A) GST-TTC9A binds to Tm5NM-1-(His)6. COS-7 cells were transfected with Tm5NM-1-(His)6 expression vector or control vector and total cell lysates were collected at 48 h post-transfection. 60 μg GST-TTC9A protein was immobilized onto Glutathione Sepharose 4B gel (Amersham Biosciences) and 300 μg total cell lysates were used for Tm5NM-1-(His)6 pull-down. The proteins bound to the beads were eluted with 2 × SDS-PAGE sample buffer and were separated on an SDS-PAGE gel. Tm5NM-1-(His)6 was detected using anti-His antibody (Amersham Biosciences). GST protein expressed by empty pGEX-5X-3 vector was included as a negative control. 15 μg total cell lysates (5% of input) were loaded in the first lane to indicate the position of Tm5NM-1-(His)6 band. (B) Tm5NM-1-(His)6 pull-down by GST-TTC9A is concentration-dependent. GST-pull down assay was carried out with 2 μg or 20 μg GST-TTC9A as bait protein. The amount of Tm5NM-1-(His)6 pulled down was proportional to the amount of bait protein used. (C) TTC9A-flag interacted with Tm5NM-1-(His)6. Expression vectors for TTC9A-flag and Tm5NM-1-(His)6 were co-transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-flag agarose beads (Sigma-Aldrich) and Tm5NM-1 was detected by anti-His antibody (Amersham Biosciences). Upper panel: Tm5NM-1-(His)6 was expressed at similar level in control vector and TTC9A-flag transfected COS-7 cells; lower panel: Tm5NM-1-(His)6 was pulled down by TTC9A-flag. (D) TTC9A-flag interacted with endogenous Tm5NM-1-(His)6. Expression vector for TTC9A-flag was transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-Tm5NM-1/2 (Chemicon) antibody and TTC9A was detected by anti-flag antibody (Sigma-Aldrich). Co-immunoprecipitation with goat pre-immune serum was included as a negative control.

Mentions: The binding of TTC9A to Tm5NM-1 was further examined by GST-pull down assay. In Fig. 2A, Tm5NM-1-(His)6 expression vector was transfected into COS-7 cells and GST-TTC9A was used as a "bait" to pull down the cellular expressed Tm5NM-1 protein. As shown in the figure, Tm5NM-1 was pulled down by GST-TTC9A, but not by the GST-tag, suggesting that GST-TTC9A did interact with cellular expressed Tm5NM-1. The specificity of the interaction was further confirmed by GST pull-down assay with different amount of bait protein. Fig. 2B showed that the protein amount of Tm5NM-1 pulled down by GST-TTC9A increased proportionally to the amount of bait protein used in the assay. However, bands other than the expected Tm5NM-1 were also observed in Fig. 2B and these non-specific bands were not observed in sample with GST protein. These unexpected bands could be due to non-specific pull-down or degraded GST-TTC9A.


Tetratricopeptide repeat domain 9A is an interacting protein for tropomyosin Tm5NM-1.

Cao S, Ho GH, Lin VC - BMC Cancer (2008)

TTC9A binds to Tm5NM-1. (A) GST-TTC9A binds to Tm5NM-1-(His)6. COS-7 cells were transfected with Tm5NM-1-(His)6 expression vector or control vector and total cell lysates were collected at 48 h post-transfection. 60 μg GST-TTC9A protein was immobilized onto Glutathione Sepharose 4B gel (Amersham Biosciences) and 300 μg total cell lysates were used for Tm5NM-1-(His)6 pull-down. The proteins bound to the beads were eluted with 2 × SDS-PAGE sample buffer and were separated on an SDS-PAGE gel. Tm5NM-1-(His)6 was detected using anti-His antibody (Amersham Biosciences). GST protein expressed by empty pGEX-5X-3 vector was included as a negative control. 15 μg total cell lysates (5% of input) were loaded in the first lane to indicate the position of Tm5NM-1-(His)6 band. (B) Tm5NM-1-(His)6 pull-down by GST-TTC9A is concentration-dependent. GST-pull down assay was carried out with 2 μg or 20 μg GST-TTC9A as bait protein. The amount of Tm5NM-1-(His)6 pulled down was proportional to the amount of bait protein used. (C) TTC9A-flag interacted with Tm5NM-1-(His)6. Expression vectors for TTC9A-flag and Tm5NM-1-(His)6 were co-transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-flag agarose beads (Sigma-Aldrich) and Tm5NM-1 was detected by anti-His antibody (Amersham Biosciences). Upper panel: Tm5NM-1-(His)6 was expressed at similar level in control vector and TTC9A-flag transfected COS-7 cells; lower panel: Tm5NM-1-(His)6 was pulled down by TTC9A-flag. (D) TTC9A-flag interacted with endogenous Tm5NM-1-(His)6. Expression vector for TTC9A-flag was transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-Tm5NM-1/2 (Chemicon) antibody and TTC9A was detected by anti-flag antibody (Sigma-Aldrich). Co-immunoprecipitation with goat pre-immune serum was included as a negative control.
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Figure 2: TTC9A binds to Tm5NM-1. (A) GST-TTC9A binds to Tm5NM-1-(His)6. COS-7 cells were transfected with Tm5NM-1-(His)6 expression vector or control vector and total cell lysates were collected at 48 h post-transfection. 60 μg GST-TTC9A protein was immobilized onto Glutathione Sepharose 4B gel (Amersham Biosciences) and 300 μg total cell lysates were used for Tm5NM-1-(His)6 pull-down. The proteins bound to the beads were eluted with 2 × SDS-PAGE sample buffer and were separated on an SDS-PAGE gel. Tm5NM-1-(His)6 was detected using anti-His antibody (Amersham Biosciences). GST protein expressed by empty pGEX-5X-3 vector was included as a negative control. 15 μg total cell lysates (5% of input) were loaded in the first lane to indicate the position of Tm5NM-1-(His)6 band. (B) Tm5NM-1-(His)6 pull-down by GST-TTC9A is concentration-dependent. GST-pull down assay was carried out with 2 μg or 20 μg GST-TTC9A as bait protein. The amount of Tm5NM-1-(His)6 pulled down was proportional to the amount of bait protein used. (C) TTC9A-flag interacted with Tm5NM-1-(His)6. Expression vectors for TTC9A-flag and Tm5NM-1-(His)6 were co-transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-flag agarose beads (Sigma-Aldrich) and Tm5NM-1 was detected by anti-His antibody (Amersham Biosciences). Upper panel: Tm5NM-1-(His)6 was expressed at similar level in control vector and TTC9A-flag transfected COS-7 cells; lower panel: Tm5NM-1-(His)6 was pulled down by TTC9A-flag. (D) TTC9A-flag interacted with endogenous Tm5NM-1-(His)6. Expression vector for TTC9A-flag was transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-Tm5NM-1/2 (Chemicon) antibody and TTC9A was detected by anti-flag antibody (Sigma-Aldrich). Co-immunoprecipitation with goat pre-immune serum was included as a negative control.
Mentions: The binding of TTC9A to Tm5NM-1 was further examined by GST-pull down assay. In Fig. 2A, Tm5NM-1-(His)6 expression vector was transfected into COS-7 cells and GST-TTC9A was used as a "bait" to pull down the cellular expressed Tm5NM-1 protein. As shown in the figure, Tm5NM-1 was pulled down by GST-TTC9A, but not by the GST-tag, suggesting that GST-TTC9A did interact with cellular expressed Tm5NM-1. The specificity of the interaction was further confirmed by GST pull-down assay with different amount of bait protein. Fig. 2B showed that the protein amount of Tm5NM-1 pulled down by GST-TTC9A increased proportionally to the amount of bait protein used in the assay. However, bands other than the expected Tm5NM-1 were also observed in Fig. 2B and these non-specific bands were not observed in sample with GST protein. These unexpected bands could be due to non-specific pull-down or degraded GST-TTC9A.

Bottom Line: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins.The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role.In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore. ashleycsl@gmail.com

ABSTRACT

Background: Tetratricopeptide repeat domain 9A (TTC9A) protein is a recently identified protein which contains three tetratricopeptide repeats (TPRs) on its C-terminus. In our previous studies, we have shown that TTC9A was a hormonally-regulated gene in breast cancer cells. In this study, we found that TTC9A was over-expressed in breast cancer tissues compared with the adjacent controls (P < 0.00001), suggesting it might be involved in the breast cancer development process. The aim of the current study was to further elucidate the function of TTC9A.

Methods: Breast samples from 25 patients including the malignant breast tissues and the adjacent normal tissues were processed for Southern blot analysis. Yeast-two-hybrid assay, GST pull-down assay and co-immunoprecipitation were used to identify and verify the interaction between TTC9A and other proteins.

Results: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins. The interaction between TTC9A and Tm5NM-1 was further confirmed by GST pull-down assay and co-immunoprecipitation in mammalian cells. TTC9A domains required for the interaction were also characterized in this study. The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role.

Conclusion: Since the primary function of tropomyosin is to stabilize actin filament, its interaction with TTC9A may play a role in cell shape and motility. In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading. We speculate that TTC9A acts as a chaperone protein to facilitate the function of tropomyosins in stabilizing microfilament and it may play a role in cancer cell invasion and metastasis.

Show MeSH
Related in: MedlinePlus