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Interaction between NBS1 and the mTOR/Rictor/SIN1 complex through specific domains.

Wang JQ, Chen JH, Chen YC, Chen MY, Hsieh CY, Teng SC, Wu KJ - PLoS ONE (2013)

Bottom Line: Knockdown of NBS1 decreased the levels of phosphorylated Akt and its downstream targets.Ionizing radiation (IR) increased the NBS1 levels and activated Akt activity.These results demonstrate that NBS1 interacts with the mTOR/Rictor/SIN1 complex through the a.a. 221-402 domain and contributes to the activation of Akt activity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome. The NBS gene product, NBS1 (p95 or nibrin), is a part of the Mre11-Rad50-NBS1 complex. SIN1 is a component of the mTOR/Rictor/SIN1 complex mediating the activation of Akt. Here we show that NBS1 interacted with mTOR, Rictor, and SIN1. The specific domains of mTOR, Rictor, or SIN1 interacted with the internal domain (a.a. 221-402) of NBS1. Sucrose density gradient showed that NBS1 was located in the same fractions as the mTOR/Rictor/SIN1 complex. Knockdown of NBS1 decreased the levels of phosphorylated Akt and its downstream targets. Ionizing radiation (IR) increased the NBS1 levels and activated Akt activity. These results demonstrate that NBS1 interacts with the mTOR/Rictor/SIN1 complex through the a.a. 221-402 domain and contributes to the activation of Akt activity.

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Interaction between NBS1 and the mTOR/Rictor/SIN1 complex.A & B. Co-immunoprecipitation assays showed the interaction between NBS1 and mTOR in 293T cells overexpressing both proteins. C & D. Co-immunoprecipitation assays showed the interaction between NBS1 and Rictor in 293T cells overexpressing both proteins. E & F. Co-immunoprecipitation assays showed the interaction between NBS1 and SIN1β in 293T cells overexpressing both proteins.
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pone-0065586-g001: Interaction between NBS1 and the mTOR/Rictor/SIN1 complex.A & B. Co-immunoprecipitation assays showed the interaction between NBS1 and mTOR in 293T cells overexpressing both proteins. C & D. Co-immunoprecipitation assays showed the interaction between NBS1 and Rictor in 293T cells overexpressing both proteins. E & F. Co-immunoprecipitation assays showed the interaction between NBS1 and SIN1β in 293T cells overexpressing both proteins.

Mentions: We previously demonstrated that NBS1 interacted with the p110α subunit of the PI 3-kinase to induce PI 3-kinase/Akt activity [12]. However, it is possible that NBS1 may also interact with Akt to induce Akt activity. To test whether there is interaction between NBS1 and the mTOR/Rictor/SIN1 complex that is responsible for the activation of Akt activity, co-immunoprecipitation assays were performed between NBS1 and each component of the mTOR/Rictor/SIN1 complex. The results showed that the anti-NBS1 antibody pulled down mTOR in 293T cells overexpressing both NBS1 and mTOR (Fig. 1A). In addition, the anti-mTOR antibody also pulled down NBS1 (Fig. 1B), demonstrating their interaction when both proteins were overexpressed in 293T cells. Similar assays were performed to test the interaction between NBS1 and Rictor and the results showed that NBS1 interacted with Rictor in 293T cells overexpressing both proteins (Fig. 1C–D). Finally, similar assays were performed to test the interaction between NBS1 and SIN1β, which also showed the interaction between these two proteins (Fig. 1E–F). All the results demonstrated that NBS1 interacted with the mTOR/Rictor/SIN1 complex.


Interaction between NBS1 and the mTOR/Rictor/SIN1 complex through specific domains.

Wang JQ, Chen JH, Chen YC, Chen MY, Hsieh CY, Teng SC, Wu KJ - PLoS ONE (2013)

Interaction between NBS1 and the mTOR/Rictor/SIN1 complex.A & B. Co-immunoprecipitation assays showed the interaction between NBS1 and mTOR in 293T cells overexpressing both proteins. C & D. Co-immunoprecipitation assays showed the interaction between NBS1 and Rictor in 293T cells overexpressing both proteins. E & F. Co-immunoprecipitation assays showed the interaction between NBS1 and SIN1β in 293T cells overexpressing both proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675082&req=5

pone-0065586-g001: Interaction between NBS1 and the mTOR/Rictor/SIN1 complex.A & B. Co-immunoprecipitation assays showed the interaction between NBS1 and mTOR in 293T cells overexpressing both proteins. C & D. Co-immunoprecipitation assays showed the interaction between NBS1 and Rictor in 293T cells overexpressing both proteins. E & F. Co-immunoprecipitation assays showed the interaction between NBS1 and SIN1β in 293T cells overexpressing both proteins.
Mentions: We previously demonstrated that NBS1 interacted with the p110α subunit of the PI 3-kinase to induce PI 3-kinase/Akt activity [12]. However, it is possible that NBS1 may also interact with Akt to induce Akt activity. To test whether there is interaction between NBS1 and the mTOR/Rictor/SIN1 complex that is responsible for the activation of Akt activity, co-immunoprecipitation assays were performed between NBS1 and each component of the mTOR/Rictor/SIN1 complex. The results showed that the anti-NBS1 antibody pulled down mTOR in 293T cells overexpressing both NBS1 and mTOR (Fig. 1A). In addition, the anti-mTOR antibody also pulled down NBS1 (Fig. 1B), demonstrating their interaction when both proteins were overexpressed in 293T cells. Similar assays were performed to test the interaction between NBS1 and Rictor and the results showed that NBS1 interacted with Rictor in 293T cells overexpressing both proteins (Fig. 1C–D). Finally, similar assays were performed to test the interaction between NBS1 and SIN1β, which also showed the interaction between these two proteins (Fig. 1E–F). All the results demonstrated that NBS1 interacted with the mTOR/Rictor/SIN1 complex.

Bottom Line: Knockdown of NBS1 decreased the levels of phosphorylated Akt and its downstream targets.Ionizing radiation (IR) increased the NBS1 levels and activated Akt activity.These results demonstrate that NBS1 interacts with the mTOR/Rictor/SIN1 complex through the a.a. 221-402 domain and contributes to the activation of Akt activity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome. The NBS gene product, NBS1 (p95 or nibrin), is a part of the Mre11-Rad50-NBS1 complex. SIN1 is a component of the mTOR/Rictor/SIN1 complex mediating the activation of Akt. Here we show that NBS1 interacted with mTOR, Rictor, and SIN1. The specific domains of mTOR, Rictor, or SIN1 interacted with the internal domain (a.a. 221-402) of NBS1. Sucrose density gradient showed that NBS1 was located in the same fractions as the mTOR/Rictor/SIN1 complex. Knockdown of NBS1 decreased the levels of phosphorylated Akt and its downstream targets. Ionizing radiation (IR) increased the NBS1 levels and activated Akt activity. These results demonstrate that NBS1 interacts with the mTOR/Rictor/SIN1 complex through the a.a. 221-402 domain and contributes to the activation of Akt activity.

Show MeSH
Related in: MedlinePlus