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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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Co-immunoprecipitation assays and sucrose density gradient analysis showed the interaction between NBS1 and the mTOR/Rictor/SIN1 complex in vivo.A. Co-immunoprecipitation assays showed the invivo interaction of NBS1 with the mTOR/Rictor/SIN1 complex using extracts from H1299 cell line. B. Sucrose density gradient analysis showed the co-localization in the same fractions between NBS1 and the mTOR/Rictor/SIN1 complex.
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pone-0065586-g005: Co-immunoprecipitation assays and sucrose density gradient analysis showed the interaction between NBS1 and the mTOR/Rictor/SIN1 complex in vivo.A. Co-immunoprecipitation assays showed the invivo interaction of NBS1 with the mTOR/Rictor/SIN1 complex using extracts from H1299 cell line. B. Sucrose density gradient analysis showed the co-localization in the same fractions between NBS1 and the mTOR/Rictor/SIN1 complex.

Mentions: In order to test whether NBS1 indeed interacts with the mTOR/Rictor/SIN1 complex, co-immunoprecipitation assays using extracts from a lung cancer cell line H1299 were used. The result showed that the anti-NBS1 antibody pulled down mTOR, Rictor, and SIN1β (Fig. 5A, left panel), supporting their interaction in vivo. Co-immunoprecipitation experiment using the anti-Raptor antibody did not pull down the whole mTOR/Rictor/SIN1 complex or NBS1 (Fig. 5A, right panel), indicating that NBS1 did not interact with the mTOR/Raptor complex. Finally, a sucrose density gradient experiment using extracts from H1299 cells was performed to test whether these proteins were localized in the same fraction. The result showed that indeed NBS1, mTOR, Rictor, and SIN1β were localized in the same fraction (fraction 4 is the major fraction) (Fig. 5B). All the results indicated that NBS1 interacted with the mTOR/Rictor/SIN1 complex invivo.


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)

Co-immunoprecipitation assays and sucrose density gradient analysis showed the interaction between NBS1 and the mTOR/Rictor/SIN1 complex in vivo.A. Co-immunoprecipitation assays showed the invivo interaction of NBS1 with the mTOR/Rictor/SIN1 complex using extracts from H1299 cell line. B. Sucrose density gradient analysis showed the co-localization in the same fractions between NBS1 and the mTOR/Rictor/SIN1 complex.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675082&req=5

pone-0065586-g005: Co-immunoprecipitation assays and sucrose density gradient analysis showed the interaction between NBS1 and the mTOR/Rictor/SIN1 complex in vivo.A. Co-immunoprecipitation assays showed the invivo interaction of NBS1 with the mTOR/Rictor/SIN1 complex using extracts from H1299 cell line. B. Sucrose density gradient analysis showed the co-localization in the same fractions between NBS1 and the mTOR/Rictor/SIN1 complex.
Mentions: In order to test whether NBS1 indeed interacts with the mTOR/Rictor/SIN1 complex, co-immunoprecipitation assays using extracts from a lung cancer cell line H1299 were used. The result showed that the anti-NBS1 antibody pulled down mTOR, Rictor, and SIN1β (Fig. 5A, left panel), supporting their interaction in vivo. Co-immunoprecipitation experiment using the anti-Raptor antibody did not pull down the whole mTOR/Rictor/SIN1 complex or NBS1 (Fig. 5A, right panel), indicating that NBS1 did not interact with the mTOR/Raptor complex. Finally, a sucrose density gradient experiment using extracts from H1299 cells was performed to test whether these proteins were localized in the same fraction. The result showed that indeed NBS1, mTOR, Rictor, and SIN1β were localized in the same fraction (fraction 4 is the major fraction) (Fig. 5B). All the results indicated that NBS1 interacted with the mTOR/Rictor/SIN1 complex invivo.

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