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Rapamycin inhibits mSin1 phosphorylation independently of mTORC1 and mTORC2.

Luo Y, Liu L, Wu Y, Singh K, Su B, Zhang N, Liu X, Shen Y, Huang S - Oncotarget (2015)

Bottom Line: Here we found that rapamycin inhibited phosphorylation of mSin1 potently and rapidly.Furthermore, disruption of mTORC1 and mTORC2 by silencing raptor and rictor, respectively, or downregulation of S6K1 or Akt did not induce the dephosphorylation of mSin1 as rapamycin did.However, silencing mTOR or mLST8 mimicked the effect of rapamycin, inhibiting mSin1 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, People's Republic of China.

ABSTRACT
Current knowledge indicates that the mammalian target of rapamycin (mTOR) functions as two complexes, mTORC1 and mTORC2, regulating cell growth, proliferation, survival, differentiation, and motility. Recently mSin1 has been identified as a critical component of mTORC2, which is essential for phosphorylation of Akt and other signaling molecules. Studies have shown that rapamycin inhibits phosphorylation of mSin1. However, the underlying mechanism is unknown. Here we found that rapamycin inhibited phosphorylation of mSin1 potently and rapidly. Expression of rapamycin-resistant mutant of mTOR (mTOR-T), but not rapamycin-resistant and kinase dead mutant of mTOR (mTOR-TE), prevented rapamycin from inhibiting mSin1 phosphorylation, suggesting that rapamycin-induced dephosphorylation of mSin1 is mTOR-dependent. Surprisingly, ectopic expression of rapamycin-resistant and constitutively active p70 S6 kinase 1 (S6K1) did not confer resistance to rapamycin-induced dephosphorylation of mSin1. Furthermore, disruption of mTORC1 and mTORC2 by silencing raptor and rictor, respectively, or downregulation of S6K1 or Akt did not induce the dephosphorylation of mSin1 as rapamycin did. However, silencing mTOR or mLST8 mimicked the effect of rapamycin, inhibiting mSin1 phosphorylation. Our findings suggest that rapamycin inhibits mSin1 phosphorylation, which is independent of mTORC1 and mTORC2, but is possibly dependent on a new mTOR complex, which at least contains mTOR and mLST8.

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Related in: MedlinePlus

mLST8 is essential for mTOR-mediated phosphorylation of mSin1Rh30 (A) or HeLa cells (B) infected with lentiviral shRNAs to GFP and mLST8, respectively, were serum-starved for 24 h. The cells were then pretreated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, and further stimulated with or without IGF-1 (10 ng/ml) for 10 h, followed by Western blotting with indicated antibodies.
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Figure 7: mLST8 is essential for mTOR-mediated phosphorylation of mSin1Rh30 (A) or HeLa cells (B) infected with lentiviral shRNAs to GFP and mLST8, respectively, were serum-starved for 24 h. The cells were then pretreated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, and further stimulated with or without IGF-1 (10 ng/ml) for 10 h, followed by Western blotting with indicated antibodies.

Mentions: As mLST8 is an essential component for all mTOR complexes identified so far [1, 2], next we investigated whether the phosphorylation of mSin1 requires the involvement of mLST8. For this, Rh30 cells were infected with lentiviral shRNA to mLST8 or GFP (as a control). As illustrated in Figure 7A, mLST8 was downregulated by ~80%, in Rh30 cells by the lentiviral shRNA to mLST8. Silencing mLST8, like silencing mTOR (Figure 3B), inhibited the basal and IGF-1-stimulated phosphorylation of mSin1, even in the absence of rapamycin. Similar results were observed in HeLa cells (Figure 7B). The results indicate that mLST8 is necessary for mTOR-mediated phosphorylation of mSin1.


Rapamycin inhibits mSin1 phosphorylation independently of mTORC1 and mTORC2.

Luo Y, Liu L, Wu Y, Singh K, Su B, Zhang N, Liu X, Shen Y, Huang S - Oncotarget (2015)

mLST8 is essential for mTOR-mediated phosphorylation of mSin1Rh30 (A) or HeLa cells (B) infected with lentiviral shRNAs to GFP and mLST8, respectively, were serum-starved for 24 h. The cells were then pretreated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, and further stimulated with or without IGF-1 (10 ng/ml) for 10 h, followed by Western blotting with indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414190&req=5

Figure 7: mLST8 is essential for mTOR-mediated phosphorylation of mSin1Rh30 (A) or HeLa cells (B) infected with lentiviral shRNAs to GFP and mLST8, respectively, were serum-starved for 24 h. The cells were then pretreated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, and further stimulated with or without IGF-1 (10 ng/ml) for 10 h, followed by Western blotting with indicated antibodies.
Mentions: As mLST8 is an essential component for all mTOR complexes identified so far [1, 2], next we investigated whether the phosphorylation of mSin1 requires the involvement of mLST8. For this, Rh30 cells were infected with lentiviral shRNA to mLST8 or GFP (as a control). As illustrated in Figure 7A, mLST8 was downregulated by ~80%, in Rh30 cells by the lentiviral shRNA to mLST8. Silencing mLST8, like silencing mTOR (Figure 3B), inhibited the basal and IGF-1-stimulated phosphorylation of mSin1, even in the absence of rapamycin. Similar results were observed in HeLa cells (Figure 7B). The results indicate that mLST8 is necessary for mTOR-mediated phosphorylation of mSin1.

Bottom Line: Here we found that rapamycin inhibited phosphorylation of mSin1 potently and rapidly.Furthermore, disruption of mTORC1 and mTORC2 by silencing raptor and rictor, respectively, or downregulation of S6K1 or Akt did not induce the dephosphorylation of mSin1 as rapamycin did.However, silencing mTOR or mLST8 mimicked the effect of rapamycin, inhibiting mSin1 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, People's Republic of China.

ABSTRACT
Current knowledge indicates that the mammalian target of rapamycin (mTOR) functions as two complexes, mTORC1 and mTORC2, regulating cell growth, proliferation, survival, differentiation, and motility. Recently mSin1 has been identified as a critical component of mTORC2, which is essential for phosphorylation of Akt and other signaling molecules. Studies have shown that rapamycin inhibits phosphorylation of mSin1. However, the underlying mechanism is unknown. Here we found that rapamycin inhibited phosphorylation of mSin1 potently and rapidly. Expression of rapamycin-resistant mutant of mTOR (mTOR-T), but not rapamycin-resistant and kinase dead mutant of mTOR (mTOR-TE), prevented rapamycin from inhibiting mSin1 phosphorylation, suggesting that rapamycin-induced dephosphorylation of mSin1 is mTOR-dependent. Surprisingly, ectopic expression of rapamycin-resistant and constitutively active p70 S6 kinase 1 (S6K1) did not confer resistance to rapamycin-induced dephosphorylation of mSin1. Furthermore, disruption of mTORC1 and mTORC2 by silencing raptor and rictor, respectively, or downregulation of S6K1 or Akt did not induce the dephosphorylation of mSin1 as rapamycin did. However, silencing mTOR or mLST8 mimicked the effect of rapamycin, inhibiting mSin1 phosphorylation. Our findings suggest that rapamycin inhibits mSin1 phosphorylation, which is independent of mTORC1 and mTORC2, but is possibly dependent on a new mTOR complex, which at least contains mTOR and mLST8.

Show MeSH
Related in: MedlinePlus