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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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Rapamycin-induced dephosphorylation of mSin1 is not by inhibiting S6K1Rh30 cells, infected with recombinant adenoviruses expressing GFP (Ad-GFP) and HA-tagged rapamycin-resistant and constitutively active S6K1 (Ad-S6K1-ca) (A) or with lentiviral shRNAs to GFP and S6K1 (B) 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 4: Rapamycin-induced dephosphorylation of mSin1 is not by inhibiting S6K1Rh30 cells, infected with recombinant adenoviruses expressing GFP (Ad-GFP) and HA-tagged rapamycin-resistant and constitutively active S6K1 (Ad-S6K1-ca) (A) or with lentiviral shRNAs to GFP and S6K1 (B) 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: Both rictor and mSin1 are the components of mTORC2 [1, 2], and S6K1 has recently been identified as the kinase that phosphorylates rictor (Thr1135) [37, 46, 47]. Particularly, it has been described that mSin1 can be phosphorylated on T86 and T398 by S6K1 in a cellular context-dependent manner [48]. Therefore, at the very beginning, we hypothesized that rapamycin may inhibit mSin1 phosphorylation by suppressing the activity of S6K1. To test this hypothesis, Rh30 cells were infected with recombinant adenovirus encoding constitutively active and rapamycin-resistant HA-tagged S6K1 mutant (F5A-E389-R3A) (Ad-S6K1-ca) and the control virus encoding GFP (Ad-GFP), respectively. In agreement with our previous data [49], expression of constitutively active and rapamycin-resistant (HA-S6K1-ca), but not GFP, increased the basal phosphorylation level of S6 ribosomal protein, a substrate of S6K1, and conferred resistance to rapamycin inhibition of S6 phosphorylation (Figure 4A), suggesting that the S6K1-ca was functional in the cells. However, expression of the rapamycin-resistant constitutively active S6K1 failed to prevent rapamycin from inhibiting the phosphorylation of mSin1 (Figure 4A). The results suggest that rapamycin-induced dephosphorylation of mSin1 is probably not by inhibiting S6K1 activity.


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)

Rapamycin-induced dephosphorylation of mSin1 is not by inhibiting S6K1Rh30 cells, infected with recombinant adenoviruses expressing GFP (Ad-GFP) and HA-tagged rapamycin-resistant and constitutively active S6K1 (Ad-S6K1-ca) (A) or with lentiviral shRNAs to GFP and S6K1 (B) 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 4: Rapamycin-induced dephosphorylation of mSin1 is not by inhibiting S6K1Rh30 cells, infected with recombinant adenoviruses expressing GFP (Ad-GFP) and HA-tagged rapamycin-resistant and constitutively active S6K1 (Ad-S6K1-ca) (A) or with lentiviral shRNAs to GFP and S6K1 (B) 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: Both rictor and mSin1 are the components of mTORC2 [1, 2], and S6K1 has recently been identified as the kinase that phosphorylates rictor (Thr1135) [37, 46, 47]. Particularly, it has been described that mSin1 can be phosphorylated on T86 and T398 by S6K1 in a cellular context-dependent manner [48]. Therefore, at the very beginning, we hypothesized that rapamycin may inhibit mSin1 phosphorylation by suppressing the activity of S6K1. To test this hypothesis, Rh30 cells were infected with recombinant adenovirus encoding constitutively active and rapamycin-resistant HA-tagged S6K1 mutant (F5A-E389-R3A) (Ad-S6K1-ca) and the control virus encoding GFP (Ad-GFP), respectively. In agreement with our previous data [49], expression of constitutively active and rapamycin-resistant (HA-S6K1-ca), but not GFP, increased the basal phosphorylation level of S6 ribosomal protein, a substrate of S6K1, and conferred resistance to rapamycin inhibition of S6 phosphorylation (Figure 4A), suggesting that the S6K1-ca was functional in the cells. However, expression of the rapamycin-resistant constitutively active S6K1 failed to prevent rapamycin from inhibiting the phosphorylation of mSin1 (Figure 4A). The results suggest that rapamycin-induced dephosphorylation of mSin1 is probably not by inhibiting S6K1 activity.

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