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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 dependent on mTOR kinase activityRh30 cells, infected with recombinant adenoviruses expressing GFP (Ad-GFP), FLAG-tagged rapamycin-resistant and kinase active mTOR (S2035T, Ad-mTOR-T), and rapamycin-resistant and kinase dead mTOR (S2035T/D2357E, Ad-mTOR-TE) (A) or with lentiviral shRNAs to GFP and mTOR (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 3: Rapamycin-induced dephosphorylation of mSin1 is dependent on mTOR kinase activityRh30 cells, infected with recombinant adenoviruses expressing GFP (Ad-GFP), FLAG-tagged rapamycin-resistant and kinase active mTOR (S2035T, Ad-mTOR-T), and rapamycin-resistant and kinase dead mTOR (S2035T/D2357E, Ad-mTOR-TE) (A) or with lentiviral shRNAs to GFP and mTOR (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: It has been described that rapamycin inhibits skeletal myogenesis in an mTOR kinase activity-independent manner [42, 43], although this remains controversial [44, 45]. To determine whether rapamycin inhibition of mSin1 phosphorylation depends on the kinase activity of mTOR, serum-starved Rh30 cells were infected with recombinant adenoviruses expressing empty vector (GFP), FLAG-tagged rapamycin-resistant kinase active mTOR (S2035T, mTOR-T), and rapamycin-resistant kinase-dead mTOR (S2035T/D2357E, mTOR-TE), respectively. Subsequently, the cells were pre-treated with or without rapamycin for 2 h, and then stimulated with IGF-1 (10 ng/ml) for 10 h. Consistent with our previous observations [20, 21], expression of mTOR-T, but not mTOR-TE or GFP, potently prevented rapamycin from inhibiting phosphorylation of 4E-BP1 and S6K1 (Figure 3A), two best-characterized substrates of mTOR [1, 2]. Of note, the phosphorylation state of 4E-BP1 was detected with an antibody to 4E-BP1. Rapamycin inhibited phosphorylation of 4E-BP1, as indicated by the decrease in the intensity of the uppermost band γ and by the increase in the higher mobility band α and β that corresponds to a less phosphorylated form of 4E-BP1. The results indicate that mTOR-T functioned as a rapamycin-resistant and kinase active mutant, and mTOR-TE as a kinase-dead mutant in Rh30 cells. Interestingly, expression of mTOR-T, but not mTOR-TE or GFP, conferred high resistance to rapamycin inhibition of mSin1 phosphorylation (Figure 3A), suggesting that rapamycin inhibits phosphorylation of mSin1 in an mTOR kinase activity-dependent manner.


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 dependent on mTOR kinase activityRh30 cells, infected with recombinant adenoviruses expressing GFP (Ad-GFP), FLAG-tagged rapamycin-resistant and kinase active mTOR (S2035T, Ad-mTOR-T), and rapamycin-resistant and kinase dead mTOR (S2035T/D2357E, Ad-mTOR-TE) (A) or with lentiviral shRNAs to GFP and mTOR (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 3: Rapamycin-induced dephosphorylation of mSin1 is dependent on mTOR kinase activityRh30 cells, infected with recombinant adenoviruses expressing GFP (Ad-GFP), FLAG-tagged rapamycin-resistant and kinase active mTOR (S2035T, Ad-mTOR-T), and rapamycin-resistant and kinase dead mTOR (S2035T/D2357E, Ad-mTOR-TE) (A) or with lentiviral shRNAs to GFP and mTOR (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: It has been described that rapamycin inhibits skeletal myogenesis in an mTOR kinase activity-independent manner [42, 43], although this remains controversial [44, 45]. To determine whether rapamycin inhibition of mSin1 phosphorylation depends on the kinase activity of mTOR, serum-starved Rh30 cells were infected with recombinant adenoviruses expressing empty vector (GFP), FLAG-tagged rapamycin-resistant kinase active mTOR (S2035T, mTOR-T), and rapamycin-resistant kinase-dead mTOR (S2035T/D2357E, mTOR-TE), respectively. Subsequently, the cells were pre-treated with or without rapamycin for 2 h, and then stimulated with IGF-1 (10 ng/ml) for 10 h. Consistent with our previous observations [20, 21], expression of mTOR-T, but not mTOR-TE or GFP, potently prevented rapamycin from inhibiting phosphorylation of 4E-BP1 and S6K1 (Figure 3A), two best-characterized substrates of mTOR [1, 2]. Of note, the phosphorylation state of 4E-BP1 was detected with an antibody to 4E-BP1. Rapamycin inhibited phosphorylation of 4E-BP1, as indicated by the decrease in the intensity of the uppermost band γ and by the increase in the higher mobility band α and β that corresponds to a less phosphorylated form of 4E-BP1. The results indicate that mTOR-T functioned as a rapamycin-resistant and kinase active mutant, and mTOR-TE as a kinase-dead mutant in Rh30 cells. Interestingly, expression of mTOR-T, but not mTOR-TE or GFP, conferred high resistance to rapamycin inhibition of mSin1 phosphorylation (Figure 3A), suggesting that rapamycin inhibits phosphorylation of mSin1 in an mTOR kinase activity-dependent manner.

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