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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

Rapamycin inhibits phosphorylation of mSin1(A) Indicated cells were serum-starved for 24 h, pretreated with rapamycin (Rapa, 100 ng/ml) for 2 h, and then stimulated with or without IGF-1 (10 ng/ml) for 22 h, followed by Western blotting with mSin1 antibody. (B) Rh30 cells were treated as described in (A). The whole cell lysates were treated with or without shrimp alkaline phosphatase (SAP) for 30 min, and then analyzed by Western blotting with mSin1 antibody. (C) Rh30 cells were labeled with [32P] orthophosphate, followed by autoradiography, as described in “Materials and methods”. (D) Quantification data for panel (C). aP < 0.05, difference vs control group; bP < 0.05, difference vs IGF-1 group.
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Figure 1: Rapamycin inhibits phosphorylation of mSin1(A) Indicated cells were serum-starved for 24 h, pretreated with rapamycin (Rapa, 100 ng/ml) for 2 h, and then stimulated with or without IGF-1 (10 ng/ml) for 22 h, followed by Western blotting with mSin1 antibody. (B) Rh30 cells were treated as described in (A). The whole cell lysates were treated with or without shrimp alkaline phosphatase (SAP) for 30 min, and then analyzed by Western blotting with mSin1 antibody. (C) Rh30 cells were labeled with [32P] orthophosphate, followed by autoradiography, as described in “Materials and methods”. (D) Quantification data for panel (C). aP < 0.05, difference vs control group; bP < 0.05, difference vs IGF-1 group.

Mentions: To determine the effect of rapamycin on phosphorylation of mSin1, Rh1 cells, a rapamycin-sensitive human Ewing sarcoma cell line [40, 41], were initially selected for the study. As there were no antibodies against phospho-mSin1 available commercially, at the beginning, we detected phosphorylation of mSin1 according to the electrophoretic mobility of mSin1 by Western blot analysis, as described by other groups [38, 39]. As shown in Figure 1A, serum starvation for 24 h did not increase the electrophoretic mobility of mSin1 in Rh1 cells, but treatment of the serum-starved cells with rapamycin (100 ng/ml) for 24 h increased the electrophoretic mobility of mSin1 obviously, regardless of stimulation with or without IGF-1 (10 ng/ml), suggesting that rapamycin might inhibit phosphorylation of mSin1. Furthermore, similar results were also observed in other cell lines, including human cervical cancer (HeLa), prostate cancer (PC-3), rhabdomyosarcoma (Rh30) cells and mouse embryonic fibroblasts (MEF) (Figure 1A and 1B), demonstrating that rapamycin inhibition of mSin1 phosphorylation is not cell line-dependent. To verify the electrophoretic mobility of mSin1 is correlated to its phosphorylation status, shrimp alkaline phosphatase (SAP) was used. We found that treatment of Rh30 cell lysates with SAP for 30 min reversed the mobility of mSin1 in the control (data not shown) or IGF-1-treated cells to that of rapamycin-treated cells (Figure 1B), revealing that the increased mSin1 mobility shift was indeed due to its dephosphorylation. In addition, rapamycin inhibition of mSin1 phosphorylation was further confirmed by 32P-labeling (Figure 1C, 1D).


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 inhibits phosphorylation of mSin1(A) Indicated cells were serum-starved for 24 h, pretreated with rapamycin (Rapa, 100 ng/ml) for 2 h, and then stimulated with or without IGF-1 (10 ng/ml) for 22 h, followed by Western blotting with mSin1 antibody. (B) Rh30 cells were treated as described in (A). The whole cell lysates were treated with or without shrimp alkaline phosphatase (SAP) for 30 min, and then analyzed by Western blotting with mSin1 antibody. (C) Rh30 cells were labeled with [32P] orthophosphate, followed by autoradiography, as described in “Materials and methods”. (D) Quantification data for panel (C). aP < 0.05, difference vs control group; bP < 0.05, difference vs IGF-1 group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Rapamycin inhibits phosphorylation of mSin1(A) Indicated cells were serum-starved for 24 h, pretreated with rapamycin (Rapa, 100 ng/ml) for 2 h, and then stimulated with or without IGF-1 (10 ng/ml) for 22 h, followed by Western blotting with mSin1 antibody. (B) Rh30 cells were treated as described in (A). The whole cell lysates were treated with or without shrimp alkaline phosphatase (SAP) for 30 min, and then analyzed by Western blotting with mSin1 antibody. (C) Rh30 cells were labeled with [32P] orthophosphate, followed by autoradiography, as described in “Materials and methods”. (D) Quantification data for panel (C). aP < 0.05, difference vs control group; bP < 0.05, difference vs IGF-1 group.
Mentions: To determine the effect of rapamycin on phosphorylation of mSin1, Rh1 cells, a rapamycin-sensitive human Ewing sarcoma cell line [40, 41], were initially selected for the study. As there were no antibodies against phospho-mSin1 available commercially, at the beginning, we detected phosphorylation of mSin1 according to the electrophoretic mobility of mSin1 by Western blot analysis, as described by other groups [38, 39]. As shown in Figure 1A, serum starvation for 24 h did not increase the electrophoretic mobility of mSin1 in Rh1 cells, but treatment of the serum-starved cells with rapamycin (100 ng/ml) for 24 h increased the electrophoretic mobility of mSin1 obviously, regardless of stimulation with or without IGF-1 (10 ng/ml), suggesting that rapamycin might inhibit phosphorylation of mSin1. Furthermore, similar results were also observed in other cell lines, including human cervical cancer (HeLa), prostate cancer (PC-3), rhabdomyosarcoma (Rh30) cells and mouse embryonic fibroblasts (MEF) (Figure 1A and 1B), demonstrating that rapamycin inhibition of mSin1 phosphorylation is not cell line-dependent. To verify the electrophoretic mobility of mSin1 is correlated to its phosphorylation status, shrimp alkaline phosphatase (SAP) was used. We found that treatment of Rh30 cell lysates with SAP for 30 min reversed the mobility of mSin1 in the control (data not shown) or IGF-1-treated cells to that of rapamycin-treated cells (Figure 1B), revealing that the increased mSin1 mobility shift was indeed due to its dephosphorylation. In addition, rapamycin inhibition of mSin1 phosphorylation was further confirmed by 32P-labeling (Figure 1C, 1D).

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