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. 2011 Feb 25;286(8):6791-800.
doi: 10.1074/jbc.M110.172882. Epub 2010 Dec 22.

PI3K-mTORC1 attenuates stress response by inhibiting cap-independent Hsp70 translation

Jun Sun  1 , Crystal S Conn, Yan Han, Vincent Yeung, Shu-Bing Qian
Affiliations

PI3K-mTORC1 attenuates stress response by inhibiting cap-independent Hsp70 translation

"VSports注册入口" Jun Sun et al. J Biol Chem. .

Abstract

Protein synthesis is a key regulated cellular process that links nutrient availability and organismal growth. It has long been known that some cellular proteins continue to be synthesized under conditions where global translation is severely compromised. One prominent example is the selective translation of heat shock proteins (Hsps) under stress conditions. Although the transcriptional regulation of Hsp genes has been well established, neither the specific translation-promoting features nor the regulatory mechanism of the translation machinery have been clearly defined VSports手机版. Here we show that the stress-induced preferential translation of Hsp70 mRNA is negatively regulated by PI3K-mTORC1 signaling. Despite the transcriptional up-regulation, the translation of Hsp70 mRNA is deficient in cells lacking tuberous sclerosis complex 2. Conversely, Hsp70 synthesis is enhanced under the reduced PI3K-mTORC1 signaling. We found that the 5' UTR of Hsp70 mRNA contributes to cap-independent translation without exhibiting typical features of internal ribosome entry site. Our findings imply a plausible mechanism for how persistent PI3K-mTORC1 signaling favors the development of age-related pathologies by attenuating stress resistance. .

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Figures

FIGURE 1.
FIGURE 1.
TSC2 null cells are defective in heat shock-induced Hsp70 expression. A, TSC2 wild type (WT) and knock-out (KO) cells were heat shocked at 42 °C for 1 h and recovered at 37 °C for the times as indicated. HSF1 activity was measured by using a F-Luc reporter driven by the HspA1a promoter. The experiments were repeated 5 times. Error bar, S.E. *, p < 0.01; **, p < 0.001 (Student's t test, two tails). B, general protein synthesis in cells as A was determined by using a control F-Luc reporter driven by the CMV promoter. The experiments were repeated 5 times. C, molecular chaperone levels in cells as A were determined by immunoblotting analysis using the antibodies as indicated. D, TSC2 WT and TSC2 KO cells were transfected with plasmids encoding Hsp70 with different doses (0, 0.1, 0.5, and 2.5 μg) in a 6-well plate. 24 h after transfection, whole cell lysates were immunoblotted with antibodies as indicated.
FIGURE 2.
FIGURE 2.
Deficient Hsp70 mRNA translation in TSC2 null cells after heat shock. A, ribosome profiling of TSC2 WT cells before and after heat shock. Cell lysates were sedimented on a 15–45% sucrose gradient followed by fractionation. The positions of the 40 S, 60 S, 80 S, and polysomal peaks were indicated. Total RNA was extracted from each fraction and subject to RT-PCR and qPCR analysis. Hsp70 RT-PCR results were shown in the middle without concentration normalization. The Hsp70 mRNA levels in whole cell lysates before sucrose gradient are indicated by the triangle. qPCR results of Hsp70 (black bar) and β-actin (grey bar) were normalized based on RNA concentration of each fraction. The highest level was arbitrarily set as 100 and the relative mRNA levels were presented in all polysome fractions. B, ribosome profiling of TSC2 KO cells before and after heat shock. RT-PCR and qPCR were performed as described under A.
FIGURE 3.
FIGURE 3.
PI3K-mTORC1 negatively regulates Hsp70 mRNA translation. A, TSC2 WT cells were transfected with plasmids encoding β-Gal or Rheb. 48 h after transfection, cells were incubated at 42 °C for the times as indicated. Whole cell lysates were immunoblotted using Hsp70 and β-actin antibodies. Relative Hsp70 levels were quantitated by densitometry. n = 3, error bar, S.E.; **, p < 0.01; *, p < 0.05. B, TSC2 WT cells were transfected with siRNA targeting Raptor or GFP as control. 48 h after transfection, cells were incubated at 42 °C for the times as indicated. Whole cell lysates were immunoblotted using Hsp70 and β-actin antibodies. Relative Hsp70 levels were quantitated by densitometry. n = 3, error bar, S.E.; **, p < 0.01; *, p < 0.05. C, TSC2 WT cells were incubated at 42 °C for the times as indicated in the presence of 20 nm rapamycin or DMSO as control. Whole cell lysates were immunoblotted using Hsp70 and β-actin antibodies. Relative Hsp70 levels were quantitated by densitometry. n = 3, error bar, S.E. D, TSC2 WT cells were incubated at 42 °C for the times as indicated in the presence of 50 μm LY294002 or DMSO as control. Whole cell lysates were immunoblotted using Hsp70 and β-actin antibodies. Relative Hsp70 levels were quantitated by densitometry. n = 3, error bar, S.E.; *, p < 0.05.
FIGURE 4.
FIGURE 4.
Hsp70 5′ UTR responds to the PI3K-mTORC1 signaling. A, luciferase mRNA (Luc) was synthesized using in vitro transcription followed by 5′ end capping and 3′ end polyadenylation. mRNA transfection was performed on TSC2 WT and TSC2 KO cells. Real time luciferase activity was recorded immediately after mRNA transfection (left panel). Relative Luc expression (3 h) in TSC2 KO cells was normalized against the wild type (right panel). n = 5, error bar, S.E. B, Luc mRNA transfection was performed on TSC2 WT cells treated with 50 μm LY294002 or DMSO as control. Real time luciferase activity was recorded immediately after mRNA transfection (left panel). Relative Luc expression (3 h) after LY294002 treatment was normalized against the DMSO control (right panel). n = 5, error bar, S.E. *, p < 0.01. C, Luc mRNA bearing the Hsp70 5′ UTR was synthesized using in vitro transcription followed by 5′ end capping and 3′ end polyadenylation. mRNA transfection was performed in cells as in A. n = 5, error bar, S.E. *, p < 0.01. D, Hsp70 5′ UTR Luc mRNA transfection was performed on TSC2 WT cells treated with 50 μm LY294002 (LY) or DMSO as control. n = 5, error bar, S.E. *, p < 0.01.
FIGURE 5.
FIGURE 5.
Hsp70 5′-UTR differs from IRES in mediating cap-independent translation. A, bicistronic Luc mRNA driven by polIRES was synthesized using in vitro transcription followed by 5′ end capping and 3′ end polyadenylation. mRNA transfection was performed on TSC2 WT cells treated with 50 μm LY294002 or DMSO. Real time luciferase activity was recorded immediately after mRNA transfection. B, bicistronic Luc mRNA driven by Hsp70 5′ UTR was synthesized using in vitro transcription followed by 5′ end capping and 3′ end polyadenylation. mRNA transfection and real time luciferase measurements were the same as A. C, Luc expression after a 3-h transfection of mRNAs containing polIRES or Hsp70 5′ UTR in the presence or absence of LY294002. Error bar, S.E. D, in vitro synthesized Luc mRNA was capped at the 5′ end with a non-functional analog (ApppG) followed by 3′ end polyadenylation. mRNA transfection and real time luciferase measurements were the same as A. E, in vitro synthesized Luc mRNA bearing Hsp70 5′ UTR was capped at the 5′ end with a non-functional analog ApppG followed by 3′ end polyadenylation. mRNA transfection and real time luciferase measurements were the same as A. F, Luc expression after a 3-h transfection of ApppG-capped mRNAs in the presence or absence of LY294002. Error bar, S.E.
FIGURE 6.
FIGURE 6.
Hsp70 5′ UTR-mediated cap-independent translation is sensitive to 4E-BP1. A, in vitro synthesized Luc mRNA was capped at the 5′ end with a non-functional analog ApppG followed by 3′ end polyadenylation. mRNA transfection was performed on TSC2 WT cells pre-transfected with plasmids encoding 4E-BP1 (S37A/S46A), 4E-BP1, or GFP. Real time luciferase activity was recorded immediately after mRNA transfection. B, in vitro synthesized Luc mRNA bearing the Hsp70 5′ UTR was capped at the 5′ end with non-functional analog ApppG followed by 3′ end polyadenylation. mRNA transfection and real time luciferase measurements were the same as A. C, Luc expression after a 3-h transfection of m7G-capped mRNAs in cells transfected with plasmids encoding 4E-BP1 (S37A/S46A), 4E-BP1, or GFP. Error bar, S.E. D, in vitro synthesized Luc mRNA was capped at the 5′ end with non-functional analog ApppG followed by 3′ end polyadenylation. mRNA transfection and real time luciferase measurements were the same as A. E, in vitro synthesized Luc mRNA bearing the Hsp70 5′ UTR was capped at the 5′ end with non-functional analog ApppG followed by 3′ end polyadenylation. mRNA transfection and real time luciferase measurements were the same as A. F, Luc expression after a 3-h transfection of ApppG-capped mRNAs in cells transfected with plasmids encoding 4E-BP1 (S37A/S46A) (37/46AA), 4E-BP1, or GFP. Error bar, S.E.
FIGURE 7.
FIGURE 7.
Deficient Hsp70 translation contributes to the attenuation of stress resistance in TSC2 null cells. A, TSC2 WT, TSC2 KO, and adenovirus (AdV)-infected TSC2 KO cells were incubated at 45 °C for various times as indicated. Cell viability was measured by trypan blue counting. n = 4, error bar, S.E. *, p < 0.05 (Student's t test, two tails). B, AdV-infected TSC2 WT and TSC2 KO cells were incubated at 45 °C for various times followed by immunoblotting using antibodies as indicated. C, a schematic model for PI3K-mTORC1-controlled translational balance between cap-dependent and -independent mechanisms.
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References

    1. Ma X. M., Blenis J. (2009) Nat. Rev. Mol. Cell. Biol. 10, 307–318 - PubMed (VSports注册入口)
    1. Holcik M., Sonenberg N. (2005) Nat. Rev. Mol. Cell. Biol. 6, 318–327 - "VSports注册入口" PubMed
    1. Proud C. G. (2007) Biochem. J. 403, 217–234 - PubMed
    1. Wullschleger S., Loewith R., Hall M. N. (2006) Cell 124, 471–484 - PubMed
    1. Sarbassov D. D., Ali S. M., Sabatini D. M. (2005) Curr. Opin. Cell Biol. 17, 596–603 - PubMed

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