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Nuclear PRAS40 couples the Akt/mTORC1 signaling axis to the RPL11-HDM2-p53 nucleolar stress response pathway.

Havel JJ, Li Z, Cheng D, Peng J, Fu H - Oncogene (2014)

Bottom Line: This effect is rescued by wild-type PRAS40, but not by the RPL11-binding- PRAS40T246A mutant.We found that PRAS40 negatively regulates the RPL11-HDM2-p53 nucleolar stress response pathway and suppresses induction of p53-mediated cellular senescence.These findings may help to explain the protumorigenic effect of PRAS40 and identify the PRAS40-RPL11 complex as a promising target for p53-restorative anticancer drug discovery.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA [2] Graduate Program in Molecular and Systems Pharmacology, Emory University, Atlanta, GA, USA.

ABSTRACT
The ribosomal protein (RP)-HDM2-p53 pathway has been shown to have key roles in oncogene-induced apoptosis and senescence, but the mechanism regulating this pathway remains elusive. The proline-rich Akt substrate of 40 kDa (PRAS40) has recently been identified as a binding partner and inhibitor of the mechanistic (formerly referred to as mammalian) target of rapamycin complex 1 (mTORC1). Although other inhibitors of mTORC1 are known tumor suppressors, PRAS40 promotes cell survival and tumorigenesis. Here we demonstrate that Akt- and mTORC1-mediated phosphorylation of PRAS40 at T246 and S221, respectively, promotes nuclear-specific association of PRAS40 with ribosomal protein L11 (RPL11). Importantly, silencing of PRAS40 induces upregulation of p53 in a manner dependent on RPL11. This effect is rescued by wild-type PRAS40, but not by the RPL11-binding- PRAS40T246A mutant. We found that PRAS40 negatively regulates the RPL11-HDM2-p53 nucleolar stress response pathway and suppresses induction of p53-mediated cellular senescence. This work identifies nuclear PRAS40 as a dual-input signaling checkpoint that links cell growth and proliferation to inhibition of cellular senescence. These findings may help to explain the protumorigenic effect of PRAS40 and identify the PRAS40-RPL11 complex as a promising target for p53-restorative anticancer drug discovery.

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PRAS40 associates with RPL11 in a nuclear-specific, non-mTORC1 complexA) Putative PRAS40 binding partners identified via Flag-PRAS40 IP and mass spectrometry analysis. Background-corrected average spectral counts of putative binding partners (“prey”) in each subcellular fraction were normalized to those of PRAS40 (“bait”) from the same fraction and used to determine normalized nuclear to cytoplasmic binding ratios. B) HeLa cells were transfected and fractionated as indicated. Input and Flag-immunocomplexes were resolved by SDS-PAGE and analyzed by Western Blotting (WB). C) Endogenous PRAS40 was IPed from nuclear and cytoplasmic HeLa extract using a PRAS40-specific monocolonal antibody. Non-specific, species-matched IgG or PRAS40 antibody pre-blocked with a PRAS40 peptide were used as negative controls. D) U2OS cells growing on poly-D-Lys-coated cover slips were transfected as indicated, fixed, processed for immunofluorescent detection of Flag-PRAS40, and mounted on glass slides. Images were obtained via confocal microscopy. E) HeLa nuclear extract was resolved via gel filtration chromatography using a Superose® 6 column (GE Healthcare). 0.5 mL fractions were collected.
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Figure 2: PRAS40 associates with RPL11 in a nuclear-specific, non-mTORC1 complexA) Putative PRAS40 binding partners identified via Flag-PRAS40 IP and mass spectrometry analysis. Background-corrected average spectral counts of putative binding partners (“prey”) in each subcellular fraction were normalized to those of PRAS40 (“bait”) from the same fraction and used to determine normalized nuclear to cytoplasmic binding ratios. B) HeLa cells were transfected and fractionated as indicated. Input and Flag-immunocomplexes were resolved by SDS-PAGE and analyzed by Western Blotting (WB). C) Endogenous PRAS40 was IPed from nuclear and cytoplasmic HeLa extract using a PRAS40-specific monocolonal antibody. Non-specific, species-matched IgG or PRAS40 antibody pre-blocked with a PRAS40 peptide were used as negative controls. D) U2OS cells growing on poly-D-Lys-coated cover slips were transfected as indicated, fixed, processed for immunofluorescent detection of Flag-PRAS40, and mounted on glass slides. Images were obtained via confocal microscopy. E) HeLa nuclear extract was resolved via gel filtration chromatography using a Superose® 6 column (GE Healthcare). 0.5 mL fractions were collected.

Mentions: Although mTORC1 is thought to function mainly in the cytoplasm, it has been reported that PRAS40 and other mTORC1 components are also found in the nucleus.32–41 To rigorously test the subcellular localization of PRAS40, we purified nuclei from HeLa cells by centrifuging through a sucrose cushion. The resultant nuclear fraction is void of cytosolic and endoplasmic reticulum contamination as evidenced by lack of the marker proteins GAPDH and calnexin, respectively. As expected, PRAS40 is abundant in the post-nuclear fraction. Importantly, we also observe a distinct population of PRAS40 in the nuclear fraction, albeit in lower abundance (Fig. 1A). Similarly, exogenously expressed Venus- and Flag-tagged-PRAS40 are detected in both the cytoplasm and nuclei of U2OS and HeLa cells via confocal microscopy (Fig. 1B–E and Fig. 2D). To test if an active nuclear shuttling process is responsible for the nucleocytoplasmic concentration differential of PRAS40, we treated HeLa and U2OS cells expressing Venus-PRAS40 with the nuclear export inhibitor Leptomycin B (LMB). PRAS40 accumulates in the nuclei of cells treated with LMB, but not vehicle control, suggesting that PRAS40 subcellular localization is controlled at least in part by active, dynamic nucleocytoplasmic shuttling (Fig. 1B–D). In all LMB experiments a fraction of Ven-PRAS40 remains in the cytoplasm regardless of treatment time, suggesting that only a sub-population of PRAS40 is involved in shuttling. Importantly, no difference is observed between wild-type (WT) Venus-PRAS40 and Raptor-binding Venus-PRAS40F129A in response to LMB treatment, suggesting that PRAS40 nucleocytoplasmic shuttling does not require interaction with mTORC1 (Fig. 1E).


Nuclear PRAS40 couples the Akt/mTORC1 signaling axis to the RPL11-HDM2-p53 nucleolar stress response pathway.

Havel JJ, Li Z, Cheng D, Peng J, Fu H - Oncogene (2014)

PRAS40 associates with RPL11 in a nuclear-specific, non-mTORC1 complexA) Putative PRAS40 binding partners identified via Flag-PRAS40 IP and mass spectrometry analysis. Background-corrected average spectral counts of putative binding partners (“prey”) in each subcellular fraction were normalized to those of PRAS40 (“bait”) from the same fraction and used to determine normalized nuclear to cytoplasmic binding ratios. B) HeLa cells were transfected and fractionated as indicated. Input and Flag-immunocomplexes were resolved by SDS-PAGE and analyzed by Western Blotting (WB). C) Endogenous PRAS40 was IPed from nuclear and cytoplasmic HeLa extract using a PRAS40-specific monocolonal antibody. Non-specific, species-matched IgG or PRAS40 antibody pre-blocked with a PRAS40 peptide were used as negative controls. D) U2OS cells growing on poly-D-Lys-coated cover slips were transfected as indicated, fixed, processed for immunofluorescent detection of Flag-PRAS40, and mounted on glass slides. Images were obtained via confocal microscopy. E) HeLa nuclear extract was resolved via gel filtration chromatography using a Superose® 6 column (GE Healthcare). 0.5 mL fractions were collected.
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Figure 2: PRAS40 associates with RPL11 in a nuclear-specific, non-mTORC1 complexA) Putative PRAS40 binding partners identified via Flag-PRAS40 IP and mass spectrometry analysis. Background-corrected average spectral counts of putative binding partners (“prey”) in each subcellular fraction were normalized to those of PRAS40 (“bait”) from the same fraction and used to determine normalized nuclear to cytoplasmic binding ratios. B) HeLa cells were transfected and fractionated as indicated. Input and Flag-immunocomplexes were resolved by SDS-PAGE and analyzed by Western Blotting (WB). C) Endogenous PRAS40 was IPed from nuclear and cytoplasmic HeLa extract using a PRAS40-specific monocolonal antibody. Non-specific, species-matched IgG or PRAS40 antibody pre-blocked with a PRAS40 peptide were used as negative controls. D) U2OS cells growing on poly-D-Lys-coated cover slips were transfected as indicated, fixed, processed for immunofluorescent detection of Flag-PRAS40, and mounted on glass slides. Images were obtained via confocal microscopy. E) HeLa nuclear extract was resolved via gel filtration chromatography using a Superose® 6 column (GE Healthcare). 0.5 mL fractions were collected.
Mentions: Although mTORC1 is thought to function mainly in the cytoplasm, it has been reported that PRAS40 and other mTORC1 components are also found in the nucleus.32–41 To rigorously test the subcellular localization of PRAS40, we purified nuclei from HeLa cells by centrifuging through a sucrose cushion. The resultant nuclear fraction is void of cytosolic and endoplasmic reticulum contamination as evidenced by lack of the marker proteins GAPDH and calnexin, respectively. As expected, PRAS40 is abundant in the post-nuclear fraction. Importantly, we also observe a distinct population of PRAS40 in the nuclear fraction, albeit in lower abundance (Fig. 1A). Similarly, exogenously expressed Venus- and Flag-tagged-PRAS40 are detected in both the cytoplasm and nuclei of U2OS and HeLa cells via confocal microscopy (Fig. 1B–E and Fig. 2D). To test if an active nuclear shuttling process is responsible for the nucleocytoplasmic concentration differential of PRAS40, we treated HeLa and U2OS cells expressing Venus-PRAS40 with the nuclear export inhibitor Leptomycin B (LMB). PRAS40 accumulates in the nuclei of cells treated with LMB, but not vehicle control, suggesting that PRAS40 subcellular localization is controlled at least in part by active, dynamic nucleocytoplasmic shuttling (Fig. 1B–D). In all LMB experiments a fraction of Ven-PRAS40 remains in the cytoplasm regardless of treatment time, suggesting that only a sub-population of PRAS40 is involved in shuttling. Importantly, no difference is observed between wild-type (WT) Venus-PRAS40 and Raptor-binding Venus-PRAS40F129A in response to LMB treatment, suggesting that PRAS40 nucleocytoplasmic shuttling does not require interaction with mTORC1 (Fig. 1E).

Bottom Line: This effect is rescued by wild-type PRAS40, but not by the RPL11-binding- PRAS40T246A mutant.We found that PRAS40 negatively regulates the RPL11-HDM2-p53 nucleolar stress response pathway and suppresses induction of p53-mediated cellular senescence.These findings may help to explain the protumorigenic effect of PRAS40 and identify the PRAS40-RPL11 complex as a promising target for p53-restorative anticancer drug discovery.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA [2] Graduate Program in Molecular and Systems Pharmacology, Emory University, Atlanta, GA, USA.

ABSTRACT
The ribosomal protein (RP)-HDM2-p53 pathway has been shown to have key roles in oncogene-induced apoptosis and senescence, but the mechanism regulating this pathway remains elusive. The proline-rich Akt substrate of 40 kDa (PRAS40) has recently been identified as a binding partner and inhibitor of the mechanistic (formerly referred to as mammalian) target of rapamycin complex 1 (mTORC1). Although other inhibitors of mTORC1 are known tumor suppressors, PRAS40 promotes cell survival and tumorigenesis. Here we demonstrate that Akt- and mTORC1-mediated phosphorylation of PRAS40 at T246 and S221, respectively, promotes nuclear-specific association of PRAS40 with ribosomal protein L11 (RPL11). Importantly, silencing of PRAS40 induces upregulation of p53 in a manner dependent on RPL11. This effect is rescued by wild-type PRAS40, but not by the RPL11-binding- PRAS40T246A mutant. We found that PRAS40 negatively regulates the RPL11-HDM2-p53 nucleolar stress response pathway and suppresses induction of p53-mediated cellular senescence. This work identifies nuclear PRAS40 as a dual-input signaling checkpoint that links cell growth and proliferation to inhibition of cellular senescence. These findings may help to explain the protumorigenic effect of PRAS40 and identify the PRAS40-RPL11 complex as a promising target for p53-restorative anticancer drug discovery.

Show MeSH
Related in: MedlinePlus