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. 2008 Jun 6;283(23):15619-27.
doi: 10.1074/jbc.M800723200. Epub 2008 Mar 27.

Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORC1)-mediated phosphorylation

Lifu Wang  1 , Thurl E Harris, John C Lawrence Jr
Affiliations

Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORC1)-mediated phosphorylation

Lifu Wang et al. J Biol Chem. .

Abstract

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.

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Figures

FIGURE 1.
FIGURE 1.
Identification of Ser-212 and Ser-221 as in vitro mTORC1-catalyzed phosphorylation sites in PRAS40. 3T3-L1 adipocytes were incubated with insulin (60 nm) for 30 min (A and D). A, immune complex kinase assays were performed with [γ-32P]ATP and PRAS40 wild type (wt) and the indicated mutative proteins as substrates after conducting immunoprecipitations (IP) of raptor, Akt1, and S6K1. After SDS-PAGE, a phosphor image of 32P-labeled PRAS40 (left) and immunoblots of mTOR, raptor, PRAS40, Akt1, and S6K1 (right) were prepared, and substrates were stained with 0.5% Ponceau S, as shown with substrate level. The effect on 32P incorporation into PRAS40 (corrected for substrate level) is expressed as a percentage relative to wild type (mean ± S.E. from three experiments). B, mTOR wild type, kinase-dead (S2338A, KD), and constitutive active (deleting 2433–2451, ΔRD) mutants were co-expressed with raptor in HEK293 cells. mTOR complexes were isolated with antibodies toward FLAG-tagged mTOR, and then mTOR kinase assays were performed with PRAS40 as substrate. C, PRAS40 wild type and mutants as indicated were phosphorylated in vitro by mTORC1 immune complexes (raptor IP) from insulin-stimulated HEK293 cells. After SDS-PAGE and transferring, 32P-incorporated PRAS40 was excised from the polyvinylidene difluoride membrane, digested with trypsin, and then subjected to two-dimensional phosphopeptide mapping. The results were visualized by a PhosphorImager. The pattern of migration for the three identified sites is shown in the last panel. D, phosphorylation of the indicated PRAS40 mutants in kinase assays with mTORC1 and Akt1 immune complexes were assessed by 32P incorporation. The effect on 32P incorporation into PRAS40 (corrected for substrate level) is expressed as a percentage relative to wild type (mean ± S.E. from three experiments). E, washed raptor antibody immune complexes isolated from HEK293 cells were incubated in kinase reaction mixtures containing no additions (None) for 20 min or the following: 10 μm LY294002 (LY), 5 μm rapamycin (rapa), 5 μm GST-FKBP12 (BP12), or 5 μm GST-FKBP12 plus 5 μm rapamycin (rapa + BP12). Phosphor images of 32P-labeled PRAS40 wild type and mutants of S212A and S183A/S221A are presented.
FIGURE 2.
FIGURE 2.
Phosphorylation of PRAS40 by mTORC1 in vivo. PRAS40 and mutants were transfected in HEK293 cells (A and B), and cells were labeled with [32P]orthophosphate for 3 h followed by insulin treatment (200 nm) for 30 min. 32P-Labeled PRAS40 was immunoprecipitated by HA antibody, and trypsin-digested peptides were resolved by two-dimensional phosphopeptide mapping. A, a variety of PRAS40 mutants were used to assess phosphopeptides. Major peptide spots are indicated by a–g, and phosphorylation sites are indicated in the last panel. B, in vitro and in vivo phosphorylated PRAS40 were mixed after trypsin digestion. 1, 2, and 3 indicate phosphopeptides obtained from in vitro phosphorylation by mTORC1 as described in Fig. 1C. The phosphopeptides in vivo are indicated by a–c in S202A/S203A (SS202,3AA) mutants and d–f in the T246A mutant. C, HEK293 cells were treated without or with rapamycin (rapa; 20 nm) for 20 min before insulin treatment. The circles indicate the spots that were altered after rapamycin treatment. D, HEK293T cells with lentivirus expressing either scramble or mTOR short hairpin RNA (shRNA) were transfected with PRAS40 mutant S202A/S203A. The circles indicate the spots that were altered in mTOR knockdown cells. wt, wild type.
FIGURE 3.
FIGURE 3.
Disassociation of mTOR and raptor by Triton X-100 and disruption of the TOS motif in PRAS40 are unable to eliminate phosphorylation of PRAS40. 3T3-L1 adipocytes were incubated with insulin (60 nm) for 30 min. A, mTORC1 and Akt1 were isolated from adipocyte extracts in the presence of 0.2% Tween 20 by using raptor and Akt1 antibodies. The indicated detergents (0.2% final concentration) were added to the washed complexes, which were then incubated with [γ-32P]ATP and PRAS40 or 4E-BP1. 32P-Labeled PRAS40 and 4E-BP1 were detected by phosphorimaging. The amounts of incorporated 32P in PRAS40 and 4E-BP1 relative to the level of phosphorylation in the presence of Tween 20 with insulin treatment (%TW) were determined (mean ± S.E. from three experiments). TW-20, Tween 20; TX-100, Triton X-100. B, mTORC1 and Akt1 kinase assays were conducted using PRAS40 wild type (wt) and the indicated mutative proteins as substrates. The effect on 32P incorporation into PRAS40 (corrected for substrate level) is expressed as a percentage relative to wild type (mean ± S.E. from three experiments), and a t test was conducted between wild type and mutants. IP, immunoprecipitation.
FIGURE 4.
FIGURE 4.
In addition to Thr-246, phosphorylation at Ser-221 regulates the affinity of PRAS40 binding to 14-3-3. HEK293E cells were transfected with 3 μg of HA-tagged PRAS40 plasmid or the indicated mutants. 36 h later, serum-starved cells were treated with or without rapamycin (20 nm)(A) followed by insulin (200 nm) for 30 min (A and B). Cells were lysed in 0.2% Triton X-100. A, the extracts were incubated with glutathione beads coupled with GST-tagged 14-3-3 or FKBP12. Immunoblots of HA-PRAS40 and phospho-Thr-389 of S6K1 were prepared, and the coupled proteins were stained with 0.5% Ponceau S. B, one-half of the extracts containing PRAS40 wild type and mutants were subjected to GST-14-3-3 pulldown assays, and in parallel the other half were subjected to HA immunoprecipitations. Immunoblots of HA-PRAS40 and phospho-Thr-246 of PRAS40 are presented.
FIGURE 5.
FIGURE 5.
Phosphorylation at Ser-221 modulates PRAS40 association with mTORC1 and its inhibitory effect on mTORC1 activity. A, PRAS40 wild type (wt) and various mutants as indicated were transfected into HEK293E cells. Cell extracts were prepared and immunoprecipitated (IP) with anti-HA antibodies. Endogenous mTOR and raptor co-immunoprecipitated with HA-PRAS40 were identified by immunoblotting. Co-immunoprecipitated mTOR and raptor (corrected by HA-PRAS40) from three experiments were calculated by densitometry and statistically analyzed by t test (mean ± S.E.). HEK293E cells were co-transfected with mTOR rapamycin-resistant mutant S2035W (SW), HA-S6K1 (B), or myc-4E-BP1 (C) without or with PRAS40 wild type or the indicated mutant with amounts of 0.5, 0.5, 1, and 2 μg. 36 h later, cells were treated with rapamycin (20 nm) for 20 min and then insulin (200 nm) for 30 min. B, HA-S6K1 was isolated by HA immunoprecipitation and phospho-Thr-389 of S6K1, total HA-S6K1, and HA-PRAS40 were detected by immunoblotting. C, phospho-Thr-36/45 of 4E-BP1, total 4E-BP1, and HA-PRAS40 were analyzed by immunoblotting.
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References

    1. Wullschleger, S., Loewith, R., and Hall, M. N. (2006) Cell 124 471–484 - PubMed
    1. Yang, Q., and Guan, K. L. (2007) Cell Res. 17 666–681 - PubMed
    1. Brunn, G. J., Fadden, P., Haystead, T. A., and Lawrence, J. C., Jr. (1997) J. Biol. Chem. 272 32547–32550 - PubMed
    1. Burnett, P. E., Barrow, R. K., Cohen, N. A., Snyder, S. H., and Sabatini, D. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95 1432–1437 - PMC - PubMed
    1. Schalm, S. S., and Blenis, J. (2002) Curr. Biol. 12 632–639 - PubMed

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