The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1

Abstract

The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser(183)). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe(129) to Ala). Immediately carboxyl-terminal to Phe(129) are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser(183), to Asp. PRAS40 (Ser(183)) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser(183)) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser(183)) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.

FIGURE 1. Identification of PRAS40 as a raptor-binding protein

A, isolation of the proteins interacting with raptor. HEK293 cells were transfected with FLAG-tagged raptor or the empty vector, and the cell lysates were subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitated (IP) proteins were applied to SDS-PAGE on 8% gel and visualized by silver staining. FLAG-tagged raptor, mTOR, and a 40-kDa protein (p40) are indicated by arrows. The positions of the molecular mass markers are shown in kDa. B, association of raptor and PRAS40 in the cells. HEK293 cells transfected with FLAG-tagged raptor (top) or without transfection (bottom) were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids (AA) in the presence or absence of rapamycin (Rap). The cell lysates were subjected to immunoprecipitation with the anti-FLAG (top) or anti-PRAS40 (bottom) antibodies. The immunoprecipitate by normal rabbit globulin (NRG) was employed as a control. Immunoblot was carried out with the anti-FLAG, anti-PRAS40, and anti-raptor antibodies, and the blots were scanned. A representative experiment is shown in the insets. In the upper experiments, the OD of the PRAS40 blot was divided by the OD of the corresponding FLAG-raptor blot, whereas in the lower experiments, the OD of the raptor blot was divided by the OD of the corresponding PRAS40 blot. In both sets of experiments, this ratio for the amino acid minus (AA–) condition was set to 100% and divided into the ratio for each of the other conditions. The bar graphs summarize the results of four (upper) or 12 (lower) experiments. **, a reduction as compared with AA–, p < 0.01; the plus symbol in the upper bar graph indicates that the AA+ values are less than those of the corresponding Rap values, p < 0.01; the dagger in the lower bar graph indicates that the Rap values are lower than the corresponding AA+ values, p < 0.01. C, HEK293 cells transfected with His₆-tagged PRAS40 or mock-transfected were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids in the presence or absence of rapamycin. The cell lysates were subjected to His₆ pull down with Ni²⁺-nitrilotriacetic acid beads. The proteins bound to the resin were washed with the buffer containing either 0.3% CHAPS or 1% Nonidet P-40. Immunoblot was carried out with the anti-mTOR, anti-raptor, and anti-PRAS40 antibodies.

FIGURE 2. The involvement of a PRAS40 (Phe¹²⁹), located in a TOS motif-like sequence, in PRAS40 binding to raptor

HEK293 cells transfected with Myc-tagged wild-type PRAS40 (WT), Myc-tagged PRAS40 (F129A) mutant, or the empty vector (–) were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids in the presence or absence of rapamycin. The cell lysates were subjected to immunoprecipitation with the anti-Myc antibody. Immunoblot was carried out with the anti-mTOR, anti-raptor, and anti-Myc antibodies. The longer exposure reveals the persistence of a minor amount of raptor associated with the PRAS40(F129A) polypeptide, best shown by virtue of the slightly greater sensitivity of the anti-mTOR as compared with the anti-raptor antibody.

FIGURE 3. Phosphorylation of PRAS40 by mTOR in vitro

A, mTOR immunoprecipitated from HEK293 cells was used to phosphorylate GST, GST-PRAS40, MBP-PRAS40, GST-S6K1, and GST-4E-BP1 (1 μg each) as substrates. The immunoprecipitate by the normal mouse globulin (NMG) was employed as a control. The top panel shows autoradiography, and the lower two panels show immunoblot by using the anti-mTOR and anti-GST antibodies, respectively. The white arrowheads in the bottom panel indicate GST and GST fusion proteins. The stoichiometry of ³²P incorporation (mol of P/mol of polypeptide) into the specific band was estimated as follows: GST, 0.01; GST-PRAS40, 0.14; MBP-PRAS40, 0.11; GST-S6K1, 0.20; GST-4E-BP1, 0.12. B, HEK293 cells were transfected with HA-tagged wild-type mTOR (WT), kinase-negative mTOR (N2343K) mutant (NK), or the empty vector (–). The immunoprecipitates were used to phosphorylate GST-PRAS40 and GST as substrates. The top panel shows autoradiography, and the lower two panels show immunoblot with anti-HA and anti-GST antibodies, respectively.

FIGURE 4. Identification of an in vitro mTORC1-catalyzed phosphorylation site in PRAS40

A, mutation of PRAS40 Ser¹⁸³ to Ala reduces PRAS40 phosphorylation by mTOR in vitro. mTOR immunoprecipitated from HEK293 cells was used to phosphorylate GST, GST-PRAS40, and GST-PRAS40 (S183A). The immunoprecipitate by the NMG was employed as a control. The top panel shows autoradiography, and the lower three panels show immunoblot by using the anti-mTOR, anti-GST, and phospho-specific anti-PRAS40 (Ser(P)¹⁸³) antibodies, respectively. B, mTORC1-specific phosphorylation of PRAS40 in vitro. mTORC1 and mTORC2 were immunoprecipitated from HEK293 cells by the anti-raptor and anti-rictor antibodies, respectively, and the immunoprecipitates were used to phosphorylate GST-PRAS40 in vitro. The immunoprecipitate by the NRG was employed as a control. Immunoblot was carried out with the anti-mTOR, anti-rictor, anti-raptor, anti-PRAS40 (Ser(P)¹⁸³), and anti-GST antibodies.

FIGURE 5. Phosphorylation of PRAS40 at Ser¹⁸³ in vivo

A, effects of amino acids and rapamycin on phosphorylation of PRAS40 at Ser¹⁸³. HEK293 cells were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids (AA) in the presence or absence of rapamycin (Rap). Where indicated, the amino acid mixture readded lacked branched-chain amino acids (ΔBC). The cell lysates were subjected to immunoprecipitation with the anti-PRAS40 antibody. The immunoprecipitate by the NRG was employed as a control. Immunoblot was carried out with the anti-PRAS40 (Ser(P)¹⁸³), anti-PRAS40, anti-raptor, and anti-mTOR antibodies, and the blots were scanned. A representative experiment is shown in the inset. The OD of the PRAS40 (Ser(P)¹⁸³) blot was divided by the OD of the corresponding PRAS40 blot. This ratio for the AA+ condition was set to 100% and divided into the ratio for each of the other conditions. The bar graphs summarize the results of three experiments. The double asterisks indicate a reduction as compared with AA+, p < 0.01. B, effect of ATP depletion on phosphorylation of PRAS40 at Ser¹⁸³. HEK293 cells were deprived of serum and incubated in the absence of glucose (Gluc) alone or with added 2-deoxyglucose (2-DG). The cell lysates were subjected to immunoprecipitation with the anti-PRAS40 antibody. The immunoprecipitate by the NRG was employed as a control. Immunoblot was carried out with the anti-PRAS40 (Ser(P)¹⁸³), anti-PRAS40, and anti-raptor antibodies. The aliquots of the cell lysates were subjected to immunoblot by the anti-AMPK (Thr(P)¹⁷²) antibody. A representative experiment is shown in the insets. The OD of the PRAS40 (Ser(P)¹⁸³) blot was divided by the OD of the corresponding PRAS40 blot. This ratio for the Gluc+ condition was set to 100% and divided into the ratio for each of the other conditions. The bar graphs summarize the results of four experiments. The double or single asterisks indicate a reduction as compared with Gluc+; p < 0.01 or p < 0.05, respectively. C, an active, rapamycin-resistant mTOR mutant overcomes rapamycin-induced dephosphorylation of PRAS40 (Ser¹⁸³) in vivo. HEK293 cells co-transfected with Myc-tagged PRAS40 and either FLAG-tagged wild-type mTOR (WT), rapamycin-resistant mTOR (S2035T) mutant (ST), or rapamycin-resistant/kinase-negative mTOR (S2035T/N2343K) mutant (ST/NK) were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids in the presence or absence of rapamycin. The cell lysates were subjected to immunoprecipitation with the anti-Myc antibody. Immunoblot was carried out with the anti-PRAS40 (Ser(P)¹⁸³), anti-PRAS40 (Thr(P)²⁴⁶), and anti-Myc antibodies. The aliquots of the cell lysates were subjected to immunoblot by the anti-FLAG antibody. D, mutation of PRAS40 (Ser¹⁸³) to Asp abolishes association with raptor. HEK293 cells transfected with Myc-tagged PRAS40, its mutants, or the empty vector were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids in the presence or absence of rapamycin. The cell lysates were subjected to immunoprecipitation with the anti-Myc antibody. Immunoblot was carried out with the anti-mTOR, anti-raptor, and anti-Myc antibodies.

FIGURE 6. Effects of TSC1/2 and Rheb on phosphorylation of PRAS40 at Ser¹⁸³

A, effect of TSC1/2 on phosphorylation of PRAS40. HEK293 cells cotransfected with Myc-tagged PRAS40 and either FLAG-tagged TSC1/2 or the empty vector were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids. The cells transfected with the empty vectors were employed as a control. The cell lysates were subjected to immunoprecipitation with the anti-Myc antibody. Immunoblot was carried out with the anti-PRAS40 (Ser(P)¹⁸³) and anti-Myc antibodies. The aliquots of the cell lysates were subjected to immunoblot by the anti-FLAG antibody. B, effect of Rheb on phosphorylation of PRAS40. HEK293 cells cotransfected with FLAG-tagged PRAS40 and either Myc-tagged Rheb or the empty vector were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids. The cells transfected with the empty vectors were employed as a control. The cell lysates were subjected to immunoprecipitation with the anti-FLAG antibody. Immunoblot was carried out with the anti-PRAS40 (Ser(P)¹⁸³) and anti-FLAG antibodies. The aliquots of the cell lysates were subjected to immunoblot by the anti-Myc antibody.

FIGURE 7. Competitive effects among mTORC1 substrates for raptor binding and mTORC1 phosphorylation in vivo

A, effect of overexpression of an mTORC1 substrate on phosphorylation of the other two substrates. In the experiments shown in the four panels on the upper left, HEK293 cells were co-transfected with FLAG-tagged 4E-BP1 and either empty vector, Myc-tagged PRAS40 wild type, or the PRAS40 mutants indicated. The cells were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids. The aliquots of the cell lysates were subjected to immunoblot by the anti-Myc antibody to verify comparable expression of the PRAS40 variants. An anti-FLAG immunoprecipitate was immunoblotted for 4E-BP1 (Thr(P)^37/46) and FLAG. The upper three panels show the immunoblots from a representative experiment. The 4E-BP1 (Thr(P)^37/46) blots were scanned, and the OD was divided by the OD of the corresponding FLAG-4E-BP1 blot. This ratio for the FLAG immunoprecipitate from cells that did not receive Myc-PRAS40 (vector only) was set to 100% and divided into the ratio obtained for each of the other FLAG immunoprecipitates. The bar graph summarizes the results from three experiments. The double or single asterisks indicate a reduction as compared with vector only; p < 0.01 or p <0.05, respectively. In the three panels shown at the lower left, HA-tagged S6K1 was co-expressed with either empty vector, Myc-tagged PRAS40 wild type, or the PRAS40 mutants indicated. The aliquots of the cell lysates were subjected to immunoblot by the anti-Myc antibody to verify comparable expression of the PRAS40 variants. An anti-HA immunoprecipitate was immunoblotted for S6K1 (Thr(P)³⁸⁹) and HA, and the blots were scanned. The OD of the S6K1 (Thr(P)³⁸⁹) blot was divided by the OD of the corresponding HA-S6K1 blot. This ratio for the HA immunoprecipitate from cells that did not receive Myc-PRAS40 (vectoronly) was set to 100% and divided into the ratio obtained for each of the other HA immunoprecipitates. These values of one experiment are shown below the bottom panel; a second experiment gave similar results. The panels on the right show the effects of 4E-BP1 (upper) and kinase-negative S6K1 (lower) co-expression on PRAS40 (Ser¹⁸³) phosphorylation; cells were treated in a manner similar to the experiments on the left. B, effect of RNA interference-induced PRAS40 depletion on the phosphorylation of endogenous S6K1 and 4E-BP1. HEK293 cells (left) and HeLa cells (right) transfected with indicated siRNA were deprived of serum, further incubated without amino acids, and stimulated by the readdition of amino acids in the presence or absence of rapamycin. The cell lysates were subjected to immunoblot by the anti-PRAS40, anti-S6K1 (Thr(P)³⁸⁹), anti-S6K1, anti-4E-BP1 (Ser(P)⁶⁵), anti-4E-BP1 (Thr(P)^37/46), and anti-β-actin antibodies. C, effect of overexpression of 4E-BP1 and S6K1 on the association of endogenous PRAS40 with overexpressed recombinant raptor. HEK293 cells co-transfected with FLAG-tagged raptor and either GST, GST-fused 4E-BP1, or GST-fused S6K1 were deprived of serum and further incubated without amino acids. The cell lysates were subjected to immunoprecipitation with the anti-FLAG antibody. Immunoblot was carried out with the anti-FLAG, anti-GST, and anti-PRAS40 antibodies. The aliquots of the cell lysates were subjected to immunoblot by the anti-GST antibody.