Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins

Abstract

Signaling through the mammalian target of rapamycin (mTOR) controls cell size and growth as well as other functions, and it is a potential therapeutic target for graft rejection, certain cancers, and disorders characterized by inappropriate cell or tissue growth. mTOR signaling is positively regulated by hormones or growth factors and amino acids. mTOR signaling regulates the phosphorylation of several proteins, the best characterized being ones that control mRNA translation. Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) undergoes phosphorylation at multiple sites. Here we show that amino acids regulate the N-terminal phosphorylation sites in 4E-BP1 through the RAIP motif in a rapamycin-insensitive manner. Several criteria indicate this reflects a rapamycin-insensitive output from mTOR. In contrast, the insulin-stimulated phosphorylation of the C-terminal site Ser64/65 is generally sensitive to rapamycin, as is phosphorylation of another well-characterized target for mTOR signaling, S6K1. Our data imply that it is unlikely that mTOR directly phosphorylates Thr69/70 in 4E-BP1. Although 4E-BP1 and S6K1 bind the mTOR partner, raptor, our data indicate that the outputs from mTOR to 4E-BP1 and S6K1 are distinct. In cells, efficient phosphorylation of 4E-BP1 requires it to be able to bind to eIF4E, whereas phosphorylation of 4E-BP1 by mTOR in vitro shows no such preference. These data have important implications for understanding signaling downstream of mTOR and the development of new strategies to impair mTOR signaling.

FIG. 1.

Amino acid withdrawal and rapamycin have distinct effects on 4E-BP1 phosphorylation. (A) The graphic depicts the regulatory RAIP and TOS motifs in 4E-BP1, the eIF4E binding site, and the phosphorylation sites studied here (showing the differences in numbering between human and rodent). (B and C) HEK293 (B) or CHO (C) cells were first starved of serum overnight and then, where indicated, also starved of amino acids (−AA) for 1 h or treated with rapamycin and/or insulin. In some cases (−AA/+AA), amino acids were resupplied to cells that had been deprived of them for 60 min. Times are indicated in minutes. Samples of cell lysate were analyzed by Western blotting with the indicated antisera for 4E-BP1 (total BP1), specific phosphorylation sites in 4E-BP1, or S6K1 (multiple arrows indicate that several differently phosphorylated species are resolved) or an antibody that recognizes S6K1 only when it is phosphorylated at Thr389 or at Thr421 and Ser424 (the two bands correspond to the 70- and 85-kDa species of S6K1). (D) CHO cells were starved of serum overnight and, where indicated, were also starved of amino acids (-AA for times shown) or resupplied with amino acids (−AA/+AA; times shown). Where added, rapamycin was added for 40 min before lysis of the cells. (E) CHO cells, which had been starved of serum and, in some cases, also of amino acids (−AA) or that had been resupplied with amino acids (-AA/+AA), as described above, were in some cases (+) treated with insulin. Samples of cell lysate were analyzed by Western blotting using anti-(P)PKB (Thr308) or anti-(P)Erk, which recognizes the phosphorylated active forms of both Erk1 and Erk2.

FIG. 2.

Sequence and regulation of hamster 4E-BP1. (A) Amino acid sequences of rat (Rattus norvegicus [Rn]) and Chinese hamster (Cricetulus griseus [Cg]) 4E-BP1 and of rat 4E-BP2 (C). The RAIP and TOS motifs are underlined, the eIF4E-binding motif is italicized, and phosphorylation sites in 4E-BP1 or the corresponding residues in 4E-BP2 are shown in boldface. Residues that are different in hamster 4E-BP1 compared to those of the rat protein are also in boldface and are highlighted by an arrowhead. (B) HEK293 cells were transfected with vectors for hamster 4E-BP1 or human 4E-BP1, both with N-terminal his/myc tags, or with a vector for rat 4E-BP1 with C-terminal tags. After overnight serum starvation, cells were treated with rapamycin and, where indicated, insulin. Samples of lysate were analyzed by SDS-PAGE and Western blotting using anti-myc (for the total 4E-BP1) or the indicated phosphospecific antisera. (C) CHO cells were treated as indicated (using the notation employed for the other panels), and samples of lysate were subjected to affinity chromatography on m⁷GTP-Sepharose prior to analysis of the bound material by SDS-PAGE and Western blotting using the indicated antisera. The signal for eIF4E serves as a loading control.

FIG. 3.

Effects of PI 3-kinase inhibitors or PTEN on the phosphorylation of specific sites in 4E-BP1. (A and B) CHO cells were starved of serum and then, in some cases (−AA), were also starved of amino acids (60 min). Amino acids were then added back to some plates for 30 min (−AA/+AA). Where indicated, cells were treated with LY294002 or wortmannin (WM) (at the indicated concentrations, for 40 min) and then, where indicated, with insulin (Ins). Samples of lysates were analyzed by Western blotting using the indicated antisera for 4E-BP1 or specific phosphorylation sites in 4E-BP1 (A) or for (P)PKB (Thr308) or S6K1 (B), as indicated. (C) Serum-starved HEK293 cells were treated with insulin or with PI 3-kinase inhibitors as described for panels A and B for CHO cells. (D) HEK293 cells were transfected with a vector encoding rat 4E-BP1, plus vectors for wild-type PTEN, the indicated PTEN point mutants, or the empty vector (pcDNA). Samples of lysate (20 μg of protein) were analyzed by SDS-PAGE and Western blotting using antisera for 4E-BP1, specific sites in 4E-BP1, PTEN, or phosphorylated PKB, as indicated.

FIG. 4.

Effects of proteins that regulate mTOR signaling on the phosphorylation of 4E-BP1. (A) HEK293 cells were transfected with vectors for rat 4E-BP1 and TSC1 and TSC2, TSC1 and the SATA mutant of TSC2, or the corresponding empty vectors. Cells were starved of serum overnight and then treated with insulin (Ins). Samples of cell lysate were analyzed by SDS-PAGE and Western blotting with the indicated antisera (anti-FLAG was used to detect overexpressed TSC1 and TSC2). (B) HEK293 cells were transfected with vectors for rat 4E-BP1 and either Rheb or the corresponding empty vector. Cells were starved of serum overnight (S-St), deprived of amino acids (−AA), or transferred to the medium used for amino acid starvation but containing amino acids (+AA). Samples of cell lysate were analyzed with the indicated antisera (anti-myc was used to detect both 4E-BP1 and Rheb). S6(P) indicates an antibody that detects rpS6 when it is phosphorylated at Ser235.

FIG. 5.

Approaches to impair mTOR signaling. (A) HeLa cells were transfected with 60 nM siRNA directed against mTOR (mTOR-1) or were mock transfected. Forty-eight hours after the first transfection, cells were starved of serum for 4 h and, in some cases, were then treated with insulin (Ins) (100 nM, 30 min). Lysates were then analyzed by SDS-PAGE and Western blotting for actin (normalization control), mTOR, S6K1, total 4E-BP1, or the indicated phosphorylation sites in 4E-BP1. (B) HEK293 cells were transfected with vectors encoding wild-type or kinase-dead FLAG-mTOR along with rat 4E-BP1. Twenty-four hours later, cells were starved of serum overnight and, in some cases, were then treated with insulin (100 nM, 30 min). Lysates were analyzed as described for panel A (but not here for actin levels). (C) Targeted ES cells (53) were treated with HTNC or (as negative control) phosphate-buffered saline (PBS). After the times indicated, lysates were prepared and analyzed by SDS-PAGE and Western blotting using the indicated antisera.

FIG. 6.

Features of 4E-BP2 and 4E-BP1 required for efficient phosphorylation in vivo. (A) HEK293 cells were transfected with vectors for wild-type 4E-BP2 or a mutant in which the RAIP motif was altered to AAAP. Cells were starved of serum overnight and, in some cases, also of amino acids (60 min). Where indicated, cells were treated with rapamycin or insulin. Samples were analyzed by SDS-PAGE and Western blotting using anti-myc (to detect 4E-BP2) or anti-4E-BP1 (Thr36/45 phosphospecific antibody). (B and C) HEK293 cells were transfected with vectors for WT 4E-BP1 or the LM/AA mutant, each as N-terminally his/myc-tagged proteins. After serum starvation overnight, cells were treated with rapamycin or insulin as indicated. (B) Samples of lysate were subjected to affinity chromatography on m⁷GTP-Sepharose prior to analysis of the bound material by SDS-PAGE and Western blotting using the indicated antisera. (C) Samples of lysate were analyzed by SDS-PAGE and Western blotting using anti-myc and the indicated phosphospecific antisera for 4E-BP1. For (P)Ser64, both the endogenous (endog.; serves as a positive control) and recombinant (his/myc) 4E-BP1 proteins can be seen. (D to F) HEK293 cells were transfected with vectors encoding FLAG-tagged mTOR and myc-raptor. (D) Western blot demonstrating that mTOR and myc-raptor are efficiently expressed. (E) GST-4E-BP1 and eIF4E were mixed and pulled down on glutathione-Sepharose (pull down), and the bound material was analyzed by SDS-PAGE and Western blotting using antisera for eIF4E and 4E-BP1. Purified eIF4E and GST-4E-BP1 (input) were run as controls. (F) Recombinant 4E-BP1 was incubated with immunoprecipitated mTOR/raptor in the presence of [γ-³²P]ATP, MnCl₂, and, where indicated, recombinant eIF4E and/or GST-FKBP12 plus rapamycin.

FIG. 7.

Model for regulation of the phosphorylation of 4E-BP1, integrating the findings of the present report. Our data are consistent with mTOR directly phosphorylating Thr37/46. This requires the RAIP motif in 4E-BP1, which may interact with a (putative) partner protein, X, that could also recruit mTOR to 4E-BP1. The kinase acting at Thr70 is unknown but appears unlikely to be mTOR itself, based on insensitivity to LY294002 and the lack of effect of deletion of the most C-terminal residues of mTOR. The identity of the kinase acting at Ser65 is also unknown, but this phosphorylation event is stimulated by insulin (Ins) and blocked by rapamycin (rapa). The kinase might be mTOR itself. In cells where the phosphorylation of Thr70 is low under serum-starved conditions and stimulated by insulin, this effect too is blocked by rapamycin. This suggests rapamycin may interfere with signaling from mTOR to 4E-BP1 rather than with their direct phosphorylation by mTOR. Amino acids primarily influence the phosphorylation of Thr37/46. The requirement for binding of 4E-BP1 to eIF4E for its efficient phosphorylation in vivo is not shown, for reasons of clarity.