Target of Rapamycin (TOR)-signaling and RAIP Motifs Play Distinct Roles in the Mammalian TOR-dependent Phosphorylation of Initiation Factor 4E-binding Protein 1

Abstract

The translational repressor protein eIF4E-binding protein 1 (4E-BP1, also termed PHAS-I) is regulated by phosphorylation through the rapamycin-sensitive mTOR (mammalian target of rapamycin) pathway. Recent studies have identified two regulatory motifs in 4E-BP1, an mTOR-signaling (TOS) motif in the C terminus of 4E-BP1 and an RAIP motif (named after its sequence) in the N terminus. Other recent work has shown that the protein raptor binds to mTOR and 4E-BP1. We show that raptor binds to full-length 4E-BP1 or a Cterminal fragment containing the TOS motif but not to an N-terminal fragment containing the RAIP motif. Mutation of several residues within the TOS motif abrogates binding to raptor, indicating that the TOS motif is required for this interaction. 4E-BP1 undergoes phosphorylation at multiple sites in intact cells. The effects of removal or mutation of the RAIP and TOS motifs differ. The RAIP motif is absolutely required for phosphorylation of sites in the N and C termini of 4E-BP1, whereas the TOS motif primarily affects phosphorylation of Ser-64/65, Thr-69/70, and also the rapamycin-insensitive site Ser-101. Phosphorylation of N-terminal sites that are dependent upon the RAIP motif is sensitive to rapamycin. The RAIP motif thus promotes the mTOR-dependent phosphorylation of multiple sites in 4E-BP1 independently of the 4E-BP1/raptor interaction.Eukaryotic initiation factor (eIF)-4E 1 plays a key role in mRNA translation in mammalian cells and in its regulation (1). eIF4E binds the 5Ј-cap structure of the mRNA, which contains the 7-methyl-GTP (m 7 GTP) moiety. It also interacts with the scaffold protein eIF4G and thereby recruits 40 S ribosomal subunits to the mRNA (2). A large body of evidence shows that eIF4E plays key roles in cell proliferation (1) and cell survival/ apoptosis (3). For example, overexpression of eIF4E can transform cells (4,5), and many human tumor cells express high levels of this protein (6). Nuclear eIF4E seems to be important in the export of certain mRNAs to the cytoplasm such as that for cyclin D1 (7), which is important in cell cycle progression.The transforming abilities of eIF4E may be linked to this effect (8,9).The availability of eIF4E for binding to eIF4G is regulated by its interaction with heat-stable proteins termed eIF4E-binding proteins (4E-BPs). 4E-BP1 is by far the best understood of these. Phosphorylation of 4E-BP1 leads to its release from eIF4E, allowing eIF4E to form initiation complexes with eIF4G (1). Expression of 4E-BP1 reverses the transforming ability of increased levels of eIF4E (10). Phosphorylation of 4E-BP1 induced by insulin or other agents is blocked by the immunosuppressant rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR) (11). 4E-BP1 undergoes phosphorylation at up to seven sites in intact cells; phosphorylation is complex, hierarchical, and, at some sites, is required for the subsequent modification of others (12-15).Regulatory motifs have been identified in the N and C termini of 4E-BP1. The N terminus contains the so-c...

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

“…Our work showing partial reversal of sT-mediated foci formation indicates that the 4E-BP1 phosphorylation site S83 contributes to that MCV sT cell transformation activity. sT transformation is eliminated by a raptor-binding mutant of 4E-BP1 (38)(39)(40), suggesting that mTOR and CDK1 cooperatively promote sT-induced cell transformation. These findings hint at the possibility that this residue is also permissive for cell transformation induced by other oncoproteins that promote mitogenesis.…”

“…4A). Unlike 4E-BP1.S83A, phosphorylation-defective and raptor-binding mutants (T37A/T46A and I15A/F114A) efficiently inhibited eIF4G binding to eIF4E and in vitro cap-dependent translation (32,(38)(39)(40), suggesting that S83 phosphorylation does not play a significant role in general cap-dependent translation regulation.…”

The eukaryotic initiation factor 4E-binding proteins and apoptosis

“…70 However, strikingly, phosphorylation of this fragment within cells is greatly reduced versus intact 4E-BP1, suggesting that the amino terminal region contains a feature required for normal phosphorylation of 4E-BP1. Further analysis revealed that this involves the four amino-acid motif Arg-Ala-Ile-Pro (RAIP), which is required for phosphorylation of Thr37/46/70 and Ser65 70,71 and appears to mediate a rapamycin-insensitive input from mTOR, which is dependent upon amino acids (especially leucine). A major consequence of this modification is that eIF4E becomes sequestered by D4E-BP1 (Figure 2), leading to decreased levels of eIF4F complexes and inhibition of cap-dependent translation (while IRES-driven translation can continue).…”

Altered protein synthesis is a trigger for long-term memory formation

“…Akt phosphorylates TSC2, decreasing its Rheb-GAP function (Garami et al, 2003, Inoki et al, 2005a, Inoki et al, 2005b) that results in mTOR activation. mTOR exists in two distinct complexes, one of which contains the adaptor protein Raptor that recruits S6K1/2 and 4E-BP, the two mTOR effectors involved in translation control (Beugnet et al, 2003, Choi et al, 2003, Schalm et al, 2003. The drug rapamycin disrupts mTOR-Raptor interactions, thereby preventing mTOR from phosphorylating S6K1/2 and 4E-BP (Kim et al, 2002, Oshiro et al, 2004.…”