PMID- 19272448 OWN - NLM STAT- MEDLINE DCOM- 20090603 LR - 20211203 IS - 1873-3913 (Electronic) IS - 0898-6568 (Linking) VI - 21 IP - 7 DP - 2009 Jul TI - Mammalian target of rapamycin complex 1-mediated phosphorylation of eukaryotic initiation factor 4E-binding protein 1 requires multiple protein-protein interactions for substrate recognition. PG - 1073-84 LID - 10.1016/j.cellsig.2009.02.024 [doi] AB - The mammalian target of rapamycin (mTOR) pathway is implicated in a number of human diseases, but the pathway details are not fully understood. Here we elucidate the interactions between various proteins involved in mTOR complex 1 (mTORC1). An in vitro mTORC1 kinase assay approach was used to probe the role of the mTORC1 component Raptor and revealed that certain Raptor mutations disrupt binding to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and prevent its subsequent phosphorylation by mTOR. Interestingly, we show that a point mutation in the highly conserved Raptor RNC domain still allows binding to mTOR but prevents Raptor association and mTOR-dependent phosphorylation of 4E-BP1, indicating that this Raptor domain facilitates substrate recognition by mTORC1. This Raptor RNC domain mutant also dominantly inhibits mTORC1 signalling to 4E-BP1, S6K1 and HIF1alpha in vivo. We further characterise the functions of the mTORC1 signalling (TOS) and RAIP motifs of 4E-BP1, which are involved in substrate recognition by Raptor and phosphorylation by mTORC1. We show that an mTOR mutant, L1460P, responds to insulin even in nutrient-deprived conditions and is resistant to inhibition by inactive RagB-RagC heterodimers that mimic nutrient withdrawal suggesting that this region of mTOR is involved in sensing the permissive amino acid input. We found that FKBP38 inhibits mTOR(L1460P), while the mTOR(E2419K) kinase domain mutant was resistant to FKBP38 inhibition. Finally, we show that activation of mTORC1 by both Rheb and RhebL1 is impaired by FKBP38. Our work demonstrates the value of an in vitro mTORC1 kinase assay to characterise cell signalling components of mTORC1 involved in recognition and phosphotransfer to mTORC1 substrates. FAU - Dunlop, Elaine A AU - Dunlop EA AD - Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, Wales, UK. FAU - Dodd, Kayleigh M AU - Dodd KM FAU - Seymour, Lyndsey A AU - Seymour LA FAU - Tee, Andrew R AU - Tee AR LA - eng GR - 06-0914/AICR_/Worldwide Cancer Research/United Kingdom PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20090309 PL - England TA - Cell Signal JT - Cellular signalling JID - 8904683 RN - 0 (Carrier Proteins) RN - 0 (Mutant Proteins) RN - 0 (Neuropeptides) RN - 0 (Phosphoproteins) RN - 0 (Transcription Factors) RN - EC 2.7.- (Protein Kinases) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.1.1 (mTOR protein, mouse) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) RN - EC 3.6.5.2 (ras Proteins) RN - EC 5.2.1.- (Tacrolimus Binding Proteins) SB - IM MH - Amino Acid Motifs MH - Amino Acid Sequence MH - Amino Acid Substitution MH - Animals MH - Carrier Proteins/metabolism MH - Cell Line MH - Genes, Dominant MH - Humans MH - Mice MH - Models, Biological MH - Molecular Sequence Data MH - Mutant Proteins/metabolism MH - Neuropeptides/metabolism MH - Phosphoproteins/chemistry/*metabolism MH - Phosphorylation MH - Protein Binding MH - Protein Kinases/metabolism MH - Signal Transduction MH - Substrate Specificity MH - TOR Serine-Threonine Kinases MH - Tacrolimus Binding Proteins/metabolism MH - Transcription Factors/antagonists & inhibitors/chemistry/*metabolism MH - ras Proteins/metabolism EDAT- 2009/03/11 09:00 MHDA- 2009/06/06 09:00 CRDT- 2009/03/11 09:00 PHST- 2009/01/08 00:00 [received] PHST- 2009/02/20 00:00 [revised] PHST- 2009/02/22 00:00 [accepted] PHST- 2009/03/11 09:00 [entrez] PHST- 2009/03/11 09:00 [pubmed] PHST- 2009/06/06 09:00 [medline] AID - S0898-6568(09)00098-9 [pii] AID - 10.1016/j.cellsig.2009.02.024 [doi] PST - ppublish SO - Cell Signal. 2009 Jul;21(7):1073-84. doi: 10.1016/j.cellsig.2009.02.024. Epub 2009 Mar 9.