title: ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding.
abstract: Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.
authors: Institute of Medical Genetics, Cardiff University, Cardiff, UK.
authors: Elaine A
authors: EA
authors: Dunlop
authors: David K
authors: DK
authors: Hunt
authors: Hugo A
authors: HA
authors: Acosta-Jaquez
authors: Diane C
authors: DC
authors: Fingar
authors: Andrew R
authors: AR
authors: Tee
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000071189:Autophagy-Related Protein-1 Homolog; D018797:Cell Cycle Proteins; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D000076223:Regulatory-Associated Protein of mTOR; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D058570:TOR Serine-Threonine Kinases
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011506:Proteins; C463659:RPTOR protein, human; D000076223:Regulatory-Associated Protein of mTOR; D000071189:Autophagy-Related Protein-1 Homolog; D000076222:Mechanistic Target of Rapamycin Complex 1; D017346:Protein Serine-Threonine Kinases; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; C484754:ULK1 protein, human; C541703:Ulk2 protein, human; C448901:ribosomal protein S6 kinase, 70kD, polypeptide 1