title: DAPK2 is a novel regulator of mTORC1 activity and autophagy.
abstract: Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca(2+)-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction. DAPK2 knockdown reduced autophagy triggered either by amino acid deprivation or by increases in intracellular Ca(2+) levels. At the molecular level, DAPK2 depletion interfered with mTORC1 inhibition caused by these two stresses, as reflected by the phosphorylation status of mTORC1 substrates, ULK1 (unc-51-like kinase 1), p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1. An increase in mTORC1 kinase activity was also apparent in unstressed cells that were depleted of DAPK2. Immunoprecipitated mTORC1 from DAPK2-depleted cells showed increased kinase activity in vitro, an indication that DAPK2 regulation of mTORC1 is inherent to the complex itself. Indeed, we found that DAPK2 associates with components of mTORC1, as demonstrated by co-immunoprecipitation with mTOR and its complex partners, raptor (regulatory-associated protein of mTOR) and ULK1. DAPK2 was also able to interact directly with raptor, as shown by recombinant protein-binding assay. Finally, DAPK2 was shown to phosphorylate raptor in vitro. This phosphorylation was mapped to Ser721, a site located within a highly phosphorylated region of raptor that has previously been shown to regulate mTORC1 activity. Thus, DAPK2 is a novel kinase of mTORC1 and is a potential new member of this multiprotein complex, modulating mTORC1 activity and autophagy levels under stress and steady-state conditions.
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Y
authors.initials: Y
authors.lastname: Ber
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: R
authors.initials: R
authors.lastname: Shiloh
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Y
authors.initials: Y
authors.lastname: Gilad
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: N
authors.initials: N
authors.lastname: Degani
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: S
authors.initials: S
authors.lastname: Bialik
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: A
authors.initials: A
authors.lastname: Kimchi
mesh_terms: D001343:Autophagy / Q000502:physiology*; D045744:Cell Line, Tumor; D064550:Death-Associated Protein Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000378:metabolism*; D010766:Phosphorylation; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D014162:Transfection
chemical_list: D046912:Multiprotein Complexes; C579104:DAPK2 protein, human; D064550:Death-Associated Protein Kinases; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases