Peptide combinatorial libraries identify TSC2 as a death-associated protein kinase (DAPK) death domain-binding protein and reveal a stimulatory role for DAPK in mTORC1 signaling.
Death-associated protein kinase (DAPK) is a multidomain enzyme that plays a central role in autophagic and apoptotic signaling, although the protein-protein interactions regulating DAPK functions are not well defined. Peptide aptamer libraries were used to identify the tumor suppressor protein tuberin (TSC2) as a novel DAPK death domain-binding protein, and we evaluated whether DAPK is a positive or negative effector of the TSC2-regulated mammalian target of rapamycin (mTORC1) signaling pathway. Binding studies using death domain miniproteins in vitro and deletion analysis in vivo determined that the death domain of DAPK is the major site for the interaction with TSC2. Recombinant DAPK phosphorylates TSC2 in vitro, and DAPK kinase activity is stimulated by growth factor signaling. Transfection of DAPK promotes phosphorylation of TSC2 in vivo, whereas short interfering RNA-mediated attenuation of DAPK reduces growth factor-stimulated phosphorylation of TSC2. DAPK-dependent phosphorylation leads to TSC1-TSC2 complex dissociation, and consequently manipulation of DAPK by transfection or short interfering RNA demonstrated that DAPK is a positive regulator of mTORC1 in response to growth factor activation. Epistatic studies suggest that DAPK functions downstream from the RAS-MEK-ERK and phosphatidylinositol 3-kinase-AKT growth factor signaling pathways. DAPK(+/-) mouse embryo fibroblasts have attenuated mTORC1 signaling compared with DAPK+/+ counterparts, and overexpression of DAPK in DAPK(+/-) MEFs stimulates mTORC1 activity. These data uncover a novel interaction between DAPK and TSC2 proteins that has revealed a positive link between growth factor stimulation of DAPK and mTORC1 signaling that may ultimately affect autophagy, cell survival, or apoptosis.
Stevens
Craig
C
Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom and the CRUK Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom.
Lin
Yao
Y
Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom and the CRUK Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom.
Harrison
Ben
B
Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom and the CRUK Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom.
Burch
Lindsay
L
Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom and the CRUK Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom.
Ridgway
Rachel A
RA
Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom and the CRUK Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom.
Sansom
Owen
O
Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom and the CRUK Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom.
Hupp
Ted
T
Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom and the CRUK Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom. Electronic address: ted.hupp@ed.ac.uk.
D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D064550:Death-Associated Protein Kinases; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D019151:Peptide Library; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D046912:Multiprotein Complexes; D019151:Peptide Library; D011506:Proteins; D034741:RNA, Small Interfering; C000624653:TSC2 protein, human; D014157:Transcription Factors; C000624654:Tsc2 protein, mouse; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D064550:Death-Associated Protein Kinases; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases