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
Authors: Yao Lin
Owen Sansom
Ben Harrison
Lindsay Burch
Rachel A Ridgway
Ted Hupp
Craig Stevens
Journal: Journal of Biological Chemistry (2009)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Death-associated protein kinase (DAPK); TSC2; mTORC1 signaling pathway; protein-protein interactions; autophagy
Affiliations: 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., 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: .

Abstract:  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. 

Retracted: No

PubPeer comments:
 Figure 5. Much more similar than expected. ![file] , 
 #1 The authors agree that these data look very similar. The original blots are no longer available for resolving this issue so the original data from the paper was downloaded from the journal web site. The purpose of this figure 5 was to define the effects of DAPK manipulation on S6 phosphorylation and S6K phosphorylation. Actin was used as the loading control. If the actin loading control images were duplicated this would not alter the conclusion of either figure because the more robust loading control for changes in S6 or S6K phosphorylation are blotting for total S6 protein and total S6K protein (as in Fig 5A and 5B lanes 3 vs 4 or Fig 5B lanes 2 vs 3). Neverthless, we think these blots are not duplicated images. The attached analysis (below in png format) appears to reveal differences in both blots which would lead us to conclude that they are not identical. https: pubpeer.com storage image-1642072712447.png ,