Tuberous sclerosis-2 (TSC2) regulates the stability of death-associated protein kinase-1 (DAPK) through a lysosome-dependent degradation pathway.
We previously identified a novel interaction between tuberous sclerosis-2 (TSC2) and death-associated protein kinase-1 (DAPK), the consequence being that DAPK catalyses the inactivating phosphorylation of TSC2 to stimulate mammalian target of rapamycin complex 1 (mTORC1) activity. We now report that TSC2 binding to DAPK promotes the degradation of DAPK. We show that DAPK protein levels, but not gene expression, inversely correlate with TSC2 expression. Furthermore, altering mTORC1 activity does not affect DAPK levels, excluding indirect effects of TSC2 on DAPK protein levels through changes in mTORC1 translational control. We provide evidence that the C-terminus regulates TSC2 stability and is required for TSC2 to reduce DAPK protein levels. Importantly, using a GTPase-activating protein-dead missense mutation of TSC2, we demonstrate that the effect of TSC2 on DAPK is independent of GTPase-activating protein activity. TSC2 binds to the death domain of DAPK and we show that this interaction is required for TSC2 to reduce DAPK protein levels and half-life. Finally, we show that DAPK is regulated by the lysosome pathway and that lysosome inhibition blocks TSC2-mediated degradation of DAPK. Our study therefore establishes important functions of TSC2 and the lysosomal-degradation pathway in the control of DAPK stability, which taken together with our previous findings, reveal a regulatory loop between DAPK and TSC2 whose balance can either promote: (a) TSC2 inactivation resulting in mTORC1 stimulation, or (b) DAPK degradation via TSC2 signalling under steady-state conditions. The fine balance between DAPK and TSC2 in this regulatory loop may have subtle but important effects on mTORC1 steady-state function.
University of Edinburgh, Institute of Genetics and Molecular Medicine, Edinburgh, UK.
Yao
Y
Lin
Paul
P
Henderson
Susanne
S
Pettersson
Jack
J
Satsangi
Ted
T
Hupp
Craig
C
Stevens
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology; D064550:Death-Associated Protein Kinases; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics; D046912:Multiprotein Complexes; D020125:Mutation, Missense / Q000502:physiology; D009479:Neuropeptides / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D011506:Proteins / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000235:genetics; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D017384:Sequence Deletion / Q000502:physiology; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D014162:Transfection; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D007976:Leupeptins; D046912:Multiprotein Complexes; D009479:Neuropeptides; D061988:Proteasome Inhibitors; D011506:Proteins; C490211:RHEB protein, human; D034741:RNA, Small Interfering; C463659:RPTOR protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; C490213:Rheb protein, mouse; C540162:Rptor protein, mouse; C000624653:TSC2 protein, human; C000624654:Tsc2 protein, mouse; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D002738:Chloroquine; C579096:DAPK1 protein, human; C579106:Dapk1 protein, mouse; D064550:Death-Associated Protein Kinases; D000076222:Mechanistic Target of Rapamycin Complex 1; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D046988:Proteasome Endopeptidase Complex; D020559:Monomeric GTP-Binding Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D020123:Sirolimus