Báo cáo khoa học: Tuberous sclerosis-2 (TSC2) regulates the stability of death-associated protein kinase-1 (DAPK) through a lysosome-dependent degradation pathway doc

Tuberous sclerosis-2 (TSC2) regulates the stability of death-associated protein kinase-1 (DAPK) through a lysosome-dependent degradation pathway Yao Lin1, Paul Henderson1,2, Susanne Pettersson1, Jack Satsangi1, Ted Hupp1 and Craig Stevens1 University of Edinburgh, Institute of Genetics and Molecular Medicine, UK Department of Child Life and Health, University of Edinburgh, UK Keywords DAPK; degradation; lysosome; mTORC1; TSC2 Correspondence C Stevens, University of Edinburgh, Institute of Genetics and Molecular Medicine, Edinburgh, EH4 2XR, UK Fax: +44 131 651 1085 Tel: +44 131 651 1025 E-mail: craig.stevens@ed.ac.uk (Received 28 July 2010, revised October 2010, accepted 11 November 2010) doi:10.1111/j.1742-4658.2010.07959.x 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 (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 Structured digital abstract l MINT-8057232: DAPK (uniprotkb:P53355) physically interacts (MI:0915) with TSC2 (uniprotkb:P49815) by anti tag coimmunoprecipitation (MI:0007) l MINT-8057213: TSC1 (uniprotkb:Q92574) physically interacts (MI:0914) with DAPK (uniprotkb:P53355) and TSC2 (uniprotkb:P49815) by anti bait coimmunoprecipitation (MI:0006) l MINT-8057200: TSC1 (uniprotkb:Q92574) physically interacts (MI:0915) with TSC2 (uniprotkb:P49815) by anti bait coimmunoprecipitation (MI:0006) Abbreviations DAPK, death-associated protein kinase-1; GAP, GTPase-activating protein; IFN, interferon; 3-MA, 3-methyladenine; MEF, mouse embryonic fibroblast; mTORC1, mammalian target of rapamycin complex 1; siRNA, short interfering RNA; TNF, tumour necrosis factor; TSC2, tuberous sclerosis-2 354 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Lin et al TSC2 promotes the degradation of DAPK Introduction Death-associated protein kinase-1 (DAPK) is the prototypic member of a family of death-related kinases that includes DAPK-1-related protein (DRP-1, also named DAPK-2), Zipper interacting kinase (also named DAPK-3), DRAK1 (DAPK kinase-related apoptosis-inducing protein kinase 1) and DRAK2 [1] DAPK is a large 160 kDa serine ⁄ threonine protein kinase composed of several functional domains including a kinase domain, a calmodulin regulatory domain, eight consecutive ankyrin repeats, two putative nucleotide-binding domains (P-loops), a cytoskeletal binding domain and a death domain [1] Recent advances have established an important role for DAPK in a diverse range of signal transduction pathways including growth factor signalling, apoptosis, autophagy and membrane blebbing [2,3] DAPK was originally identified as a factor that regulates apoptosis in response to the death-inducing cytokine interferon (IFN)-c [4], and has subsequently been shown to function as a positive mediator of apoptosis induced by various stimuli including the transforming oncogenes c-myc and E2F1, transforming growth factor-beta and ceramide [2] In accordance with its proapoptotic activity, evidence suggests that DAPK functions as a tumour suppressor having been shown to suppress transformation in vitro [5] and block tumour metastasis in murine models [6] Furthermore, DAPK gene expression is frequently lost in human cancers due to promoter hypermethylation [7] and a loss of DAPK gene expression correlates with the development of chronic lymphocytic leukaemia [8] DAPK has also recently been shown to play a role in survival pathways reflected in its autophagy-signalling activity [9,10] and its ability to counter tumour necrosis factor (TNF)-mediated apoptosis [11,12] Post-transcriptional mechanisms regulating protein translation, stabilization and turnover are also critical for modulating DAPK activities For example, translational repression of DAPK occurs in response to IFN-c treatment mediated by the IFN-c-activated inhibitor of translation complex [13] Central to protein stability, the control of protein degradation by the ubiquitin– proteasome system is a key regulator of many cellular processes [14] In this pathway, proteins are tagged with ubiquitin through the concerted action of E1-ubiquitin-activating enzyme, E2-conjugating enzyme, E3-ubiquitin ligase enzyme and finally degraded by the proteasome [14] To date, it has been demonstrated that the post-translational control of DAPK protein levels are regulated by at least three distinct E3-ubiquitin ligase family members [11,15–19] In addition, work from our own group has shown that the lysosomal protease cathepsin B negatively regulates protein levels of DAPK [12] and that a small, alternatively spliced form of DAPK (s-DAPK) destabilizes DAPK in a proteasome-independent manner [20] In a previous study [21], we performed a proteininteraction screen to identify novel DAPK deathdomain-interacting proteins and identified tuberous sclerosis-2 (TSC2) as one such protein We demonstrated that the consequence of this interaction between TSC2 and DAPK was phosphorylation of TSC2 by DAPK This led to inactivation of the TSC complex to stimulate mTORC1 activity in an epidermal growth factor-dependent manner [21] The TSC complex, formed by two proteins – tuberous sclerosis-1 (TSC1) and TSC2 – is a major regulator of the mTORC1-signalling pathway [22], with mutations in either the TSC1 or TSC2 gene, resulting in the autosomal-dominant disease tuberous sclerosis TSC2 contains a GTPase-activating protein (GAP) domain in its C-terminus, and through GTP hydrolysis of the small protein Rheb antagonizes the mTORC1-signalling pathway [23] TSC2 is phosphorylated and regulated by various kinases to integrate signals such as nutrient availability, energy, hormones and growth factors with mTORC1 activity [24] mTORC1 directly controls cell growth by regulating the phosphorylation of components of the protein translational machinery In particular, phosphorylation and activation of eukaryotic initiation factor 4E binding protein-1 (4EBP-1) and ribosomal protein S6 kinase-1 (S6K) are stimulated by serum, insulin and growth factors in an mTORC1-dependent manner [24] The pathway that regulates autophagy also acts through mTORC1 Autophagy is a membrane system that sequesters proteins and organelles into a structure called the autophagosome, which then fuses with a lysosome where cargo is degraded The resulting degradation products are then released back into the cytosol where they can be recycled to sustain the growth requirements of the cell The lipophilic macrolide antibiotic rapamycin forms a complex with FK506-binding protein 12, which then binds to and inactivates mTORC1, leading to an upregulation of autophagy [25] Thus mTORC1 acts as a central regulator balancing anabolic and catabolic pathways within the cell [24] In this report, we extend our previous studies [12,20,21] and describe a novel function for TSC2 in promoting the lysosome-dependent degradation of DAPK We suggest that the TSC2–DAPK protein complex forms a regulatory feedback loop whose balance may influence the extent of mTORC1 signalling by either stimulating TSC2 inactivation via DAPK FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 355 TSC2 promotes the degradation of DAPK Y Lin et al activation in epidermal growth factor-treated cells, or stimulating DAPK degradation via TSC2 signalling under steady-state conditions Results DAPK protein but not mRNA levels inversely correlate with TSC2 expression We recently identified TSC2 as a novel DAPK deathdomain-interacting protein [21] During the course of that study, we observed that the abundance of DAPK inversely correlated with TSC2 expression (Stevens, C; Lin, Y; Harrison, B; Burch, L; Ridgway, R.A; Sansom, O and Hupp, T unpublished results) To further investigate this observation, we first evaluated the effect of overexpressing increasing amounts of TSC2 on the levels of endogenous DAPK protein TSC2 overexpression led to a significant reduction in the level of DAPK protein in a dose-dependent manner (Fig 1A and quantified in Fig 1B) Because the overexpression of TSC2 reduced DAPK protein levels, we anticipated that silencing of TSC2 expression with short interfering RNA (siRNA) would have the opposite effect and lead to an increase in DAPK Indeed, DAPK protein was increased in TSC2 siRNA-treated cells compared with control siRNA-treated cells (Fig 1C) As expected, inhibition of TSC2 function and concomitant activation of mTORC1 resulted in an increase in phosphorylation of S6K on T389 (Fig 1C) To add physiological relevance to these findings, we took advantage of TSC2 mouse embryonic fibroblasts (MEFs) deficient for TSC2 TSC2-null MEFs undergo early-onset senescence because of a dramatic p53-dependent induction of p21, thus to circumvent senescence, p53 is also knocked-out in these cells Immunoblotting of DAPK protein from TSC2 (+ ⁄ +) and TSC2 () ⁄ )) MEFs revealed that DAPK protein was elevated in TSC2 () ⁄ )) cells (Fig 1D) As expected, loss of TSC2 function results in activation of mTORC1 and increased phosphorylation of S6K on T389 (Fig 1D) To confirm that loss of TSC2 was responsible for the higher level of DAPK protein observed in TSC2 () ⁄ )) MEFs, we reconstituted TSC2 by transfection and determined the levels of DAPK by western blot Overexpression of TSC2 led to a clear reduction in DAPK protein to a level comparable with TSC2 (+ ⁄ +) control cells (Fig 1E), confirming that the elevated levels of DAPK observed are a direct result of TSC2 loss DAPK activity is auto-inhibited by auto-phosphorylation on Ser308 within its calmodulin regulatory-binding domain [1] To determine whether the TSC2 regulatory effect on DAPK is important functionally, we assessed DAPK activity by 356 immunoblotting DAPK protein from TSC2 (+ ⁄ +) and TSC2 () ⁄ )) MEFs with phospho-Ser308 antibodies and compared the abundance of the phosphorylated inactive form relative to the total level of DAPK Again, DAPK protein was elevated in TSC2 () ⁄ )) cells compared with TSC2 (+ ⁄ +) control cells (Fig 1F), however, a decrease in the level of phosphorylated DAPK was observed in TSC2 () ⁄ )) cells (Fig 1F), thus both DAPK level and activity are elevated in the absence of TSC2 Next, we assessed whether TSC2 was mediating its effect on DAPK at the transcriptional level Real-time PCR revealed that TSC2 overexpression did not significantly alter the level of DAPK mRNA (Fig 1G), demonstrating that the coincident reduction in DAPK protein observed (Fig 1H) is independent of changes in DAPK gene expression Taken together, these results demonstrate that TSC2 can regulate the abundance and activity of DAPK via a post-transcriptional mechanism DAPK protein levels are not affected by mTORC1 activity Because mTORC1 is an important regulator of protein translation, it was necessary to determine whether the effect of TSC2 on DAPK might be indirect, through changes in mTORC1 activity To examine whether mTORC1 was involved, we used the mTORC1 inhibitor rapamycin Rapamycin treatment efficiently inhibited mTORC1 translational activity, as measured by phosphorylation of S6K on T389 and phosphorylation of the S6K-substrate ribosomal protein S6 on S235 ⁄ 236, but had no effect on the levels of DAPK protein (Fig 2A) The elevated level of DAPK protein observed in TSC2 () ⁄ )) MEFs may result from increased mTORC1 activity in these cells (Fig 1D), therefore we investigated the effect of rapamycin on DAPK level in TSC2 () ⁄ )) cells A timecourse of rapamycin treatment resulted in the efficient inhibition of mTORC1 activity, as measured by the phosphorylation of S6 on S235 ⁄ 236 (Fig 2B), however no change in DAPK levels was observed (Fig 2B), suggesting that the increased level of DAPK in these cells is not a result of increased mTORC1 activity Several studies have recently demonstrated that rapamycin does not inhibit all functions of mTORC1 [26], therefore the effects of Rheb and the mTORC1 component Raptor were also evaluated in TSC2 () ⁄ )) cells First, we investigated the effect of Rheb overexpression in serum-starved cells Rheb overexpression resulted in a pronounced increase in phosphorylation of S6 on S235 ⁄ 236, but no change in the level of DAPK was observed (Fig 2C) To exclude any direct effects of FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Lin et al TSC2 promotes the degradation of DAPK A B DAPK Actin FLAG–TSC2 (µg) 0.25 0.5 0.75 1.0 1.0 Relative DAPK level FLAG–TSC2 2.0 ** 0.8 0.6 0.4 0.2 0.0 0.25 0.50 0.75 1.0 2.0 Concentration of FLAG–TSC2 (µg) C D TSC2 TSC2 DAPK DAPK S6K P-T389 S6K P-T389 S6K S6K Actin siRNA + siRNA TSC2 – Actin – + E TSC2 MEF (+/+) (–/–) F TSC2 (–/–) MEF FLAG – DAPK Actin FLAG–TSC2 DAPK P-S308 DAPK TSC2 Actin TSC2 MEF + (+/+) (–/–) NS DAPK : actin mRNA ratio G H 1.5 FLAG–TSC2 1.0 DAPK 0.5 Actin FLAG–TSC2 (µg) 0.0 0 2.0 2.0 Concentration of FLAG–TSC2 (µg) Fig DAPK protein level inversely correlates with TSC2 expression (A) A549 cells were transfected with increasing amounts of FLAG– TSC2 Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK and actin or FLAG antibodies to detect TSC2 (B) Quantification of DAPK protein levels from (A) Results are reported as the mean ± SD (**P < 0.01, n = 3) (C) HEK293 cells were transfected with either TSC2 siRNA or nonspecific control siRNA, as indicated, for 48 h Following transfection, cell lysates were prepared and immunoblotted with antibodies to detect endogenous TSC2, DAPK, S6K P-T389, S6K and actin (D) Cell lysates were prepared from TSC2 (+ ⁄ +) and TSC2 () ⁄ )) MEFs and immunoblotted with antibodies to detect endogenous TSC2, DAPK, S6K P-T389, S6K and actin (E) TSC2 () ⁄ )) MEFs were transfected with FLAG–TSC2 Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK and actin or FLAG antibodies to detect TSC2 (F) Cell lysates were prepared from TSC2 (+ ⁄ +) and TSC2 () ⁄ )) MEFs an assessed for DAPK activity by immunoblotting with antibodies to detect P-Ser308 DAPK, DAPK and actin (G) A549 cells were transfected with vector control or FLAG–TSC2 and the mRNA levels of DAPK determined by real-time PCR (NS, not significant, n = 3) (H) A549 cells were transfected with vector control or FLAG–TSC2 Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK and actin or FLAG antibodies to detect TSC2 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 357 TSC2 promotes the degradation of DAPK Y Lin et al A Serum-starved C DAPK DAPK S6K P-T389 FLAG–RHEB S6K S6 P-S235/236 S6 P-S235/236 S6 S6 Actin Actin – RAPA FLAG-RHEB RAPA + – – + – + – + + TSC2 (–/–) MEF B D Serum-starved DAPK DAPK S6 P-S235/236 RHEB S6 S6 P-S235/236 Actin S6 RAPA (h) 16 Actin Serum-starved E siRNA siRNA RHEB + – – + DAPK F HA Serum-starved S6 P-S235/236 S6 P-S235/236 (short exposure) Raptor S6 S6 P-S235/236 Actin – DAPK S6 – HA-Raptor HA-Raptor MT4 + – + + – – + + – + Rapamycin – – – – + + Actin siRNA + – siRNA Raptor – + Fig DAPK protein level is not affected by mTORC1 activity (A) HEK293 cells were treated with 100 nM rapamycin for h Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK, S6K P-T389, S6K, S6 P-S235 ⁄ 236, S6 and actin (B) TSC2 () ⁄ )) MEFs were treated with 100 nM rapamycin for the indicated time Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK, S6 P-S235 ⁄ 236, S6 and actin (C) HEK293 cells were transfected with control vector or FLAG–Rheb Cells were serum-starved and treated with 100 nM rapamycin for h where indicated Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK, S6 P-S235 ⁄ 236, S6 and actin or FLAG antibodies to detect Rheb (D) TSC2 () ⁄ )) MEFs were transfected with either Rheb siRNA or nonspecific control siRNA, as indicated, for 48 h Following transfection, cell lysates were prepared and immunoblotted with antibodies to detect endogenous Rheb, DAPK, S6 P-S235 ⁄ 236, S6 and actin (E) HEK293 cells were transfected with HA–Raptor or HA–Raptor mutant Cells were serum-starved and treated with 100 nM rapamycin for h where indicated Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK, S6 P-S235 ⁄ 236, S6 and actin or HA antibodies to detect Raptor (F) TSC2 () ⁄ )) MEFs were transfected with either Raptor siRNA or nonspecific control siRNA, as indicated, for 48 h Following transfection, cell lysates were prepared and immunoblotted with antibodies to detect endogenous Raptor, DAPK, S6 P-S235 ⁄ 236, S6 and actin 358 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Lin et al Rheb on DAPK level, Rheb was overexpressed in serum-starved cells in the presence of rapamycin Under these conditions, Rheb expression did not lead to a change in the phosphorylation of S6 on S235 ⁄ 236 or in DAPK level (Fig 2C) To confirm these findings, we investigated the effect of Rheb depletion with siRNA on DAPK levels in serum-starved TSC2 () ⁄ )) cells Decreased Rheb expression correlated with partial inhibition of mTORC1 activity and reduction of S6 S235 ⁄ 236 phosphorylation, likely because of functional redundancy between Rheb and RhebL1 [27], and again no change in DAPK level was observed (Fig 2D) Next, we investigated the effects of overexpressing Raptor or a mutant Raptor (Raptor mutant 4) that interferes with mTORC1 substrate recognition [28] In cells growing in full serum, expression of Raptor mutant resulted in decreased phosphorylation of S6 on S235 ⁄ 236, confirming its ability to dominantly impair mTORC1 activity (Fig 2E) In serum-starved cells, Raptor or Raptor mutant overexpression failed to alter phosphorylation of S6 on S235 ⁄ 236 (Fig 2E) Similarly, expression of Raptor or Raptor mutant in serum-starved cells treated with rapamycin resulted in no observable difference in S6 phosphorylation (Fig 2E) Importantly, no change in the level of DAPK protein was observed under any of these conditions (Fig 2E) To further confirm these findings, we investigated the effect of Raptor depletion with siRNA on DAPK levels in serum-starved TSC2 () ⁄ )) cells Decreased Raptor expression correlated with efficient inhibition of mTORC1 activity and reduction of S6 phosphorylation on S235 ⁄ 236, however no change in DAPK levels was observed (Fig 2F) Together, these results clearly demonstrate that DAPK levels are not regulated by mTORC1 activity, thus excluding indirect effects of TSC2 on DAPK protein levels through changes in mTORC1 translational control, and suggest that it is the stability of DAPK protein that is altered by TSC2 TSC2 GAP activity is not required to reduce DAPK protein levels The C-terminal domain of TSC2, which contains the GAP domain, is critical for its correct activity [23] and has recently been shown to be important for control of the protein’s stability [29] Therefore, to gain some mechanistic insight into how TSC2 might exert its effect on DAPK, we created a TSC2 truncation mutant lacking its C-terminus, TSC2 (1–1516), and compared its effect on DAPK levels with a well-characterized patient-derived GAP-dead missense mutant TSC2 (N1693K) [23] (Fig 3A) Consistent with a TSC2 promotes the degradation of DAPK previous study [29], cycloheximide treatment revealed that TSC2 is a short-lived protein with a half-life of  h under normal growth conditions (Fig 3B and quantified in Fig 3C) The GAP-dead mutant TSC2 (N1693K) exhibited a half-life similar to wildtype TSC2 (Fig 3B and quantified in Fig 3C) By contrast, TSC2 (1–1516) exhibited a significantly increased stability with a half-life of  h (Fig 3B and quantified in Fig 3C), confirming the importance of this domain in the regulation of TSC2 stability To investigate further the mechanism through which TSC2 regulates DAPK stability we compared the levels of DAPK in TSC2 () ⁄ )) MEFs reconstituted with TSC2, TSC2 (1–1516) or TSC2 (N1693K) Reconstitution of TSC2 or TSC2 (N1693K) resulted in a pronounced reduction in DAPK level that was not observed in cells reconstituted with TSC2 (1–1516) (Fig 3D) As expected, cells reconstituted with TSC2 exhibited reduced mTORC1 activity, as measured by phosphorylation of S6 on S235 ⁄ 236, whereas cells reconstituted with TSC2 (1–1516) or TSC2 (N1693K) exhibited no change in mTORC1 activity (Fig 3D) Importantly, the observation that TSC2 (N1693K) retained the ability to efficiently reduce DAPK levels demonstrates that TSC2 effect on DAPK is independent of its GAP activity, and is consistent with our previous observation that DAPK levels not correlate with changes in mTORC1 translational activity Furthermore, TSC2 (1–1516) failed to reduce DAPK levels when overexpressed in HEK293 cells, in stark contrast to the reduction observed when TSC2 or TSC2 (N1693K) were overexpressed (Fig 3E,G) The impaired ability of TSC2 (1–1516) to reduce DAPK levels is not due to altered affinity because TSC2, TSC2 (N1693K) and TSC2 (1–1516) immunoprecipitate with DAPK to a similar degree (Fig 3F,H) These results collectively demonstrate that the C-terminus of TSC2 is important for regulating its stability, and suggest that TSC2 can regulate the stability of interacting proteins such as DAPK via a mechanism that is dependent on its C-terminal domain, but independent is of its GAP activity The TSC2 (1–1516) truncation mutant forms a complex with TSC1 and DAPK Our previous findings could be explained by altered binding of the TSC2 (1–1516) truncation mutant with TSC1, therefore it was necessary to compare the relative binding of endogenous TSC1 with TSC2 and TSC2 (1– 1516) For this, we transfected cells with FLAG–TSC2 or FLAG–TSC2 (1–1516), cell extracts were then prepared and endogenous TSC1 immunoprecipitated FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 359 TSC2 promotes the degradation of DAPK Y Lin et al C A 1.0 N1693K TSC1 binding LZ CC GAP CC Relative TSC2 level TSC2 1807 TSC2 (1–1516) 1516 B FLAG–TSC2 (1–1516) FLAG–TSC2 (Wild-type) FLAG–TSC2 (N1396K) 0.8 0.6 0.4 ** 0.2 FLAG–TSC2 0.0 Actin *** Cycloheximide (h) FLAG–TSC2 (N1693K) D TSC2 (–/–) MEF Actin FLAG FLAG–TSC2 (1–1516) DAPK Actin Chx (h) S6 P-S235/236 S6 E Actin TSC2 TSC2 (1–1516) DAPK FLAG FLAG–TSC2 – FLAG–TSC2 (1–1516) – FLAG–TSC2 (N1693K) – Actin HA-FLAG–DAPK FLAG–TSC2 FLAG–TSC2 (1–1516) + – + + – + G H FLAG F IP: HA FLAG Actin HA DAPK + + FLAG–TSC2 – + + + – HA + – – – + FLAG Lysate HA – FLAG–TSC2 (N1693K) Actin + – FLAG IP: HA HA TSC2 TSC2 (1–1516) DAPK HA-FLAG–DAPK FLAG–TSC2 FLAG–TSC2 (1–1516) – – + + – – – + – + – – + Actin FLAG–TSC2 + + + – FLAG–TSC2 (N1693K) – HA-FLAG–DAPK + – + Fig The C-terminus of TSC2 but not GAP-activity is required for reduction of DAPK protein level (A) TSC2 is comprised of an N-terminal domain that mediates its interaction with TSC1 and a C-terminal GAP (GTPase-activating protein) domain LZ, leucine zipper; CC, coiled coil A TSC2 truncation mutant lacking the GAP domain TSC2 (1–1516) and a GAP-dead missense mutant TSC2 (N1693K) are described (B) HEK293 cells were transfected with FLAG–TSC2, FLAG–TSC2 (N1693K) or FLAG–TSC2 (1–1516) Following transfection, cells were treated with cycloheximide for the indicated times Cell lysates were prepared and immunoblotted with antibodies to detect actin or FLAG antibodies to detect TSC2 (C) Quantification of TSC2 protein levels from Fig 3B Results are reported as the mean ± SD (N1693K vs 1–1516 ***P < 0.001; wild-type vs 1–1516 **P < 0.01, n = 3) (D) TSC2 () ⁄ )) MEFs were transfected with FLAG–TSC2, FLAG–TSC2 (1–1516) or FLAG–TSC2 (N1693K) Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK, S6 P-S235 ⁄ 236, S6 and actin or FLAG antibodies to detect TSC2 (E) HEK293 cells were transfected with dual tagged HA–FLAG–DAPK in combination with FLAG– TSC2 or FLAG–TSC2 (1–1516) as indicated Following transfection, cell lysates were prepared and immunoblotted with antibodies to detect actin or FLAG antibodies to detect DAPK and TSC2 (F) HEK293 cells were transfected with dual tagged HA–FLAG–DAPK in combination with FLAG–TSC2 or FLAG–TSC2 (1–1516) as indicated Cell lysates were prepared and DAPK was immunoprecipitated with HA antibodies Bound proteins were eluted and detected by FLAG immunoblot Lysates were immunoblotted for actin (G) HEK293 cells were transfected with HA–DAPK in combination with FLAG–TSC2 or FLAG–TSC2 (1–1516) as indicated Following transfection cell lysates were prepared and immunoblotted with antibodies to detect actin, HA antibodies to detect DAPK or FLAG antibodies to detect TSC2 (H) HEK293 cells were transfected with FLAG–TSC2, FLAG–TSC2 (N1693K) and HA–DAPK as indicated Cell lysates were prepared and exogenous DAPK was immunoprecipitated with HA–specific antibodies Bound proteins were eluted and immunoblotted with HA antibodies to detect DAPK or FLAG antibodies to detect TSC2 Direct lysate was immunoblotted with HA antibodies to detect DAPK, FLAG antibodies to detect TSC2 and actin with anti-TSC1-specific IgG Both TSC2 and TSC2 (1–1516) coprecipitated with TSC1 to a similar extent (Fig 4A) These results are consistent with a previous 360 study that mapped the TSC1-binding domain to the N-terminal region of TSC2 [30] (Fig 3A) To further demonstrate that TSC2 (1–1516) interacts normally FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Lin et al TSC2 promotes the degradation of DAPK A TSC2 TSC2 (1–1516) IB: FLAG IP: TSC1 IB: TSC1 TSC2 TSC2 (1–1516) IB: FLAG IB: TSC1 Lysate IB: Actin FLAG–TSC2 FLAG–TSC2 (1–1516) B – + + – TSC2 FLAG TSC1 TSC2 (1–1516) TSC1 FLAG Actin FLAG–TSC2 + + FLAG–TSC1 – + C TSC2 TSC2 (1–1516) IB: FLAG IB: TSC1 IP: TSC1 with TSC1, we overexpressed FLAG–TSC2 or FLAG– TSC2 (1–1516) in the presence or absence of FLAG– TSC1 Consistent with previous reports, TSC1 overexpression resulted in increased stability of TSC2 [22] and also an equivalent increase in the stability of TSC2 (1–1516) (Fig 4B) We have shown previously that DAPK overexpression results in only partial disruption of the TSC1–TSC2 complex [21] We therefore anticipated that DAPK would form a complex with both TSC1 and TSC2 proteins and we wished to determine whether TSC2 (1–1516) retained the ability to form a complex with both DAPK and TSC1 To evaluate this, cells were transfected with FLAG–TSC2 or FLAG–TSC2 (1–1516), cell extracts were then prepared and TSC1 immunoprecipitated with antiTSC1-specific IgG Once again, TSC2 and TSC2 (1– 1516) coprecipitated with TSC1 to a similar degree (Fig 4C) and endogenous DAPK was also coprecipitated with each of the TSC complexes (Fig 4C) Consistent with our previous observations, overexpression of TSC2, but not TSC2 (1–1516), resulted in a reduction in DAPK protein level (Fig 4C) Taken together, these results exclude the possibility that the loss of activity towards DAPK observed with TSC2 (1–1516) results from altered binding to TSC1 and demonstrate that DAPK binds to the TSC complex IB: DAPK TSC2 TSC2 (1–1516) Death domain binding is required for TSC2 to reduce DAPK protein levels IB: FLAG IB: TSC1 Lysate IB: DAPK IB: Actin FLAG–TSC2 FLAG–TSC2 (1–1516) – + + – Fig The TSC2 truncation mutant forms a complex with TSC1 and DAPK (A) HEK293 cells were transfected with FLAG–TSC2 or FLAG–TSC2 (1–1516) Cell lysates were prepared and endogenous TSC1 was immunoprecipitated with TSC1-specific antibodies Bound proteins were eluted and immunoblotted with antibodies to detect TSC1 or FLAG antibodies to detect TSC2 Direct lysate was immunoblotted with antibodies to detect TSC1 and actin or FLAG antibodies to detect TSC2 (B) HEK293 cells were transfected with FLAG–TSC2 or FLAG–TSC2 (1–1516) in combination with FLAG– TSC1 Cell lysates were prepared and immunoblotted with antibodies to detect actin or FLAG antibodies to detect TSC1 and TSC2 (C) HEK293 cells were transfected with FLAG–TSC2 or FLAG– TSC2 (1–1516) Cell lysates were prepared and endogenous TSC1 was immunoprecipitated with TSC1-specific antibodies Bound proteins were eluted and immunoblotted with antibodies to detect DAPK and TSC1 or FLAG antibodies to detect TSC2 Direct lysate was immunoblotted with antibodies to detect DAPK, TSC1 and actin or FLAG antibodies to detect TSC2 We have previously shown that the death domain is the major determinant for the interaction of DAPK with TSC2 [21] Therefore, we asked whether the effect of TSC2 on DAPK was a direct result of protein binding To explore this possibility, we made use of a mutant DAPK (DAPK 1–1313) lacking the C-terminal death domain (Fig 5A) First, to confirm our previous results, we overexpressed DAPK or DAPK (1–1313) in cells and evaluated the binding of endogenous TSC2 by immunoprecipitation Immunoprecipitation confirmed that TSC2 interacts with DAPK, but not the DAPK mutant lacking the death domain (Fig 5B) Next, we overexpressed DAPK or DAPK (1–1313) in the presence or absence of coexpressed TSC2 Although TSC2 overexpression led to a marked reduction in the level of DAPK, it had no effect on the DAPK (1–1313) mutant lacking the TSC2-binding domain (Fig 5C), indicating that binding to the death domain is required for TSC2 to affect DAPK levels A recent study demonstrated that the death domain module is important for the control of DAPK stability [18], therefore to gain further insight into the effect of TSC2 on DAPK levels we FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 361 TSC2 promotes the degradation of DAPK A Kinase Y Lin et al P-loops CaM D Cyto Ankyrin DD DAPK 378 641 835 378 641 835 FLAG–TSC2 1431 HA–DAPK DAPK (1–1313) 1313 Actin Chx (h) B 8 TSC2 IP: HA E HA FLAG–TSC2 TSC2 Lysate HA–DAPK (1–1313) HA Actin Chx (h) Actin HA–DAPK – HA–DAPK (1–1313) + Relative DAPK level Actin – + + + – + DAPK DAPK (+TSC2) DAPK 1–1313 DAPK 1–1313 (+TSC2) 1.0 HA + – – F FLAG–TSC2 – + – + – C HA–DAPK HA–DAPK (1–1313) FLAG–TSC2 0.8 * 0.6 0.4 * 0.2 0.0 Cycloheximide (h) Fig Death domain binding is required for TSC2 to reduce DAPK protein levels (A) DAPK is comprised of an N-terminal kinase domain, a calmodulin-binding domain, eight ankyrin repeats, two nucleotide binding domains (P-loops), a cytoskeleton binding domain and a C-terminal death domain A mutant DAPK lacking the death domain DAPK (1–1313) is described (B) A549 cells were transfected with HA–DAPK or HA–DAPK (1–1313) Cell lysates were prepared and DAPK was immunoprecipitated with HA–specific antibodies Bound proteins were eluted and immunoblotted with antibodies to detect TSC2 or HA antibodies to detect DAPK Direct lysates were also immunoblotted with antibodies to detect TSC2 and actin or HA antibodies to detect DAPK (C) A549 cells were transfected with HA–DAPK or HA–DAPK (1–1313) in the presence or absence of FLAG–TSC2 Cell lysates were prepared and immunoblotted with antibodies to detect actin, HA antibodies to detect DAPK or FLAG antibodies to detect TSC2 (D) HEK293 cells were transfected with HA–DAPK in the presence or absence of FLAG–TSC2, followed by treatment with cycloheximide for the indicated times Cell lysates were prepared and immunoblotted with antibodies to detect actin, HA antibodies to detect DAPK or FLAG antibodies to detect TSC2 (E) HEK293 cells were transfected with HA–DAPK (1–1313) in the presence or absence of FLAG–TSC2 followed by treatment with cycloheximide for the indicated times Cell lysates were prepared and immunoblotted with antibodies to detect actin, HA antibodies to detect DAPK or FLAG antibodies to detect TSC2 (F) Quantification of DAPK protein levels from (D) and (E) Results are reported as the mean ± SD (*P < 0.05, n = 3) evaluated DAPK and DAPK (1–1313) protein halflives in HEK293 cells treated with the protein synthesis inhibitor cycloheximide Cycloheximide treatment revealed that DAPK (1–1313) exhibited an extended half-life compared with DAPK (Fig 5D,E, left-hand panels and quantified in Fig 5F) Interestingly, the DAPK (1–1313) mutant also exhibited an extended half-life compared with DAPK when introduced into TSC2 () ⁄ )) MEFs (Fig S1A,B) These results are consistent with a recent study demonstrating that other factors in addition to TSC2 can control the stability of DAPK via the death domain [18] Importantly, however, the stability of the wild-type DAPK protein is increased in TSC2 () ⁄ )) cells compared with HEK293 cells, confirming that endogenous 362 TSC2 is playing an active role in regulating the level of overexpressed DAPK proteins (Fig S1A,B) Next, we compared the half-life of DAPK in the presence or absence of coexpressed TSC2 and observed that the stability of DAPK was significantly reduced when TSC2 was coexpressed (Fig 5D, right-hand panels and quantified in Fig 5F) By contrast, TSC2 overexpression had no effect on the stability of the DAPK (1–1313) mutant lacking the TSC2-binding domain (Fig 5E, right-hand panels and quantified in Fig 5F) These results clearly demonstrate that the effect of TSC2 on DAPK is dependent on binding to the death domain of DAPK and are consistent with our previous study showing that the DAPK (1–1313) mutant has lost the ability to stimulate mTORC1 activity [21] FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Lin et al TSC2 promotes the degradation of DAPK A B DAPK His-Ub pulldown p53 HA–DAPK Actin MG132 – + C Lysate DAPK Leupeptin E-64 d Chloro – + – – – – – + – – – + E DAPK Actin Chloro – + – – + – + – – + – – – D 2.0 1.5 1.0 0.5 0.0 Leupeptin + MG132 MG132 Relative DAPK level Actin HA–DAPK E64D Chloro – + – – – + G DAPK p62 LC3-I LC3-II Actin – 3-MA F Relative DAPK level Fig DAPK is regulated by the lysosome pathway (A) HEK293 cells were treated with 10 lM MG132 for h Cell extracts were prepared and immunoblotted with antibodies to detect endogenous DAPK, p53 or actin (B) HEK293 cells were transfected with HA–DAPK and His–ubiquitin Following transfection cells were treated with MG132 (10 lM) for h DAPK ubiquitination was analysed by His–ubiquitin capture on Ni-agarose beads followed by immunoblotting with HA antibodies Direct lysates were immunoblotted for DAPK with HA antibodies (C) HEK293 cells were left untreated as control or incubated in the presence of leupeptin (200 lM), E64D (10 lgỈmL-1) or chloroquine (100 lM) for 24 h Cell lysates were immunoblotted with antibodies to detect the levels of endogenous DAPK or actin (D) Quantification of DAPK protein levels from (C) Results are reported as the mean ± SD (n = 3) (E) HEK293 cells were left untreated as control or incubated in the presence of chloroquine (100 lM) for 24 h, or combined chloroquine (100 lM, 24 h) and MG132 (10 lM, h) Cell lysates were prepared and immunoblotted with antibodies to detect the levels of endogenous DAPK or actin (F) Quantification of DAPK protein levels from (E) Results are reported as the mean ± SD (n = 3) (G) HEK293 cells were treated with 10 mM 3-MA for h Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK, p62, LC3 and actin (H) HEK293 cells were transfected with 20 lM ATG7 siRNA or control siRNA for 48 h Cell lysates were prepared and immunoblotted with antibodies to detect endogenous ATG7, DAPK, p62, LC3 and actin 2.0 1.5 1.0 0.5 0.0 Chloro + H DAPK MG132 – + + – – + p62 ATG7 LC3-I LC3-II Actin siRNA + – siRNA ATG7 – + Moreover, the reduction in DAPK half-life observed upon TSC2 expression provides strong evidence that TSC2 is promoting the degradation of DAPK DAPK is regulated by the lysosomal pathway It has been reported previously that DAPK stability is regulated by the ubiquitin–proteasome pathway [11,15–19] To test this, cells were treated with the proteasome inhibitor MG132 and the levels of endogenous DAPK determined by western blot MG132 treatment did not significantly alter the level of DAPK, however, longer exposure of the film did reveal the presence of a slower migrating smear indicative of ubiquitination (Fig 6A) We used p53 as a control, with levels clearly increased upon proteasomal FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 363 TSC2 promotes the degradation of DAPK Y Lin et al inhibition (Fig 6A) To further test whether DAPK is subject to regulation via the ubiquitin–proteasome pathway, cells were transfected with HA–DAPK in combination with His–Ub, precipitated using nickel beads and western blotted with HA antibodies The addition of MG132 resulted in the linkage of multiple ubiquitin adducts to DAPK compared with untreated cells, however, little change in the stability of exogenous DAPK was observed (Fig 6B) We have previously reported that the lysosomal protease cathepsin B negatively regulates protein levels of DAPK [12] and the alternative splice variant of DAPK, s-DAPK, promotes the destabilization of DAPK in a proteasome-independent manner [20] Given the minor effects of MG132 treatment on DAPK levels, we wanted to determine whether DAPK stability is regulated via an alternate pathway, such as the lysosomal degradation pathway Cells were therefore treated with the lysosome inhibitors leupeptin, E64D and chloroquine, and the levels of endogenous DAPK were determined by western blot Treatment with each of these inhibitors led to a clear increase in DAPK protein levels (Fig 6C and quantified in Fig 6D), whereas combined proteasome and lysosome inhibition resulted in little change in DAPK levels when compared with lysosome inhibition alone (Fig 6E and quantified in Fig 6F) To determine whether DAPK is regulated by the autophagy–lysosome pathway, cells were treated with the phosphatidylinositol 3-kinase inhibitor 3-methyladenine (3-MA), an established autophagy inhibitor [31] In accordance with our previous study [32], cells were treated with 10 mm 3-MA for h, cell extracts were prepared and immunoblotted for endogenous DAPK, p62 ⁄ SQSTM1 (p62; a protein that is destroyed within the autolysosome [33]) and the conversion of LC3-I to its membrane-associated lipidated form LC3-II, which can be used to monitor autophagy levels [34] 3-MA treatment resulted in an increase in p62 levels and a reduction in LC3-II consistent with autophagy inhibition, however, no change in the level of DAPK was observed (Fig 6G) Prolonged 3-MA treatment up to 24 h had no effect on DAPK levels (data not shown) In order to further assess the effect of autophagy inhibition on DAPK levels we silenced Atg7, a critical gene required for autophagy [34] using siRNA ATG7 protein was significantly reduced by siRNA treatment, resulting in an increase in p62 levels and a reduction in LC3-II consistent with autophagy inhibition (Fig 6H) Again, no change in the level of DAPK was observed (Fig 6H) Taken together, these results suggest that DAPK stability is subject to lysosomedependent but autophagy-independent regulation 364 TSC2 promotes the degradation of DAPK through the lysosomal pathway To determine whether TSC2 promotes the degradation of DAPK through the ubiquitin–proteasome or lysosome pathway we compared the effectiveness of proteasome inhibition with that of lysosome inhibition to block TSC2 effect on DAPK First, cells coexpressing DAPK and TSC2 were cultured in the presence or absence of the proteasome inhibitor MG132 TSC2 expression in the absence of MG132 resulted in a significant reduction in DAPK protein (Fig 7A and quantified in Fig 7B) The addition of MG132 had no effect on the level of exogenous TSC2 or DAPK, and had little effect in blocking the degradation of DAPK promoted by TSC2 (Fig 7A,B) As a control, we used p53, levels of which are clearly increased upon proteasomal inhibition (Fig 7A) Next, to determine whether TSC2 promotes the degradation of DAPK via the lysosomal degradation pathway, we coexpressed DAPK and TSC2 in the presence or absence of the lysosome inhibitor chloroquine TSC2 expression in the absence of chloroquine again resulted in a clear reduction in DAPK protein (Fig 7C and quantified in Fig 7D) By contrast to MG132, the addition of chloroquine dramatically increased the levels of TSC2 and DAPK and was very effective in blocking the degradation of DAPK promoted by TSC2 (Fig 7C,D) These results further establish that DAPK is subject to regulation by the lysosome, and indicate that the lysosome is the major pathway through which TSC2 promotes the degradation of DAPK Discussion DAPK is a large protein that consists of several modular domains that enable it to function in a diverse range of signal transduction pathways such as cell survival, apoptosis and autophagy Given the size of DAPK it is not surprising that almost 20 DAPK-binding proteins have now been identified [2] From these recent studies, it is becoming increasingly clear that DAPK plays additional roles beyond cell death, and that mechanisms regulating protein stabilization and turnover are critical for modulating DAPK activities Our previous studies have identified an interaction between TSC2 and the death domain of DAPK, uncovered a role for DAPK in the control of growth factor signalling to mTORC1 and have implicated the lysosome pathway in the control of DAPK degradation [12,20,21] With this report, we have extended these previous studies and now show that TSC2 negatively regulates DAPK by promoting its lysosome-dependent FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Lin et al TSC2 promotes the degradation of DAPK A B Relative DAPK level HA–DAPK FLAG–TSC2 p53 Actin HA–DAPK + – + + – – + + – + – – MG132 + – + + + 0.5 + + – D HA–DAPK FLAG–TSC2 Actin + – – – + – + + – + – – + + + Relative DAPK level C HA–DAPK FLAG–TSC2 Chloro 1.0 0.0 HA–DAPK + FLAG–TSC2 – MG132 – + FLAG–TSC2 *** + – + + + + * 2.5 2.0 1.5 1.0 0.5 0.0 + + + HA–DAPK FLAG–TSC2 Chloro + – – + + – + – + + + + Fig TSC2 promotes the degradation of DAPK through the lysosome pathway (A) HEK293 cells were transfected with HA–DAPK and FLAG–TSC2, as indicated, and left untreated as control or incubated in the presence of MG132 (10 lM) for h Cell lysates were prepared and immunoblotted with antibodies to detect actin, HA antibodies to detect DAPK, FLAG antibodies to detect TSC2 or D01 antibodies to detect p53 (B) Quantification of DAPK protein levels from (A) Results are reported as the mean ± SD (***P < 0.001, n = 3) (C) HEK293 cells were transfected with HA–DAPK and FLAG–TSC2, as indicated, and left untreated as control or incubated in the presence of chloroquine (100 lM) for 24 h Cell lysates were prepared and immunoblotted with antibodies to detect actin, HA antibodies to detect DAPK or FLAG antibodies to detect TSC2 (D) Quantification of DAPK protein levels from (C) Results are reported as the mean ± SD (*P < 0.05, n = 3) degradation Several key lines of evidence are presented that support this conclusion First, DAPK protein, but not mRNA levels, inversely correlate with TSC2 expression Second, TSC2 affects DAPK protein levels in an mTORC1-independent manner Third, TSC2 expression significantly reduces the half-life of DAPK Finally, DAPK is stabilized by lysosome inhibitors and the effect of TSC2 on DAPK stability can be blocked by lysosome inhibition Our study therefore establishes important functions of TSC2 and the lysosomal-degradation pathway in the control of DAPK stability To date, studies investigating how DAPK is targeted for degradation at the molecular level have focused on the ubiquitin–proteasome degradation pathway with three E3-ligases having been identified that regulate DAPK ubiquitination, DIP-1 ⁄ Mib1, C-terminal HSC70-interacting protein E3-ubiquitin ligase and the KLHL20–Cul3–ROC1 E3 ligase [11,15–18] The binding of the ring finger containing E3 ligase DIP-1 ⁄ Mib1 to the ankyrin repeats region can promote the polyubiquitination and proteasomal degradation of DAPK [11,15] The U-box containing E3 ligase C-terminal HSC70-interacting protein E3-ubiquitin ligase interacts with the kinase domain of DAPK indirectly via Hsp90 to promote DAPK polyubiquitination and proteasomal degradation [16,17] In addition the cullin-3 substrate adaptor KLHL20 interacts with the death domain of DAPK to mediate DAPK polyubiquitination and proteasomal degradation to control interferon responses [18] Protein phosphatase 2A has also recently been shown to negatively regulate DAPK levels by enhancing proteasome-mediated degradation of the kinase [19] DIP1 ⁄ Mib1 and C-terminal HSC70-interacting protein E3-ubiquitin ligase have been shown to stimulate DAPK degradation in response to TNF-a and geldanamycin treatment [15,17] By contrast, KLHL20-mediated DAPK ubiquitination and degradation are suppressed in cells treated with IFN-a or IFN-c, which induces the sequestration of KLHL20 into promyelocytic leukaemia (PML) nuclear bodies resulting in the stabilization of DAPK [18] This study has revealed that the degradation of DAPK by TSC2 is constitutive and occurs in cells growing without stress At present, it is unclear whether degradation of DAPK by TSC2 can be triggered or indeed FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 365 TSC2 promotes the degradation of DAPK Y Lin et al inhibited in response to any particular signal or stress TSC2 is directly phosphorylated by AKT and by ERK, resulting in functional inactivation of the TSC1–TSC2 complex and mTORC1 activation [24] We have previously demonstrated that DAPK protein levels are unaffected by stimulation of cells with epidermal growth factor, which preferentially activates the RAS–mitogenactivated protein kinase (MEK)–extracellular signalregulated kinase (ERK) pathway, or insulin which preferentially activates the phosphatidylinositol 3-kinase– AKT signalling pathway [21] Furthermore, we observed no change in DAPK levels in response to serum-starvation or upon treatment of cells with the endoplasmic reticulum (ER)-stress inducer tunicamycin (Fig S2A,B) Thus to date, the only cellular stresses that have been shown to clearly result in altered stability of DAPK are the cytokines TNF-a and IFN-a ⁄ -c [11,18] The molecular mechanisms that control DAPK stability in response to TNF-a and IFN-a ⁄ -c have already been defined [11,18] and it will be interesting to determine whether the novel DAPK–TSC2 degradation loop we have described impinges on these pathways Consistent with the observation that DAPK has a relatively long half-life in cells growing without stress [11,12], we observed a significant increase in the DAPK level upon lysosome inhibition (Figs 6C and 7C) These results are in agreement with our previous studies [12,20] and confirm that DAPK is subject to regulation by both ubiquitin–proteasome and lysosome degradation pathways Lysosomes receive their substrates through endocytosis, phagocytosis or from within the cell via autophagy [35] Surprisingly, we did not observe any change in DAPK level upon treatment with the autophagy inhibitor 3-MA, or upon silencing of the essential autophagy gene Atg7 Furthermore, treatment of cells with rapamycin, a potent inducer of autophagy, has no observable effect on DAPK level Although these results are intriguing, a great deal of future work is required to substantiate these initial observations and determine the role that autophagy plays in the control of DAPK degradation If autophagy is not involved then the question remains how DAPK is transported to the lysosome for degradation Microtubules form an interconnected network that serves as tracks for intracellular movement of cargo to late endosomes and lysosomes [36] We have previously identified an interaction between DAPK and microtubule-associated protein 1B [32], therefore one attractive possibility is that DAPK is transported to the lysosome for degradation along the microtubule network The death domain is a signalling module present in many proapoptotic proteins The death domain of 366 DAPK is one of the key domains central to its signalling function and was identified as one of four functional domains required for DAPK to exert its growthsuppressing activity [37] The death domain of DAPK regulates its proapoptotic function, in part by interacting with the mitogen-activated protein kinase ERK [38], which is in turn abrogated by a common polymorphism in DAPK at codon 1347 [39] The death domain of DAPK also regulates its proapoptotic function by interacting with tumour necrosis superfamily members TNFR1 and FADD [40], in addition to the netrin-1 receptor UNC5H2 [41] Our identification of TSC2 as a new binding protein for DAPK uncovered additional roles for the death domain beyond the control of cell death [21] It is now becoming clear that the death domain is also important for the regulation of DAPK stability [18] and it will be of great interest to determine how this domain coordinates distinct pathways to control the balance between the degradation of DAPK with its proapoptotic and prosurvival activities Although the molecular mechanisms employed by TSC2 to target DAPK for degradation are not yet clear, our results demonstrate that binding is required for TSC2 to exert its effect on DAPK, suggesting that the proteins may be co-degraded The observation that a highly stable TSC2 mutant can retain binding to DAPK, but cannot promote DAPK degradation, adds further support to this idea It is intriguing that the death domain of DAPK is the focal point necessary for both KLHL20–Cul3–ROC1 E3-ligase and TSC2 to mediate DAPK degradation Thus, a crucial question is whether ubiquitination or modification with other small ubiquitin-like proteins such as SUMO or NEDD can signal DAPK for degradation via lysosome-dependent degradation pathways Clearly, more work is required to delineate further the role of the death domain in the control of DAPK stability In summary, we have shown that TSC2 can promote the lysosome-dependent degradation of DAPK under normal growth conditions Taken together with our previous study [21], we can conclude that the death domain of DAPK forms the key binding site that stabilizes the DAPK–TSC2 complex and leads to at least two distinct outcomes If the signalling threshold is driven towards proliferation and growth, then DAPK functions as an inhibitory kinase for TSC2, releasing mTORC1 from negative regulation by the TSC complex (Fig 8) Conversely, a constitutive DAPK inhibitory pathway is coordinated by TSC2 to drive the balance of regulation, resulting in the destabilization of DAPK protein (Fig 8) The reciprocal regulation we have identified between DAPK and FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Lin et al TSC2 promotes the degradation of DAPK Degradation TSC1 TSC2 Rheb Growth factors DAPK Phosphorylation mTORC1 IFN-γ, ER-stress Excessive growth factor signalling, IFN-γ, TGF-β, DNA damage, Oncogenes, ER-stress P -T389 S6K P -S235/236 S6 Cell growth protein synthesis (University of Cardiff, UK) For the generation of FLAGtagged TSC2 (1–1516) a stop codon was introduced at amino acid 1517 using the primers: Fwd 5¢-CGACGAGTC AAACTAGCCAATCCTGCTG-3¢; Rev 5¢-CAGCAGGAT TGGCTAGTTTGACTCGTCG-3¢ Autophagy Apoptosis Fig DAPK and TSC2 form a regulatory feedback loop Recent advances have established an important role for DAPK in a diverse range of signal-transduction pathways including growth factor signaling, apoptosis, autophagy and membrane blebbing DAPK has been shown to function as a positive mediator of apoptosis induced by various stimuli including the transforming oncogenes c-myc and E2F1, IFN-c, transforming growth factor beta, DNA damage, ER-stress and excessive growth factor signaling DAPK also plays a role in survival pathways reflected in its autophagy-signalling activity A substantial amount of research has demonstrated that the TSC proteins form a complex that inhibits mTORC1 activity leading to reduced protein synthesis and cell growth The pathway that regulates autophagy also acts through mTORC1 Our own data show that DAPK binds to and catalyses the phosphorylation of TSC2, thus inactivating the TSC complex and stimulating mTORC1 activity in a growth factor-dependent pathway Reciprocally, a constitutive DAPK inhibitory pathway is coordinated by TSC2 to drive the lysosome-dependent degradation of DAPK TSC2 proteins constitutes a feedback loop that may regulate the level of mTORC1 activity and ultimately control cell outcomes, including growth, autophagy and apoptosis all of which are relevant to the pathogenesis of many human diseases Materials and methods Plasmids Dual N-terminal FLAG–HA–DAPK vector was a gift of Adi Kimchi (Weizmann Institute, Rehovot, Israel) Generation of the FLAG–HA–DAPK (1–1313) deletion mutant has been described previously [12] FLAG–TSC1, FLAG– TSC2, FLAG–TSC2 (N1693K), FLAG–Rheb, HA–Raptor and HA–Raptor mutant were all gifts from Andrew Tee Cell culture, transfection and immunoblotting HEK293 and A549 (which express TSC2 and active DAPK) and TSC2 MEFs (a gift from Andrew Tee, University of Cardiff, UK) were grown in Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD, USA) supplemented with 10% FCS (Gibco) at 37 °C in a 5% CO2 ⁄ H2O-saturated atmosphere Cells for transient transfection were plated out 24 h before transfection at  1.5 · 106 cells per 100 mm dish or · 105 cells per 60 mm dish For Lipofectamine 2000 transfection (Invitrogen, Carlsbad, CA, USA), lL of Lipofectamine was used for every lg of DNA transfected Cells were harvested after a further incubation of 16–18 h Cells were lysed in ice-cold extraction buffer (50 mm Tris pH 7.6, 150 mm NaCl2, mm EDTA, 0.5% NP-40, mm NaF, mm sodium vanadate, · protease inhibitor cocktail) for 30 and centrifuged at 18 894 g for 15 to remove insoluble material The protein content of cell extracts was measured using Bio-Rad reagent (Bio-Rad Labs, Hercules, CA, USA) Typically, 20–30 lg of cell extract was used for immunoblot Samples were resolved by denaturing gel electrophoresis, typically 4–12% precast gels (Novex, Invitrogen, Carlsbad, CA, USA) and electrotransferred to Hybond C-extra nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK), blocked in NaCl ⁄ Pi–10% nonfat milk for 30 min, then incubated with primary antibody overnight at °C in NaCl ⁄ Pi–5% nonfat milk–0.1% Tween-20 After washing (3 · 10 min) in NaCl ⁄ Pi–Tween-20, the blot was incubated with secondary antibody, either horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Dako, Carpinteria, CA, USA; : 5000), for h at room temperature in NaCl ⁄ Pi–5% nonfat milk–0.1% Tween-20 After washing (3 · 10 min) in NaCl ⁄ Pi–Tween-20, proteins were visualized by incubation with ECL western blotting analysis system (Amersham Biosciences) or Immobilon western chemiluminescent HRP substrate (Millipore Corp., Bedford, MA, USA) Equal protein loading was confirmed with Ponceau S staining FLAG antibody (M2) and actin antibody were purchased from Sigma (St Louis, MO, USA) HA-11 antibody (Ascites) was purchased from Covance (Princeton, NJ, USA) Tuberin ⁄ TSC2 (C-20) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) DAPK antibody was purchased from BD Transduction Laboratories (Lexington, KY, USA) DAPK (clone-55, Ascites) and Phospho-DAPK (S308) were purchased from Sigma Phospho-p70 S6 kinase (Thr389), p70 S6 kinase, phospho-S6 (S235 ⁄ 236), S6, Hamartin ⁄ TSC1 (1B2), Rheb, Raptor, Bip and ATG7 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) p62 antibody was FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 367 TSC2 promotes the degradation of DAPK Y Lin et al purchased from Progen (Heidelberg, Germany) P53 (DO-1) antibody was a gift from Borek Vojtesek (Masaryk Memorial Cancer Institute, Brno, Czech Republic) Immunoprecipitation For immunoprecipitation of exogenous HA–DAPK from cells, HA-11 antibody bound to 30 lL of protein G beads (Amersham) was incubated overnight at °C with constant rotation, together with cell extract ( mg) diluted to a volume of 500 lL in extraction buffer (50 mm Tris pH 7.4, 150 mm NaCl2, mm EDTA, 0.5% NP-40, mm NaF, mm sodium vanadate, · protease inhibitor cocktail) The bead pellets were then washed five times in extraction buffer before being resuspended in · SDSloading buffer and analysed by denaturing gel electrophoresis and immunoblotting For immunoprecipitation of endogenous TSC1 from cells, the same procedure was followed except TSC1 antibodies were bound to protein G beads His–ubiquitin lysis buffer (guanidinium–HCl m, Na2HPO4 95 mm, NaH2PO4 mm, Tris ⁄ HCl 0.01 m pH 8.0, mm imidazole and 10 mm b-mercatoethanol) supplemented with 75 lL of Ni-agarose beads (Qiagen, Valencia, CA, USA) overnight at °C Beads were washed for in each of the following wash buffers: m guanidium–HCl, Na2HPO4 95 mm, NaH2PO4 mm, Tris ⁄ HCl 0.01 m pH 8.0 (buffer A); m urea, Na2HPO4 95 mm, NaH2PO4 mm, Tris ⁄ HCl 0.01 m pH 8.0 and 10 mm b-mercatoethanol (buffer B); m urea, Na2HPO4 22.5 mm, NaH2PO4 77.5 mm, Tris ⁄ HCl 0.01 m pH 6.3 and 10 mm b-mercatoethanol (buffer C); buffer C with 0.2% Triton X-100; buffer C with 0.1% Triton X-100 The Ni-bead conjugated proteins were then eluted in 75 lL elution buffer (imidazole 200 mm, SDS 5%, Tris ⁄ HCl 0.15 m pH 6.7, glycerol 30% and b-mercaptoethanol 0.72 m) for 30 at room temp Eluates were mixed in : ratio with SDS sample buffer and were analysed by 4–12% NuPAGE ⁄ immunoblot and probed with HA antibody RNA extraction and real-time PCR siRNA For the knockdown of gene expression cells were transfected with TSC2 siRNA (Cell Signaling Technology), ATG7 siRNA (Cell Signaling Technology), Rheb siRNA (Dharmacon, Lafayette, CO, USA), Raptor siRNA (Dharmacon) or a non-specific siRNA as control (Dharmacon) After 48 h, cells were lysed in ice-cold extraction buffer (50 mm Tris pH 7.6, 150 mm NaCl2, mm EDTA, 0.5% NP-40, mm NaF, mm sodium vanadate, · protease inhibitor cocktail) for 30 and centrifuged at 18 894 g for 15 to remove insoluble material Samples were resolved by denaturing gel electrophoresis followed by immunoblotting Drug treatments The following drugs were added to cell media at the indicated concentrations; MG132 (10 lm; Calbiochem, San Diego, CA, USA), leupeptin (200 lm; Calbiochem), E-64d (10 lgỈmL)1; Calbiochem), chloroquine (100 lm; Invitrogen), 3-MA (10 mm; Calbiochem), tunicamycin (1 lgỈmL)1; Sigma) and cycloheximide (10 lgỈmL)1; Supleco, Bellefonte, PA, USA) mRNA was extracted from cells using the QIAGEN RNeasy Mini kit following the manufacturer’s suggested procedures The optional step of DNase treatment using the QIAGEN RNase-free DNase set was also included One microlitre of the sample RNA was diluted in 100 lL water and loaded into a 96-well UV plate Absorbance was detected with a Bio-Tek plate reader at 260 nm After the extraction, the real-time PCR were performed in the Opticon machine using the QIAGEN QuantiTect SYBR Green one-step PCR kit Equal amounts of mRNA were loaded in each reaction The actin primers used were: L: 5¢-CTACGTCG CCCTGGACTTCGAGC-3¢, R: 5¢-GATGGAGCCGCCG ATCCACACGG-3¢ The DAPK primers were: L: 5¢-CGAGGTGATGGTG TATGGTG-3¢; R: 5¢-CTGTGCTTTGCTGGTGGA-3¢ Statistical analysis Scanning densitometry was performed using scion image software (National Institutes of Health) Results are reported as the mean ± SD with P-values calculated using an unpaired t-test in graphpad v4.03 (GraphPad Software, CA, USA) Cell-based ubiquitination assays HEK293 cells were transfected with the appropriate constructs for 24 h, then treated with MG132 (10 lm) for h prior to harvesting Twenty per cent of the cell suspension was used for direct western blot analysis with HA antibodies For the purification of His–ubiquitinated conjugates the remainder of the cell suspension was lysed in mL of 368 Acknowledgements We thank Andrew Tee and Adi Kimchi for reagents This work was funded by a CRUK Programme grant to TH (C483 ⁄ A6354) CS is funded by an MRC project grant (G0800759) PH is funded by an MRC project grant (G0800675) FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Lin et al TSC2 promotes the degradation of DAPK References Bialik S & Kimchi A (2006) The death-associated protein kinases: structure, function, and beyond Annu Rev Biochem 75, 189–210 Lin Y, Hupp TR & Stevens C (2010) Death-associated protein kinase (DAPK) and signal transduction: additional roles beyond cell death FEBS J 277, 48–57 Bovellan M, Fritzsche M, Stevens C & Charras G (2010) Death-associated protein kinase (DAPK) and signal transduction: blebbing in programmed cell death FEBS J 277, 58–65 Deiss LP, Feinstein E, Beressi H, Cohen O & Kimchi A (1995) Identification of a novel serine ⁄ threonine kinase and a novel 15-kD protein as potential mediators of the gamma-interferon induced cell death Genes Dev 9, 15–30 Raveh T, Droguett G, Horwitz MS, DePinho RA & Kimchi A (2001) DAP kinase activates a p19ARF ⁄ p53mediated apoptotic checkpoint to suppress oncogenic transformation Nat Cell Biol 3, 1–7 Inbal B, Cohen O, Polak-Charcon S, Kopolovic J, Vadai E, Eisenbach L & Kimchi A (1997) DAP kinase links the control of apoptosis to metastasis Nature 390, 180–184 Michie AM, McCaig AM, Nakagawa R & Vukovic M (2010) Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer FEBS J 277, 74–80 Raval A, Tanner SM, Byrd JC, Angerman EB, Perko JD, Chen SS, Hackanson B, Grever MR, Lucas DM, Matkovic JJ et al (2007) Downregulation of death-associated protein kinase-1 (DAPK-1) in chronic lymphocytic leukemia Cell 129, 879–890 Inbal I, Bialik S, Sabanay I, Shani G & Kimchi A (2002) DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death J Cell Biol 157, 455–468 10 Zalckvar E, Berissi H, Mizrachy L, Idelchuk Y, Koren I, Eisenstein M, Sabanay H, Pinkas-Kramarski R & Kimchi A (2009) DAP-kinase-mediated phosphorylation on the BH3 domain of beclin promotes dissociation of beclin from Bcl-XL and induction of autophagy EMBO Rep 10, 285–292 11 Jin Y, Blue E & Gallaher PJ (2006) Control of deathassociated protein kinase (DAPK) activity by phosphorylation and proteasomal degradation J Biol Chem 281, 39033–39040 12 Lin Y, Stevens C & Hupp T (2007) Identification of a dominant negative functional domain on DAPK-1 that degrades DAPK-1 protein and stimulates TNFR-1 mediated apoptosis J Biol Chem 282, 16792–16802 13 Mukhopadhyay R, Ray PS, Arif A, Brady AK, Kinter M & Fox PL (2008) DAPK–ZIPK–L13a axis 14 15 16 17 18 19 20 21 22 23 24 25 26 constitutes a negative-feedback module regulating inflammatory gene expression Mol Cell 32, 371–382 Hershko A & Ciechanover A (1998) The ubiquitin system Annu Rev Biochem 67, 425–479 Jin Y, Blue E, Dixon S, Shao Z & Gallagher PJ (2002) A death-associated protein kinase (DAPK)-interacting protein, DIP-1, is an E3 ubiquitin ligase that promotes tumour necrosis factor-induced apoptosis and regulates the cellular levels of DAPK J Biol Chem 277, 46980– 46986 Citri A, Harari D, Shohat G, Ramakrishnan P, Gan J, Lavi S, Eisenstein M, Kimchi A, Wallach D, Pietrokovski S et al (2006) Hsp90 recognises a common surface on client kinases J Biol Chem 281, 14361–14369 Zhang L, Nephew KP & Gallagher PJ (2007) Regulation of death-associated protein kinase J Biol Chem 282, 11795–11804 Lee YR, Yuan WC, Ho HC, Chen CH, Shih HM & Chen RH (2010) The cullin substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses EMBO J 29, 1748–1761 Widau RC, Jin Y, Dixon SA, Wadzinski BE & Gallagher PJ (2010) Protein phosphatase 2A (PP2A) holoenzymes regulate death-associated protein kinase (DAPK) in ceramide-induced apoptosis J Biol Chem 285, 13827–13838 Lin Y, Stevens C, Harrison B, Pathuri S, Amin E & Hupp TR (2009) The alternative splice variant of DAPK-1, s-DAPK-1, induces proteasome-independent DAPK-1 destabilization Mol Cell Biol 328, 101–107 Stevens C, Lin Y, Harrison B, Burch L, Ridgway RA, Sansom O & Hupp T (2009) Peptide combinatorial libraries identify TSC2 as a DAPK death domain binding protein and reveal a stimulatory role for DAPK in mTORC1 signalling J Biol Chem 284, 334–344 Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC & Blenis J (2002) Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling Proc Natl Acad Sci USA 99, 13571–13576 Tee AR, Manning BD, Roux PP, Cantley LC & Blenis J (2003) Tuberous sclerosis complex gene products, tuberin and hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb Curr Biol 13, 1259–1268 Proud CG (2007) Signalling to translation: how signal transduction pathways control the protein synthetic machinery Biochem J 403, 217–234 Rubinsztein DC, Gestwick JE, Murphy LO & Klionsky DJ (2007) Potential therapeutic applications of autophagy Nat Rev Drug Discov 6, 304–312 Dowling RJO, Topisirovic I, Fonseca BD & Sonenberg N (2010) Dissecting the role of mTOR: lessons from mTOR inhibitors Biochim Biophys Acta 1804, 433–439 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 369 TSC2 promotes the degradation of DAPK Y Lin et al 27 Tee AR, Blenis J & Proud CG (2005) Analysis of mTOR signaling by the small G-proteins, Rheb and RhebL1 FEBS Lett 579, 4763–4768 28 Dunlop EA, Dodd KM, Seymour LA & Tee AR (2009) Mammalian target of rapamycin complex 1-mediated phosphorylation of eukaryotic initiation factor 4E-binding protein requires multiple protein-protein interactions for substrate recognition Cell Signal 21, 1073– 1084 29 Hu J, Zacharek S, He YJ, Lee H, Shumway S, Duronio RJ & Xiong Y (2008) WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1CUL4-ROC1 ligase Genes Dev 22, 866–871 30 Hodges AK, Li S, Maynard J, Parry L, Braverman R, Cheadle JP, DeClue JE & Sampson JR (2001) Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin Hum Mol Genet 10, 2899–2905 31 Petiot A, Ogier-Denis E, Blommaart EFC, Meijer AJ & Codogno P (2000) Distinct classes of phosphatidylinositol 3-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells J Biol Chem 275, 992–998 32 Harrison B, Kraus M, Burch L, Stevens C, Craig A, Gordon-Weeks P & Hupp TR (2008) DAPK-1 binding to a linear peptide motif in MAP1B stimulates autophagy and membrane blebbing J Biol Chem 283, 9999–10014 33 Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H & Johansen T (2005) p62 ⁄ SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtininduced cell death J Cell Biol 171, 603–614 34 Van Limbergen J, Stevens C, Nimmo ER, Wilson D & Satsangi J (2009) Autophagy: from basic science to clinical application Mucosal Immunol 2, 315–330 35 Saftig P & Klumperman J (2009) Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function Nat Rev Mol Cell Biol 10, 623–635 36 Monastyrska I, Rieter E, Klionsky DJ & Reggiori F (2009) Multiple roles of the cytoskeleton in autophagy Biol Rev 84, 431–448 370 37 Cohen O, Inbal B, Kissil JL, Raveh T, Berissi H, Spivak-Kroizaman T, Feinstein E & Kimchi A (1999) DAP-kinase participates in TNF-a- and Fas-induced apoptosis and its function requires the death domain J Cell Biol 146, 141–148 38 Chen CH, Wang WJ, Kuo JC, Tsai HC, Lin JR, Chang ZF & Chen RH (2005) Bidirectional signals transduced by DAPK–ERK interaction promote the apoptotic effect of DAPK EMBO J 24, 294–304 39 Stevens C, Lin Y, Sanchez M, Amin E, Copson E, White H, Durston V, Eccles DM & Hupp T (2007) A germ line mutation in the death domain of DAPK-1 inactivates ERK-induced apoptosis J Biol Chem 282, 13791–13803 40 Henshall DC, Araki T, Schindler CK, Shinoda S, Lan JQ & Simon RP (2003) Expression of death-associated protein kinase and recruitment to the tumor necrosis factor signalling pathway following brief seizures J Neurochem 86, 1260–1270 41 Llambi F, Calheiros-Lourenco F, Gozuacik D, Guix C, Pays L, Rio G, Kimchi A & Mehlen P (2005) The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase EMBO J 24, 1192–1201 Supporting information The following supplementary material is available: Fig S1 Stability of DAPK in TSC2 () ⁄ )) MEFs Fig S2 DAPK levels in response to serum starvation and ER stress This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS ... et al TSC2 promotes the degradation of DAPK Introduction Death-associated protein kinase-1 (DAPK) is the prototypic member of a family of death-related kinases that includes DAPK-1-related protein. .. 12, which then binds to and inactivates mTORC1, leading to an upregulation of autophagy [25] Thus mTORC1 acts as a central regulator balancing anabolic and catabolic pathways within the cell [24]... Michie AM, McCaig AM, Nakagawa R & Vukovic M (2010) Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer FEBS J 277, 74–80 Raval A, Tanner SM, Byrd JC, Angerman EB,