DAPK) Through a Lysosome-Dependent Degradation Pathway Yao Lin1, Paul Henderson1,2, Susanne Pettersson1, Jack Satsangi1, Ted Hupp1 and Craig Stevens1

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

1 University of Edinburgh, Institute of Genetics and Molecular Medicine, UK 2 Department of Child Life and Health, University of Edinburgh, UK

Keywords We previously identified a novel interaction between tuberous sclerosis-2 DAPK; degradation; lysosome; mTORC1; (TSC2) and death-associated protein kinase-1 (DAPK), the consequence TSC2 being that DAPK catalyses the inactivating phosphorylation of TSC2 to stimulate mammalian target of rapamycin complex 1 (mTORC1) activity. Correspondence C. Stevens, University of Edinburgh, We now report that TSC2 binding to DAPK promotes the degradation of Institute of Genetics and Molecular DAPK. We show that DAPK protein levels, but not gene expression, Medicine, Edinburgh, EH4 2XR, UK inversely correlate with TSC2 expression. Furthermore, altering mTORC1 Fax: +44 131 651 1085 activity does not affect DAPK levels, excluding indirect effects of TSC2 on Tel: +44 131 651 1025 DAPK protein levels through changes in mTORC1 translational control. E-mail: [email protected] We provide evidence that the C-terminus regulates TSC2 stability and is required for TSC2 to reduce DAPK protein levels. Importantly, using a (Received 28 July 2010, revised 7 October 2010, accepted 11 November 2010) GTPase-activating protein–dead missense mutation of TSC2, we demonstrate that the effect of TSC2 on DAPK is independent of GTPase-activat- doi:10.1111/j.1742-4658.2010.07959.x ing 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)byanti tag coimmunoprecipitation (MI:0007) l MINT-8057213: TSC1 (uniprotkb:Q92574) physically interacts (MI:0914) with DAPK (uniprotkb:P53355) and TSC2 (uniprotkb:P49815)byanti bait coimmunoprecipitation (MI:0006) l MINT-8057200: TSC1 (uniprotkb:Q92574) physically interacts (MI:0915) with TSC2 (uniprotkb:P49815)byanti 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 pro- the lysosomal protease cathepsin B negatively regulates totypic member of a family of death-related kinases protein levels of DAPK [12] and that a small, alterna- that includes DAPK-1-related protein 1 (DRP-1, also tively spliced form of DAPK (s-DAPK) destabilizes named DAPK-2), Zipper interacting kinase (also DAPK in a proteasome-independent manner [20]. named DAPK-3), DRAK1 (DAPK kinase-related In a previous study [21], we performed a protein- apoptosis-inducing protein kinase 1) and DRAK2 [1]. interaction screen to identify novel DAPK death- DAPK is a large 160 kDa serine ⁄ threonine protein domain-interacting proteins and identified tuberous kinase composed of several functional domains includ- sclerosis-2 (TSC2) as one such protein. We demon- ing a kinase domain, a calmodulin regulatory domain, strated that the consequence of this interaction between eight consecutive ankyrin repeats, two putative nucleo- TSC2 and DAPK was phosphorylation of TSC2 by tide-binding domains (P-loops), a cytoskeletal binding DAPK. This led to inactivation of the TSC complex to domain and a death domain [1]. Recent advances have stimulate mTORC1 activity in an epidermal growth fac- established an important role for DAPK in a diverse tor-dependent manner [21]. The TSC complex, formed range of signal transduction pathways including by two proteins – tuberous sclerosis-1 (TSC1) and growth factor signalling, apoptosis, autophagy and TSC2 – is a major regulator of the mTORC1-signalling membrane blebbing [2,3]. DAPK was originally identi- pathway [22], with mutations in either the TSC1 or fied as a factor that regulates apoptosis in response to TSC2 gene, resulting in the autosomal-dominant dis- the death-inducing cytokine interferon (IFN)-c [4], and ease tuberous sclerosis. TSC2 contains a GTPase-acti- has subsequently been shown to function as a positive vating protein (GAP) domain in its C-terminus, and mediator of apoptosis induced by various stimuli through GTP hydrolysis of the small protein Rheb including the transforming oncogenes c-myc and E2F1, antagonizes the mTORC1-signalling pathway [23]. transforming growth factor-beta and ceramide [2]. In TSC2 is phosphorylated and regulated by various kin- accordance with its proapoptotic activity, evidence sug- ases to integrate signals such as nutrient availability, gests that DAPK functions as a tumour suppressor energy, hormones and growth factors with mTORC1 having been shown to suppress transformation in vitro activity [24]. mTORC1 directly controls cell growth by [5] and block tumour metastasis in murine models [6]. regulating the phosphorylation of components of the Furthermore, DAPK gene expression is frequently lost protein translational machinery. In particular, phos- in human cancers due to promoter hypermethylation phorylation and activation of eukaryotic initiation fac- [7] and a loss of DAPK gene expression correlates with tor 4E binding protein-1 (4EBP-1) and ribosomal the development of chronic lymphocytic leukaemia [8]. protein S6 kinase-1 (S6K) are stimulated by serum, DAPK has also recently been shown to play a role in insulin and growth factors in an mTORC1-dependent survival pathways reflected in its autophagy-signalling manner [24]. The pathway that regulates autophagy activity [9,10] and its ability to counter tumour necro- also acts through mTORC1. Autophagy is a membrane sis factor (TNF)-mediated apoptosis [11,12]. system that sequesters proteins and organelles into a Post-transcriptional mechanisms regulating protein structure called the autophagosome, which then fuses translation, stabilization and turnover are also critical with a lysosome where cargo is degraded. The resulting for modulating DAPK activities. For example, transla- degradation products are then released back into the tional repression of DAPK occurs in response to IFN-c cytosol where they can be recycled to sustain the growth treatment mediated by the IFN-c-activated inhibitor of requirements of the cell. The lipophilic macrolide anti- translation complex [13]. Central to protein stability, biotic rapamycin forms a complex with FK506-binding the control of protein degradation by the ubiquitin– protein 12, which then binds to and inactivates proteasome system is a key regulator of many cellular mTORC1, leading to an upregulation of autophagy processes [14]. In this pathway, proteins are tagged [25]. Thus mTORC1 acts as a central regulator balanc- with ubiquitin through the concerted action of ing anabolic and catabolic pathways within the cell [24]. E1-ubiquitin-activating enzyme, E2-conjugating In this report, we extend our previous studies enzyme, E3-ubiquitin ligase enzyme and finally [12,20,21] and describe a novel function for TSC2 in degraded by the proteasome [14]. To date, it has been promoting the lysosome-dependent degradation of demonstrated that the post-translational control of DAPK. We suggest that the TSC2–DAPK protein DAPK protein levels are regulated by at least three complex forms a regulatory feedback loop whose bal- distinct E3-ubiquitin ligase family members [11,15–19]. ance may influence the extent of mTORC1 signalling In addition, work from our own group has shown that 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 immunoblotting DAPK protein from TSC2 (+ ⁄ +) and stimulating DAPK degradation via TSC2 signalling TSC2 () ⁄ )) MEFs with phospho-Ser308 antibodies under steady-state conditions. and compared the abundance of the phosphorylated inactive form relative to the total level of DAPK. Again, DAPK protein was elevated in TSC2 () )) cells Results ⁄ compared with TSC2 (+ ⁄ +) control cells (Fig. 1F), however, a decrease in the level of phosphorylated DAPK protein but not mRNA levels inversely DAPK was observed in TSC2 () ⁄ )) cells (Fig. 1F), correlate with TSC2 expression thus both DAPK level and activity are elevated in the We recently identified TSC2 as a novel DAPK death- absence of TSC2. Next, we assessed whether TSC2 was domain-interacting protein [21]. During the course of mediating its effect on DAPK at the transcriptional that study, we observed that the abundance of DAPK level. Real-time PCR revealed that TSC2 overexpres- inversely correlated with TSC2 expression (Stevens, C; sion did not significantly alter the level of DAPK Lin, Y; Harrison, B; Burch, L; Ridgway, R.A; Sansom, mRNA (Fig. 1G), demonstrating that the coincident O and Hupp, T. unpublished results). To further inves- reduction in DAPK protein observed (Fig. 1H) is tigate this observation, we first evaluated the effect of independent of changes in DAPK gene expression. overexpressing increasing amounts of TSC2 on the lev- Taken together, these results demonstrate that TSC2 els of endogenous DAPK protein. TSC2 overexpression can regulate the abundance and activity of DAPK via a led to a significant reduction in the level of DAPK pro- post-transcriptional mechanism. tein in a dose-dependent manner (Fig. 1A and quantified in Fig. 1B). Because the overexpression of TSC2 DAPK protein levels are not affected by mTORC1 reduced DAPK protein levels, we anticipated that activity silencing of TSC2 expression with short interfering RNA (siRNA) would have the opposite effect and lead Because mTORC1 is an important regulator of protein to an increase in DAPK. Indeed, DAPK protein was translation, it was necessary to determine whether the increased in TSC2 siRNA-treated cells compared with effect of TSC2 on DAPK might be indirect, through control siRNA-treated cells (Fig. 1C). As expected, changes in mTORC1 activity. To examine whether inhibition of TSC2 function and concomitant activation mTORC1 was involved, we used the mTORC1 inhibi- of mTORC1 resulted in an increase in phosphorylation tor rapamycin. Rapamycin treatment efficiently inhib- of S6K on T389 (Fig. 1C). To add physiological rele- ited mTORC1 translational activity, as measured by vance to these findings, we took advantage of TSC2 phosphorylation of S6K on T389 and phosphorylation mouse embryonic fibroblasts (MEFs) deficient for of the S6K-substrate ribosomal protein S6 on TSC2. TSC2-null MEFs undergo early-onset senescence S235 ⁄ 236, but had no effect on the levels of DAPK because of a dramatic p53-dependent induction of p21, protein (Fig. 2A). The elevated level of DAPK protein thus to circumvent senescence, p53 is also knocked-out observed in TSC2 () ⁄ )) MEFs may result from in these cells. Immunoblotting of DAPK protein from increased mTORC1 activity in these cells (Fig. 1D), TSC2 (+ ⁄ +) and TSC2 () ⁄ )) MEFs revealed that therefore we investigated the effect of rapamycin DAPK protein was elevated in TSC2 () ⁄ )) cells on DAPK level in TSC2 () ⁄ )) cells. A timecourse of (Fig. 1D). As expected, loss of TSC2 function results in rapamycin treatment resulted in the efficient inhibition activation of mTORC1 and increased phosphorylation of mTORC1 activity, as measured by the phosphoryla- of S6K on T389 (Fig. 1D). To confirm that loss of tion of S6 on S235 ⁄ 236 (Fig. 2B), however no change TSC2 was responsible for the higher level of DAPK in DAPK levels was observed (Fig. 2B), suggesting protein observed in TSC2 () ⁄ )) MEFs, we reconsti- that the increased level of DAPK in these cells is not a tuted TSC2 by transfection and determined the levels of result of increased mTORC1 activity. Several studies DAPK by western blot. Overexpression of TSC2 led to have recently demonstrated that rapamycin does not a clear reduction in DAPK protein to a level compara- inhibit all functions of mTORC1 [26], therefore the ble with TSC2 (+ ⁄ +) control cells (Fig. 1E), confirm- effects of Rheb and the mTORC1 component Raptor ing that the elevated levels of DAPK observed are a were also evaluated in TSC2 () ⁄ )) cells. First, we direct result of TSC2 loss. DAPK activity is auto-inhib- investigated the effect of Rheb overexpression in ited by auto-phosphorylation on Ser308 within its serum-starved cells. Rheb overexpression resulted in a calmodulin regulatory-binding domain [1]. To deter- pronounced increase in phosphorylation of S6 on mine whether the TSC2 regulatory effect on DAPK is S235 ⁄ 236, but no change in the level of DAPK was important functionally, we assessed DAPK activity by observed (Fig. 2C). To exclude any direct effects of

356 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 FLAG–TSC2 1.0 ** 0.8 DAPK 0.6 Actin 0.4 FLAG–TSC2 (µg) 0.25 0.5 0.75 1.0 2.0 0.2 Relative DAPK level DAPK Relative 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 Actin siRNA con + – TSC2 MEF (+/+) (–/–) siRNA TSC2 – +

E TSC2 (–/–) MEF F

TSC2 FLAG DAPK P-S308

DAPK DAPK

Actin Actin FLAG–TSC2 – + TSC2 MEF (+/+) (–/–)

G NS

H 1.5 FLAG–TSC2

1.0 DAPK

0.5 Actin FLAG–TSC2 (µg) 02.0

DAPK : actin mRNA ratio : DAPK 0.0 0 2.0 Concentration of FLAG–TSC2 (µg)

Fig. 1. 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 C Serum-starved DAPK DAPK S6K P-T389 FLAG–RHEB S6K S6 P-S235/236 S6 P-S235/236 S6 S6 Actin Actin FLAG-RHEB – + – + RAPA – + RAPA – – + +

B TSC2 (–/–) MEF D Serum-starved DAPK DAPK S6 P-S235/236 RHEB S6 S6 P-S235/236 Actin S6 RAPA (h) 01246816 Actin E Serum-starved siRNA con + – siRNA RHEB – + DAPK

HA F Serum-starved

S6 P-S235/236 DAPK

S6 P-S235/236 Raptor (short exposure)

S6 S6 P-S235/236

Actin S6

HA-Raptor + – + – + – Actin HA-Raptor MT4 – + – + – + siRNA con + – Rapamycin – – – – ++ siRNA Raptor – +

Fig. 2. DAPK protein level is not affected by mTORC1 activity. (A) HEK293 cells were treated with 100 nM rapamycin for 6 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 2 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 4. Cells were serum-starved and treated with 100 nM rapamycin for 2 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. TSC2 promotes the degradation of DAPK

Rheb on DAPK level, Rheb was overexpressed in previous study [29], cycloheximide treatment revealed serum-starved cells in the presence of rapamycin. that TSC2 is a short-lived protein with a half-life of Under these conditions, Rheb expression did not lead 2 h under normal growth conditions (Fig. 3B and to a change in the phosphorylation of S6 on S235 ⁄ 236 quantified in Fig. 3C). The GAP-dead mutant or in DAPK level (Fig. 2C). To confirm these findings, TSC2 (N1693K) exhibited a half-life similar to wild- we investigated the effect of Rheb depletion with type TSC2 (Fig. 3B and quantified in Fig. 3C). By siRNA on DAPK levels in serum-starved TSC2 () ⁄ )) contrast, TSC2 (1–1516) exhibited a significantly cells. Decreased Rheb expression correlated with par- increased stability with a half-life of 8 h (Fig. 3B tial inhibition of mTORC1 activity and reduction of and quantified in Fig. 3C), confirming the importance S6 S235 ⁄ 236 phosphorylation, likely because of func- of this domain in the regulation of TSC2 stability. To tional redundancy between Rheb and RhebL1 [27], investigate further the mechanism through which and again no change in DAPK level was observed TSC2 regulates DAPK stability we compared the levels (Fig. 2D). Next, we investigated the effects of overex- of DAPK in TSC2 () ⁄ )) MEFs reconstituted with pressing Raptor or a mutant Raptor (Raptor mutant TSC2, TSC2 (1–1516) or TSC2 (N1693K). Reconstitu- 4) that interferes with mTORC1 substrate recognition tion of TSC2 or TSC2 (N1693K) resulted in a pro- [28]. In cells growing in full serum, expression of Rap- nounced reduction in DAPK level that was not tor mutant 4 resulted in decreased phosphorylation of observed in cells reconstituted with TSC2 (1–1516) S6 on S235 ⁄ 236, confirming its ability to dominantly (Fig. 3D). As expected, cells reconstituted with TSC2 impair mTORC1 activity (Fig. 2E). In serum-starved exhibited reduced mTORC1 activity, as measured by cells, Raptor or Raptor mutant 4 overexpression failed phosphorylation of S6 on S235 ⁄ 236, whereas cells to alter phosphorylation of S6 on S235 ⁄ 236 (Fig. 2E). reconstituted with TSC2 (1–1516) or TSC2 (N1693K) Similarly, expression of Raptor or Raptor mutant 4 in exhibited no change in mTORC1 activity (Fig. 3D). serum-starved cells treated with rapamycin resulted in Importantly, the observation that TSC2 (N1693K) no observable difference in S6 phosphorylation retained the ability to efficiently reduce DAPK levels (Fig. 2E). Importantly, no change in the level of demonstrates that TSC2 effect on DAPK is indepen- DAPK protein was observed under any of these condi- dent of its GAP activity, and is consistent with our tions (Fig. 2E). To further confirm these findings, we previous observation that DAPK levels do not corre- investigated the effect of Raptor depletion with siRNA late with changes in mTORC1 translational activity. on DAPK levels in serum-starved TSC2 () ⁄ )) cells. Furthermore, TSC2 (1–1516) failed to reduce DAPK Decreased Raptor expression correlated with efficient levels when overexpressed in HEK293 cells, in stark inhibition of mTORC1 activity and reduction of S6 contrast to the reduction observed when TSC2 or phosphorylation on S235 ⁄ 236, however no change in TSC2 (N1693K) were overexpressed (Fig. 3E,G). The DAPK levels was observed (Fig. 2F). Together, these impaired ability of TSC2 (1–1516) to reduce DAPK results clearly demonstrate that DAPK levels are not levels is not due to altered affinity because TSC2, regulated by mTORC1 activity, thus excluding indirect TSC2 (N1693K) and TSC2 (1–1516) immunoprecipi- effects of TSC2 on DAPK protein levels through tate with DAPK to a similar degree (Fig. 3F,H). These changes in mTORC1 translational control, and suggest results collectively demonstrate that the C-terminus of that it is the stability of DAPK protein that is altered TSC2 is important for regulating its stability, and sug- by TSC2. gest 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 TSC2 GAP activity is not required to reduce is of its GAP activity. DAPK protein levels The C-terminal domain of TSC2, which contains the The TSC2 (1–1516) truncation mutant forms a GAP domain, is critical for its correct activity [23] and complex with TSC1 and DAPK has recently been shown to be important for control of the protein’s stability [29]. Therefore, to gain some Our previous findings could be explained by altered mechanistic insight into how TSC2 might exert its binding of the TSC2 (1–1516) truncation mutant with effect on DAPK, we created a TSC2 truncation TSC1, therefore it was necessary to compare the relative mutant lacking its C-terminus, TSC2 (1–1516), and binding of endogenous TSC1 with TSC2 and TSC2 (1– compared its effect on DAPK levels with a well-char- 1516). For this, we transfected cells with FLAG–TSC2 acterized patient-derived GAP-dead missense mutant or FLAG–TSC2 (1–1516), cell extracts were then TSC2 (N1693K) [23] (Fig. 3A). Consistent with a 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 TSC1 binding N1693K FLAG–TSC2 (1–1516) FLAG–TSC2 (Wild-type) TSC2 0.8 FLAG–TSC2 (N1396K) 1 LZ CC CC GAP 1807 0.6 TSC2 (1–1516) 1 1516 0.4 ** B TSC2 Relative level 0.2 FLAG–TSC2 0.0 *** 0 1 2 3 4 5 6 7 8 Actin Cycloheximide (h) FLAG–TSC2 (N1693K) D TSC2 (–/–) MEF Actin FLAG FLAG–TSC2 (1–1516) DAPK Actin Chx (h) 0246 8 S6 P-S235/236

S6 E Actin TSC2 TSC2 (1–1516) FLAG FLAG–TSC2 – + –– DAPK FLAG–TSC2 (1–1516) – – + – Actin FLAG–TSC2 (N1693K) – – – +

HA-FLAG–DAPK +++ FLAG–TSC2 – + – FLAG–TSC2 (1–1516) – – + G H FLAG IP: HA FLAG HA

F IP: HA HA FLAG TSC2 TSC2 (1–1516) FLAG Actin DAPK Lysate HA Actin HA DAPK +++ Actin FLAG–TSC2 – + – HA-FLAG–DAPK +++ FLAG–TSC2 (N1693K) ––+ HA-FLAG–DAPK ++ FLAG–TSC2 – + – – FLAG–TSC2 (1–1516) – – + FLAG–TSC2 + FLAG–TSC2 (N1693K) – +

Fig. 3. 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 study that mapped the TSC1-binding domain to the (1–1516) coprecipitated with TSC1 to a similar extent N-terminal region of TSC2 [30] (Fig. 3A). To further (Fig. 4A). These results are consistent with a previous demonstrate that TSC2 (1–1516) interacts normally

360 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 with TSC1, we overexpressed FLAG–TSC2 or FLAG– TSC2 IB: FLAG TSC2 (1–1516) in the presence or absence of FLAG– TSC2 (1–1516) IP: TSC1 TSC1. Consistent with previous reports, TSC1 overex- IB: TSC1 pression resulted in increased stability of TSC2 [22] TSC2 and also an equivalent increase in the stability of IB: FLAG TSC2 (1–1516) TSC2 (1–1516) (Fig. 4B). We have shown previously Lysate that DAPK overexpression results in only partial dis- IB: TSC1 ruption of the TSC1–TSC2 complex [21]. We therefore IB: Actin anticipated that DAPK would form a complex with both TSC1 and TSC2 proteins and we wished to deter- FLAG–TSC2 – + mine whether TSC2 (1–1516) retained the ability to FLAG–TSC2 (1–1516) + – form a complex with both DAPK and TSC1. To B evaluate this, cells were transfected with FLAG–TSC2 TSC2 FLAG or FLAG–TSC2 (1–1516), cell extracts were then TSC1 prepared and TSC1 immunoprecipitated with anti- TSC2 (1–1516) FLAG TSC1-speciﬁc IgG. Once again, TSC2 and TSC2 (1– TSC1 1516) coprecipitated with TSC1 to a similar degree Actin (Fig. 4C) and endogenous DAPK was also coprecipitated with each of the TSC complexes (Fig. 4C). Con- FLAG–TSC2 ++ sistent with our previous observations, overexpression – FLAG–TSC1 + of TSC2, but not TSC2 (1–1516), resulted in a reduction in DAPK protein level (Fig. 4C). Taken together, C TSC2 these results exclude the possibility that the loss of IB: FLAG TSC2 (1–1516) activity towards DAPK observed with TSC2 (1–1516) results from altered binding to TSC1 and demonstrate IB: TSC1 IP: TSC1 that DAPK binds to the TSC complex. IB: DAPK

TSC2 IB: FLAG Death domain binding is required for TSC2 to TSC2 (1–1516) reduce DAPK protein levels IB: TSC1 Lysate We have previously shown that the death domain is IB: DAPK the major determinant for the interaction of DAPK with TSC2 [21]. Therefore, we asked whether the IB: Actin effect of TSC2 on DAPK was a direct result of pro- FLAG–TSC2 – + tein binding. To explore this possibility, we made use FLAG–TSC2 (1–1516) + – of a mutant DAPK (DAPK 1–1313) lacking the C-terminal death domain (Fig. 5A). First, to confirm Fig. 4. The TSC2 truncation mutant forms a complex with TSC1 our previous results, we overexpressed DAPK or and DAPK. (A) HEK293 cells were transfected with FLAG–TSC2 or DAPK (1–1313) in cells and evaluated the binding of FLAG–TSC2 (1–1516). Cell lysates were prepared and endogenous TSC1 was immunoprecipitated with TSC1-specific antibodies. endogenous TSC2 by immunoprecipitation. Immuno- Bound proteins were eluted and immunoblotted with antibodies to precipitation confirmed that TSC2 interacts with detect TSC1 or FLAG antibodies to detect TSC2. Direct lysate was DAPK, but not the DAPK mutant lacking the death immunoblotted with antibodies to detect TSC1 and actin or FLAG domain (Fig. 5B). Next, we overexpressed DAPK or antibodies to detect TSC2. (B) HEK293 cells were transfected with DAPK (1–1313) in the presence or absence of coex- FLAG–TSC2 or FLAG–TSC2 (1–1516) in combination with FLAG– pressed TSC2. Although TSC2 overexpression led to TSC1. Cell lysates were prepared and immunoblotted with antibod- a marked reduction in the level of DAPK, it had no ies to detect actin or FLAG antibodies to detect TSC1 and TSC2. (C) HEK293 cells were transfected with FLAG–TSC2 or FLAG– effect on the DAPK (1–1313) mutant lacking the TSC2 (1–1516). Cell lysates were prepared and endogenous TSC1 TSC2-binding domain (Fig. 5C), indicating that bind- was immunoprecipitated with TSC1-specific antibodies. Bound pro- ing to the death domain is required for TSC2 to teins were eluted and immunoblotted with antibodies to detect affect DAPK levels. A recent study demonstrated that DAPK and TSC1 or FLAG antibodies to detect TSC2. Direct lysate the death domain module is important for the control was immunoblotted with antibodies to detect DAPK, TSC1 and of DAPK stability [18], therefore to gain further actin or FLAG antibodies to detect TSC2. 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 Y Lin et al.

A P-loops D Kinase CaM Ankyrin Cyto DD

DAPK FLAG–TSC2 1 378 641 835 1431 HA–DAPK DAPK (1–1313) 1 378 641 835 1313 Actin B Chx (h) 02468 02468 TSC2 IP: HA HA E FLAG–TSC2

TSC2 HA–DAPK (1–1313) Lysate HA Actin

Actin Chx (h) 0 2 46 8 024 68

HA–DAPK – + HA–DAPK (1–1313) + – DAPK F DAPK (+TSC2) DAPK 1–1313 DAPK 1–1313 (+TSC2) C 1.0 FLAG–TSC2 0.8 HA * 0.6 Actin

HA–DAPK– + – + 0.4 HA–DAPK (1–1313) + – + – * Relative DAPK level DAPK Relative 0.2 FLAG–TSC2 – – ++ 0.0 0 1 2 3 4 5 6 7 8 Cycloheximide (h)

Fig. 5. 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 half- TSC2 is playing an active role in regulating the level of lives in HEK293 cells treated with the protein synthe- overexpressed DAPK proteins (Fig. S1A,B). Next, we sis inhibitor cycloheximide. Cycloheximide treatment compared the half-life of DAPK in the presence or revealed that DAPK (1–1313) exhibited an extended absence of coexpressed TSC2 and observed that the half-life compared with DAPK (Fig. 5D,E, left-hand stability of DAPK was significantly reduced when panels and quantified in Fig. 5F). Interestingly, the TSC2 was coexpressed (Fig. 5D, right-hand panels and DAPK (1–1313) mutant also exhibited an extended quantified in Fig. 5F). By contrast, TSC2 overexpres- half-life compared with DAPK when introduced into sion had no effect on the stability of the DAPK TSC2 () ⁄ )) MEFs (Fig. S1A,B). These results are (1–1313) mutant lacking the TSC2-binding domain consistent with a recent study demonstrating that (Fig. 5E, right-hand panels and quantified in Fig. 5F). other factors in addition to TSC2 can control the sta- These results clearly demonstrate that the effect of bility of DAPK via the death domain [18]. Impor- TSC2 on DAPK is dependent on binding to the death tantly, however, the stability of the wild-type DAPK domain of DAPK and are consistent with our previous protein is increased in TSC2 () ⁄ )) cells compared study showing that the DAPK (1–1313) mutant has with HEK293 cells, confirming that endogenous lost the ability to stimulate mTORC1 activity [21].

362 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 p53 pulldown

HA–DAPK Actin

MG132 –+ Fig. 6. DAPK is regulated by the lysosome pathway. (A) HEK293 cells were treated C Lysate with 10 lM MG132 for 6 h. Cell extracts DAPK HA–DAPK were prepared and immunoblotted with antibodies to detect endogenous DAPK, p53 or Actin MG132 –+ actin. (B) HEK293 cells were transfected Leupeptin – +– – with HA–DAPK and His–ubiquitin. Following E-64 d ––+ – D transfection cells were treated with MG132 2.0 Chloro ––– + (10 lM) for 6 h. DAPK ubiquitination was analysed by His–ubiquitin capture on Ni-aga- 1.5 rose beads followed by immunoblotting with E DAPK 1.0 HA antibodies. Direct lysates were immunoblotted for DAPK with HA antibodies. (C) 0.5 Actin

HEK293 cells were left untreated as control level DAPK Relative or incubated in the presence of leupeptin Chloro – + + 0.0 -1 Leupeptin (200 lM), E64D (10 lgÆmL ) or chloroquine – +–– MG132 – – + (100 lM) for 24 h. Cell lysates were immu- E64D ––+ – noblotted with antibodies to detect the lev- Chloro –––+ els of endogenous DAPK or actin. (D) G Quantiﬁcation of DAPK protein levels from DAPK F 2.0 (C). Results are reported as the mean ± SD p62 (n = 3). (E) HEK293 cells were left untreated 1.5 as control or incubated in the presence of LC3-I 1.0 chloroquine (100 lM) for 24 h, or combined LC3-II chloroquine (100 lM, 24 h) and MG132 Actin 0.5 (10 lM, 6 h). Cell lysates were prepared and Relative DAPK level DAPK Relative immunoblotted with antibodies to detect 3-MA – + 0.0 Chloro – + + the levels of endogenous DAPK or actin. (F) H Quantiﬁcation of DAPK protein levels from MG132 – – + DAPK (E). Results are reported as the mean ± SD (n = 3). (G) HEK293 cells were treated with p62 10 mM 3-MA for 6 h. Cell lysates were prepared and immunoblotted with antibodies to ATG7 detect endogenous DAPK, p62, LC3 and actin. (H) HEK293 cells were transfected LC3-I LC3-II with 20 lM ATG7 siRNA or control siRNA for 48 h. Cell lysates were prepared and Actin immunoblotted with antibodies to detect endogenous ATG7, DAPK, p62, LC3 and siRNA con + – actin. siRNA ATG7 –+

Moreover, the reduction in DAPK half-life observed [11,15–19]. To test this, cells were treated with the upon TSC2 expression provides strong evidence that proteasome inhibitor MG132 and the levels of endog- TSC2 is promoting the degradation of DAPK. enous DAPK determined by western blot. MG132 treatment did not signiﬁcantly alter the level of DAPK, however, longer exposure of the ﬁlm did DAPK is regulated by the lysosomal pathway reveal the presence of a slower migrating smear indic- It has been reported previously that DAPK stability ative of ubiquitination (Fig. 6A). We used p53 as a is regulated by the ubiquitin–proteasome pathway 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 TSC2 promotes the degradation of DAPK through subject to regulation via the ubiquitin–proteasome the lysosomal pathway pathway, cells were transfected with HA–DAPK in combination with His–Ub, precipitated using nickel To determine whether TSC2 promotes the degradation beads and western blotted with HA antibodies. The of DAPK through the ubiquitin–proteasome or lyso- addition of MG132 resulted in the linkage of multiple some pathway we compared the effectiveness of pro- ubiquitin adducts to DAPK compared with untreated teasome inhibition with that of lysosome inhibition to cells, however, little change in the stability of exoge- block TSC2 effect on DAPK. First, cells coexpressing nous DAPK was observed (Fig. 6B). We have previ- DAPK and TSC2 were cultured in the presence or ously reported that the lysosomal protease absence of the proteasome inhibitor MG132. TSC2 cathepsin B negatively regulates protein levels of expression in the absence of MG132 resulted in a sig- DAPK [12] and the alternative splice variant nificant reduction in DAPK protein (Fig. 7A and of DAPK, s-DAPK, promotes the destabilization of quantified in Fig. 7B). The addition of MG132 had no DAPK in a proteasome-independent manner [20]. effect on the level of exogenous TSC2 or DAPK, and Given the minor effects of MG132 treatment on had little effect in blocking the degradation of DAPK DAPK levels, we wanted to determine whether DAPK promoted by TSC2 (Fig. 7A,B). As a control, we used stability is regulated via an alternate pathway, such as p53, levels of which are clearly increased upon prote- the lysosomal degradation pathway. Cells were there- asomal inhibition (Fig. 7A). Next, to determine fore treated with the lysosome inhibitors leupeptin, whether TSC2 promotes the degradation of DAPK via E64D and chloroquine, and the levels of endogenous the lysosomal degradation pathway, we coexpressed DAPK were determined by western blot. Treatment DAPK and TSC2 in the presence or absence of the with each of these inhibitors led to a clear increase in lysosome inhibitor chloroquine. TSC2 expression in DAPK protein levels (Fig. 6C and quantified in the absence of chloroquine again resulted in a clear Fig. 6D), whereas combined proteasome and lysosome reduction in DAPK protein (Fig. 7C and quantified in inhibition resulted in little change in DAPK levels Fig. 7D). By contrast to MG132, the addition of chlo- when compared with lysosome inhibition alone roquine dramatically increased the levels of TSC2 and (Fig. 6E and quantified in Fig. 6F). To determine DAPK and was very effective in blocking the degrada- whether DAPK is regulated by the autophagy–lyso- tion of DAPK promoted by TSC2 (Fig. 7C,D). These some pathway, cells were treated with the phosphati- results further establish that DAPK is subject to regu- dylinositol 3-kinase inhibitor 3-methyladenine (3-MA), lation by the lysosome, and indicate that the lysosome an established autophagy inhibitor [31]. In accordance is the major pathway through which TSC2 promotes with our previous study [32], cells were treated with the degradation of DAPK. 10 mm 3-MA for 6 h, cell extracts were prepared and immunoblotted for endogenous DAPK, p62 SQSTM1 ⁄ Discussion (p62; a protein that is destroyed within the autolyso- some [33]) and the conversion of LC3-I to its mem- DAPK is a large protein that consists of several modu- brane-associated lipidated form LC3-II, which can be lar domains that enable it to function in a diverse used to monitor autophagy levels [34]. 3-MA treat- range of signal transduction pathways such as cell sur- ment resulted in an increase in p62 levels and a reduc- vival, apoptosis and autophagy. Given the size of tion in LC3-II consistent with autophagy inhibition, DAPK it is not surprising that almost 20 DAPK-bind- however, no change in the level of DAPK was ing proteins have now been identified [2]. From these observed (Fig. 6G). Prolonged 3-MA treatment up to recent studies, it is becoming increasingly clear that 24 h had no effect on DAPK levels (data not shown). DAPK plays additional roles beyond cell death, and In order to further assess the effect of autophagy inhi- that mechanisms regulating protein stabilization and bition on DAPK levels we silenced Atg7, a critical turnover are critical for modulating DAPK activities. gene required for autophagy [34] using siRNA. ATG7 Our previous studies have identified an interaction protein was significantly reduced by siRNA treatment, between TSC2 and the death domain of DAPK, uncov- resulting in an increase in p62 levels and a reduction ered a role for DAPK in the control of growth factor in LC3-II consistent with autophagy inhibition signalling to mTORC1 and have implicated the lyso- (Fig. 6H). Again, no change in the level of DAPK some pathway in the control of DAPK degradation was observed (Fig. 6H). Taken together, these results [12,20,21]. With this report, we have extended these suggest that DAPK stability is subject to lysosome- previous studies and now show that TSC2 negatively dependent but autophagy-independent regulation. regulates DAPK by promoting its lysosome-dependent

364 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 *** HA–DAPK 1.0 FLAG–TSC2

p53 0.5

Actin Relative DAPK level DAPK Relative 0.0 – HA–DAPK + + + –+ HA–DAPK + + + + FLAG–TSC2 – ++– ++ FLAG–TSC2 – + – + MG132 – ––+++ MG132 – – + +

C D * 2.5 HA–DAPK 2.0 1.5 FLAG–TSC2 1.0

Actin 0.5 Relative DAPK level DAPK Relative 0.0 HA–DAPK + ––+ + + HA–DAPK + + + + FLAG–TSC2 – + + – + + FLAG–TSC2 – + – + Chloro ––– + + + Chloro – – + +

Fig. 7. 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 6 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 HSC70-interacting protein E3-ubiquitin ligase interacts presented that support this conclusion. First, DAPK with the kinase domain of DAPK indirectly via Hsp90 protein, but not mRNA levels, inversely correlate with to promote DAPK polyubiquitination and proteasomal TSC2 expression. Second, TSC2 affects DAPK protein degradation [16,17]. In addition the cullin-3 substrate levels in an mTORC1-independent manner. Third, adaptor KLHL20 interacts with the death domain of TSC2 expression significantly reduces the half-life of DAPK to mediate DAPK polyubiquitination and prote- DAPK. Finally, DAPK is stabilized by lysosome asomal degradation to control interferon responses [18]. inhibitors and the effect of TSC2 on DAPK stability Protein phosphatase 2A has also recently been shown to can be blocked by lysosome inhibition. Our study negatively regulate DAPK levels by enhancing protea- therefore establishes important functions of TSC2 and some-mediated degradation of the kinase [19]. DIP- the lysosomal-degradation pathway in the control of 1 ⁄ Mib1 and C-terminal HSC70-interacting protein DAPK stability. E3-ubiquitin ligase have been shown to stimulate DAPK To date, studies investigating how DAPK is targeted degradation in response to TNF-a and geldanamycin for degradation at the molecular level have focused on treatment [15,17]. By contrast, KLHL20-mediated the ubiquitin–proteasome degradation pathway with DAPK ubiquitination and degradation are suppressed three E3-ligases having been identified that regulate in cells treated with IFN-a or IFN-c, which induces the DAPK ubiquitination, DIP-1 ⁄ Mib1, C-terminal sequestration of KLHL20 into promyelocytic leukaemia HSC70-interacting protein E3-ubiquitin ligase and the (PML) nuclear bodies resulting in the stabilization of KLHL20–Cul3–ROC1 E3 ligase [11,15–18]. The bind- DAPK [18]. ing of the ring finger containing E3 ligase DIP-1 ⁄ Mib1 This study has revealed that the degradation of to the ankyrin repeats region can promote the polyubiq- DAPK by TSC2 is constitutive and occurs in cells grow- uitination and proteasomal degradation of DAPK ing without stress. At present, it is unclear whether deg- [11,15]. The U-box containing E3 ligase C-terminal radation 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. DAPK is one of the key domains central to its signalling TSC2 is directly phosphorylated by AKT and by ERK, function and was identified as one of four functional resulting in functional inactivation of the TSC1–TSC2 domains required for DAPK to exert its growth- complex and mTORC1 activation [24]. We have previ- suppressing activity [37]. The death domain of DAPK ously demonstrated that DAPK protein levels are unaf- regulates its proapoptotic function, in part by interact- fected by stimulation of cells with epidermal growth ing with the mitogen-activated protein kinase ERK factor, which preferentially activates the RAS–mitogen- [38], which is in turn abrogated by a common poly- activated protein kinase (MEK)–extracellular signal- morphism in DAPK at codon 1347 [39]. The death regulated kinase (ERK) pathway, or insulin which pref- domain of DAPK also regulates its proapoptotic func- erentially activates the phosphatidylinositol 3-kinase– tion by interacting with tumour necrosis superfamily AKT signalling pathway [21]. Furthermore, we observed members TNFR1 and FADD [40], in addition to the no change in DAPK levels in response to serum-starva- netrin-1 receptor UNC5H2 [41]. Our identification of tion or upon treatment of cells with the endoplasmic TSC2 as a new binding protein for DAPK uncovered reticulum (ER)-stress inducer tunicamycin (Fig. S2A,B). additional roles for the death domain beyond the Thus to date, the only cellular stresses that have been control of cell death [21]. It is now becoming clear that shown to clearly result in altered stability of DAPK are the death domain is also important for the regulation the cytokines TNF-a and IFN-a ⁄ -c [11,18]. The molecu- of DAPK stability [18] and it will be of great interest lar mechanisms that control DAPK stability in response to determine how this domain coordinates distinct to TNF-a and IFN-a ⁄ -c have already been defined pathways to control the balance between the degrada- [11,18] and it will be interesting to determine whether tion of DAPK with its proapoptotic and prosurvival the novel DAPK–TSC2 degradation loop we have activities. Although the molecular mechanisms described impinges on these pathways. employed by TSC2 to target DAPK for degradation Consistent with the observation that DAPK has a are not yet clear, our results demonstrate that binding relatively long half-life in cells growing without stress is required for TSC2 to exert its effect on DAPK, sug- [11,12], we observed a significant increase in the gesting that the proteins may be co-degraded. The DAPK level upon lysosome inhibition (Figs 6C and observation that a highly stable TSC2 mutant can 7C). These results are in agreement with our previous retain binding to DAPK, but cannot promote DAPK studies [12,20] and confirm that DAPK is subject to degradation, adds further support to this idea. It is regulation by both ubiquitin–proteasome and lysosome intriguing that the death domain of DAPK is the focal degradation pathways. Lysosomes receive their sub- point necessary for both KLHL20–Cul3–ROC1 strates through endocytosis, phagocytosis or from E3-ligase and TSC2 to mediate DAPK degradation. within the cell via autophagy [35]. Surprisingly, we did Thus, a crucial question is whether ubiquitination or not observe any change in DAPK level upon treatment modification with other small ubiquitin-like proteins with the autophagy inhibitor 3-MA, or upon silencing such as SUMO or NEDD can signal DAPK for degra- of the essential autophagy gene Atg7. Furthermore, dation via lysosome-dependent degradation pathways. treatment of cells with rapamycin, a potent inducer of Clearly, more work is required to delineate further autophagy, has no observable effect on DAPK level. the role of the death domain in the control of DAPK Although these results are intriguing, a great deal of stability. future work is required to substantiate these initial In summary, we have shown that TSC2 can promote observations and determine the role that autophagy the lysosome-dependent degradation of DAPK under plays in the control of DAPK degradation. If auto- normal growth conditions. Taken together with our phagy is not involved then the question remains how previous study [21], we can conclude that the death DAPK is transported to the lysosome for degradation. domain of DAPK forms the key binding site that Microtubules form an interconnected network that stabilizes the DAPK–TSC2 complex and leads to at serves as tracks for intracellular movement of cargo to least two distinct outcomes. If the signalling threshold late endosomes and lysosomes [36]. We have previ- is driven towards proliferation and growth, then ously identified an interaction between DAPK and DAPK functions as an inhibitory kinase for TSC2, microtubule-associated protein 1B [32], therefore one releasing mTORC1 from negative regulation by the attractive possibility is that DAPK is transported to TSC complex (Fig. 8). Conversely, a constitutive the lysosome for degradation along the microtubule DAPK inhibitory pathway is coordinated by TSC2 to network. drive the balance of regulation, resulting in the The death domain is a signalling module present in destabilization of DAPK protein (Fig. 8). The recipro- many proapoptotic proteins. The death domain of cal regulation we have identified between DAPK and

366 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 (University of Cardiff, UK). For the generation of FLAG- tagged TSC2 (1–1516) a stop codon was introduced at TSC1 Growth DAPK amino acid 1517 using the primers: Fwd 5¢-CGACGAGTC TSC2 factors AAACTAGCCAATCCTGCTG-3¢; Rev 5¢-CAGCAGGAT TGGCTAGTTTGACTCGTCG-3¢. Phosphorylation Rheb Cell culture, transfection and immunoblotting Excessive growth mTORC1 factor signalling, HEK293 and A549 (which express TSC2 and active DAPK) IFN-γ, IFN-γ, TGF-β, ER-stress DNA damage, and TSC2 MEFs (a gift from Andrew Tee, University of Car- Oncogenes, ER-stress diff, UK) were grown in Dulbecco’s modiﬁed Eagle’s med- P -T389 ium (Gibco, Rockville, MD, USA) supplemented with 10% S6K FCS (Gibco) at 37 C in a 5% CO2 ⁄ H2O-saturated atmo- sphere. Cells for transient transfection were plated out 24 h 6 P -S235/236 before transfection at 1.5 · 10 cells per 100 mm dish or 5 S6 5 · 10 cells per 60 mm dish. For Lipofectamine 2000 transfection (Invitrogen, Carlsbad, CA, USA), 2 lL of Lipofecta- mine was used for every 1 lg of DNA transfected. Cells were Cell growth Autophagy Apoptosis protein synthesis harvested after a further incubation of 16–18 h. Cells were lysed in ice-cold extraction buffer (50 mm Tris pH 7.6,

Fig. 8. DAPK and TSC2 form a regulatory feedback loop. Recent 150 mm NaCl2,5mm EDTA, 0.5% NP-40, 5 mm NaF, advances have established an important role for DAPK in a diverse 1mm sodium vanadate, 1 · protease inhibitor cocktail) for range of signal-transduction pathways including growth factor sig- 30 min and centrifuged at 18 894 g for 15 min to remove naling, apoptosis, autophagy and membrane blebbing. DAPK has insoluble material. The protein content of cell extracts was been shown to function as a positive mediator of apoptosis induced measured using Bio-Rad reagent (Bio-Rad Labs, Hercules, by various stimuli including the transforming oncogenes c-myc and CA, USA). Typically, 20–30 lg of cell extract was used for E2F1, IFN-c, transforming growth factor beta, DNA damage, ER-stress and excessive growth factor signaling. DAPK also plays a immunoblot. Samples were resolved by denaturing gel elec- role in survival pathways reﬂected in its autophagy-signalling activ- trophoresis, typically 4–12% precast gels (Novex, Invitrogen, ity. A substantial amount of research has demonstrated that the Carlsbad, CA, USA) and electrotransferred to Hybond TSC proteins form a complex that inhibits mTORC1 activity leading C-extra nitrocellulose membrane (Amersham Biosciences, to reduced protein synthesis and cell growth. The pathway that Buckinghamshire, UK), blocked in NaCl ⁄ Pi–10% nonfat regulates autophagy also acts through mTORC1. Our own data milk for 30 min, then incubated with primary antibody over- show that DAPK binds to and catalyses the phosphorylation of night at 4 C in NaCl ⁄ Pi–5% nonfat milk–0.1% Tween-20. TSC2, thus inactivating the TSC complex and stimulating mTORC1 After washing (3 · 10 min) in NaCl ⁄ Pi–Tween-20, the blot activity in a growth factor-dependent pathway. Reciprocally, a con- was incubated with secondary antibody, either horseradish stitutive DAPK inhibitory pathway is coordinated by TSC2 to drive peroxidase-conjugated anti-rabbit or anti-mouse IgG (Dako, the lysosome-dependent degradation of DAPK. Carpinteria, CA, USA; 1 : 5000), for 1 h at room tempera-

ture in NaCl ⁄ Pi–5% nonfat milk–0.1% Tween-20. After washing (3 · 10 min) in NaCl ⁄ Pi–Tween-20, proteins were TSC2 proteins constitutes a feedback loop that may visualized by incubation with ECL western blotting analysis regulate the level of mTORC1 activity and ultimately system (Amersham Biosciences) or Immobilon western control cell outcomes, including growth, autophagy chemiluminescent HRP substrate (Millipore Corp., Bedford, and apoptosis all of which are relevant to the patho- MA, USA). Equal protein loading was conﬁrmed with Pon- genesis of many human diseases. ceau S staining. FLAG antibody (M2) and actin antibody were purchased from Sigma (St. Louis, MO, USA). HA-11 Materials and methods antibody (Ascites) was purchased from Covance (Princeton, NJ, USA). Tuberin ⁄ TSC2 (C-20) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Plasmids DAPK antibody was purchased from BD Transduction Lab- Dual N-terminal FLAG–HA–DAPK vector was a gift of oratories (Lexington, KY, USA). DAPK (clone-55, Ascites) Adi Kimchi (Weizmann Institute, Rehovot, Israel). Genera- and Phospho-DAPK (S308) were purchased from Sigma. tion of the FLAG–HA–DAPK (1–1313) deletion mutant Phospho-p70 S6 kinase (Thr389), p70 S6 kinase, phospho-S6 has been described previously [12]. FLAG–TSC1, FLAG– (S235 ⁄ 236), S6, Hamartin ⁄ TSC1 (1B2), Rheb, Raptor, Bip TSC2, FLAG–TSC2 (N1693K), FLAG–Rheb, HA–Raptor and ATG7 antibodies were purchased from Cell Signal- and HA–Raptor mutant 4 were all gifts from Andrew Tee ing 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) His–ubiquitin lysis buffer (guanidinium–HCl 6 m, antibody was a gift from Borek Vojtesek (Masaryk Memorial Na2HPO4 95 mm, NaH2PO4 5mm, Tris ⁄ HCl 0.01 m Cancer Institute, Brno, Czech Republic). pH 8.0, 5 mm imidazole and 10 mm b-mercatoethanol) supplemented with 75 lL of Ni-agarose beads (Qiagen, Valencia, CA, USA) overnight at 4 C. Beads were washed Immunoprecipitation for 5 min in each of the following wash buffers: 6 m guanidi- For immunoprecipitation of exogenous HA–DAPK from um–HCl, Na2HPO4 95 mm, NaH2PO4 5mm, Tris ⁄ HCl cells, HA-11 antibody bound to 30 lL of protein G beads 0.01 m pH 8.0 (buffer A); 8 m urea, Na2HPO4 95 mm, m m m (Amersham) was incubated overnight at 4 C with con- NaH2PO4 5m , Tris ⁄ HCl 0.01 pH 8.0 and 10 m b-mer- m m stant rotation, together with cell extract ( 1 mg) diluted catoethanol (buffer B); 8 urea, Na2HPO4 22.5 m , to a volume of 500 lL in extraction buffer (50 mm Tris NaH2PO4 77.5 mm, Tris ⁄ HCl 0.01 m pH 6.3 and 10 mm pH 7.4, 150 mm NaCl2,5mm EDTA, 0.5% NP-40, 5 mm b-mercatoethanol (buffer C); buffer C with 0.2% Tri- NaF, 1 mm sodium vanadate, 1 · protease inhibitor cock- ton X-100; buffer C with 0.1% Triton X-100. The Ni-bead tail). The bead pellets were then washed ﬁve times in conjugated proteins were then eluted in 75 lL elution buffer extraction buffer before being resuspended in 3 · SDS- (imidazole 200 mm, SDS 5%, Tris ⁄ HCl 0.15 m pH 6.7, loading buffer and analysed by denaturing gel electro- glycerol 30% and b-mercaptoethanol 0.72 m) for 30 min at phoresis and immunoblotting. For immunoprecipitation of room temp. Eluates were mixed in 1 : 1 ratio with SDS endogenous TSC1 from cells, the same procedure was sample buffer and were analysed by 4–12% NuPAGE ⁄ followed except TSC1 antibodies were bound to protein G immunoblot and probed with HA antibody. beads. RNA extraction and real-time PCR siRNA mRNA was extracted from cells using the QIAGEN For the knockdown of gene expression cells were transfect- RNeasy Mini kit following the manufacturer’s suggested ed with TSC2 siRNA (Cell Signaling Technology), ATG7 procedures. The optional step of DNase treatment using siRNA (Cell Signaling Technology), Rheb siRNA (Dharm- the QIAGEN RNase-free DNase set was also included. acon, Lafayette, CO, USA), Raptor siRNA (Dharmacon) One microlitre of the sample RNA was diluted in 100 lL or a non-speciﬁc siRNA as control (Dharmacon). After water and loaded into a 96-well UV plate. Absorbance was 48 h, cells were lysed in ice-cold extraction buffer (50 mm detected with a Bio-Tek plate reader at 260 nm. After the

Tris pH 7.6, 150 mm NaCl2,5mm EDTA, 0.5% NP-40, extraction, the real-time PCR were performed in the Opti- 5mm NaF, 1 mm sodium vanadate, 1 · protease inhibitor con 4 machine using the QIAGEN QuantiTect SYBR cocktail) for 30 min and centrifuged at 18 894 g for 15 min Green one-step PCR kit. Equal amounts of mRNA were to remove insoluble material. Samples were resolved loaded in each reaction. by denaturing gel electrophoresis followed by immuno- The actin primers used were: L: 5¢-CTACGTCG blotting. CCCTGGACTTCGAGC-3¢,R:5¢-GATGGAGCCGCCG ATCCACACGG-3¢. The DAPK primers were: L: 5¢-CGAGGTGATGGTG Drug treatments TATGGTG-3¢;R:5¢-CTGTGCTTTGCTGGTGGA-3¢. The following drugs were added to cell media at the indi- m cated concentrations; MG132 (10 l ; Calbiochem, San Statistical analysis Diego, CA, USA), leupeptin (200 lm; Calbiochem), E-64d (10 lgÆmL)1; Calbiochem), chloroquine (100 lm; Invitro- Scanning densitometry was performed using scion image gen), 3-MA (10 mm; Calbiochem), tunicamycin (1 lgÆmL)1; software (National Institutes of Health). Results are Sigma) and cycloheximide (10 lgÆmL)1; Supleco, Bellefonte, reported as the mean ± SD with P-values calculated using PA, USA). an unpaired t-test in graphpad v4.03 (GraphPad Software, CA, USA).

Cell-based ubiquitination assays Acknowledgements HEK293 cells were transfected with the appropriate con- structs for 24 h, then treated with MG132 (10 lm) for 6 h We thank Andrew Tee and Adi Kimchi for reagents. prior to harvesting. Twenty per cent of the cell suspension This work was funded by a CRUK Programme grant was used for direct western blot analysis with HA antibod- to TH (C483 ⁄ A6354). CS is funded by an MRC ies. For the puriﬁcation of His–ubiquitinated conjugates the project grant (G0800759). PH is funded by an MRC remainder of the cell suspension was lysed in 5 mL of project grant (G0800675).

368 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

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