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

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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 demon- strate 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 lyso- some pathway and that lysosome inhibition blocks TSC2-mediated degra- dation of DAPK. Our study therefore establishes important functions of TSC2 and the lysosomal-degradation pathway in the control of DAPK sta- bility, 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 degra- dation 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 (uni- protkb:P49815)byanti tag coimmunoprecipitation (MI:0007) l MINT-8057213: TSC1 (uniprotkb:Q92574) physically interacts (MI:0914) with DAPK (uni- protkb:P53355) and TSC2 (uniprotkb:P49815)byanti bait coimmunoprecipitation (MI:0006) l MINT-8057200: TSC1 (uniprotkb:Q92574) physically interacts (MI:0915) with TSC2 (uni- protkb: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
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