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 Lin
1
, Paul Henderson
1,2
, Susanne Pettersson
1
, Jack Satsangi
1
, Ted Hupp
1
and Craig Stevens
1
1 University of Edinburgh, Institute of Genetics and Molecular Medicine, UK
2 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:
(Received 28 July 2010, revised 7 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 1 (mTORC1) activity.
We now report that TSC2 binding to DAPK promotes the degradation of
DAPK. We show that DAPK protein levels, but not gene expression,
inversely correlate with TSC2 expression. Furthermore, altering mTORC1
activity does not affect DAPK levels, excluding indirect effects of TSC2 on
DAPK protein levels through changes in mTORC1 translational control.
We provide evidence that the C-terminus regulates TSC2 stability and is
required for TSC2 to reduce DAPK protein levels. Importantly, using a
GTPase-activating protein–dead missense mutation of TSC2, we demon-
strate that the effect of TSC2 on DAPK is independent of GTPase-activat-
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
Introduction
Death-associated protein kinase-1 (DAPK) is the pro-
totypic member of a family of death-related kinases
that includes DAPK-1-related protein 1 (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 includ-
ing a kinase domain, a calmodulin regulatory domain,
eight consecutive ankyrin repeats, two putative nucleo-
tide-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 identi-
fied 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 sug-
gests 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 necro-
sis factor (TNF)-mediated apoptosis [11,12].
Post-transcriptional mechanisms regulating protein
translation, stabilization and turnover are also critical
for modulating DAPK activities. For example, transla-
tional 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, alterna-
tively spliced form of DAPK (s-DAPK) destabilizes
DAPK in a proteasome-independent manner [20].
In a previous study [21], we performed a protein-
interaction screen to identify novel DAPK death-
domain-interacting proteins and identified tuberous
sclerosis-2 (TSC2) as one such protein. We demon-
strated 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 fac-
tor-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 dis-
ease tuberous sclerosis. TSC2 contains a GTPase-acti-
vating 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 kin-
ases 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, phos-
phorylation and activation of eukaryotic initiation fac-
tor 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 anti-
biotic 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 balanc-
ing 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 bal-
ance may influence the extent of mTORC1 signalling
by either stimulating TSC2 inactivation via DAPK
Y Lin et al. TSC2 promotes the degradation of DAPK
FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 355
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 death-
domain-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 inves-
tigate this observation, we first evaluated the effect of
overexpressing increasing amounts of TSC2 on the lev-
els of endogenous DAPK protein. TSC2 overexpression
led to a significant reduction in the level of DAPK pro-
tein in a dose-dependent manner (Fig. 1A and quanti-
fied 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 rele-
vance 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 reconsti-
tuted 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 compara-
ble with TSC2 (+ ⁄ +) control cells (Fig. 1E), confirm-
ing that the elevated levels of DAPK observed are a
direct result of TSC2 loss. DAPK activity is auto-inhib-
ited by auto-phosphorylation on Ser308 within its
calmodulin regulatory-binding domain [1]. To deter-
mine whether the TSC2 regulatory effect on DAPK is
important functionally, we assessed DAPK activity by
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 overexpres-
sion 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 inhibi-
tor rapamycin. Rapamycin treatment efficiently inhib-
ited 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 phosphoryla-
tion 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
TSC2 promotes the degradation of DAPK Y Lin et al.
356 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS
0.25 0.50 0.75 1.0 2.0
0.0
0.2
0.4
0.6
0.8
1.0
**
Concentration of FLAG–TSC2 (µg)
Relative DAPK level
B
D
DAPK
Actin
(+/+)
TSC2
S6K P-T389
S6K
(–/–)
TSC2 MEF

C
TSC2
DAPK
S6K P-T389
S6K
Actin
siRNA con
siRNA TSC2
–
+
–
+
A
FLAG–TSC2
DAPK
Actin
FLAG–TSC2 (µg)
0.25 0.5 0.75 1.0 2.0
H
DAPK
FLAG–TSC2
Actin
FLAG–TSC2 (µg)
02.0
G
0 2.0
0.0
0.5
1.0
1.5

NS
DAPK : actin mRNA ratio
Concentration of FLAG–TSC2 (µg)
FLAG
DAPK
Actin
TSC2
FLAG–TSC2 – +
E
TSC2 (–/–) MEF
F
DAPK
DAPK P-S308
Actin
(+/+) (–/–)TSC2 MEF
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 trans-
fected 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.
Y Lin et al. TSC2 promotes the degradation of DAPK
FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 357
A

DAPK

S6K P-T389
S6K
S6 P-S235/236
S6
Actin
RAPA
–
+
B
DAPK
S6 P-S235/236
S6
Actin
RAPA (h)
01246816
TSC2 (–/–) MEF
C
DAPK
Actin
S6 P-S235/236
S6
FLAG–RHEB
FLAG-RHEB
RAPA
–
+
–
+

–
–
+
+
Serum-starved
D
RHEB
DAPK
siRNA con
Serum-starved
Actin
S6 P-S235/236
S6
siRNA RHEB
–
+
–
+
E
HA-Raptor
HA-Raptor MT4
–
+
–
+
–
+
–
+
–

+
–
+
DAPK
HA
S6 P-S235/236
S6 P-S235/236
(short exposure)
Actin
S6
Serum-starved
Rapamycin
–
–
–
–
++
F
Raptor
DAPK
Serum-starved
Actin
S6 P-S235/236
S6
siRNA con
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 n
M rapamycin for the indicated time. Cell lysates were prepared and immunoblotted with antibod-
ies 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 n
M rapamycin for 2 h where indicated. Cell lysates were prepared and immunoblotted with anti-
bodies to detect endogenous DAPK, S6 P-S235 ⁄ 236, S6 and actin or FLAG antibodies to detect Rheb. (D) TSC2 () ⁄ )) MEFs were transfect-
ed 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 n
M 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.
TSC2 promotes the degradation of DAPK Y Lin et al.
358 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS
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 par-
tial inhibition of mTORC1 activity and reduction of
S6 S235 ⁄ 236 phosphorylation, likely because of func-
tional redundancy between Rheb and RhebL1 [27],
and again no change in DAPK level was observed
(Fig. 2D). Next, we investigated the effects of overex-

pressing Raptor or a mutant Raptor (Raptor mutant
4) that interferes with mTORC1 substrate recognition
[28]. In cells growing in full serum, expression of Rap-
tor mutant 4 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 4 overexpression failed
to alter phosphorylation of S6 on S235 ⁄ 236 (Fig. 2E).
Similarly, expression of Raptor or Raptor mutant 4 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 condi-
tions (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-char-
acterized patient-derived GAP-dead missense mutant
TSC2 (N1693K) [23] ( Fig. 3A). Consistent with a
previous study [29], cycloheximide treatment revealed
that TSC2 is a short-lived protein with a half-life of
 2 h under normal growth conditions (Fig. 3B and
quantified in Fig. 3C). The GAP-dead mutant
TSC2 (N1693K) exhibited a half-life similar to wild-
type TSC2 (Fig. 3B and quantified in Fig. 3C). By
contrast, TSC2 (1–1516) exhibited a significantly
increased stability with a half-life of  8 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). Reconstitu-
tion of TSC2 or TSC2 (N1693K) resulted in a pro-
nounced 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 indepen-
dent of its GAP activity, and is consistent with our
previous observation that DAPK levels do not corre-
late 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) immunoprecipi-
tate 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 sug-
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
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
Y Lin et al. TSC2 promotes the degradation of DAPK

FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 359
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
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
A
TSC1 binding
1
1807
TSC2
TSC2 (1–1516)
CC
GAPLZ
1 1516
CC
N1693K
FLAG–TSC2
Actin
FLAG–TSC2 (1–1516)
Actin
024
6
8
Chx (h)
FLAG–TSC2 (N1693K)
Actin
B
0 1 2 3 4 5 6 7 8

0.0
0.2
0.4
0.6
0.8
1.0
FLAG–TSC2 (Wild-type)
FLAG–TSC2 (N1396K)
FLAG–TSC2 (1–1516)
Cycloheximide (h)
Relative TSC2 level
**
***
C
DAPK
S6 P-S235/236
S6
Actin
FLAG
FLAG–TSC2
FLAG–TSC2 (1–1516)
FLAG–TSC2 (N1693K)
–
+
––
–
+
–
–
+

–
–
–
TSC2 (–/–) MEF
D
H
FLAG–TSC2
HA-FLAG–DAPK
FLAG–TSC2 (N1693K) –
+
+
–
++
FLAG
HA
FLAG
HA
Actin
IP: HA
Lysate
G
FLAG–TSC2
FLAG
HA DAPK
FLAG–TSC2 (N1693K)
––
+
–
+
–

+++
Actin
HA
FLAG
Actin
TSC2 (1–1516)
TSC2
DAPK
FLAG–TSC2
HA-FLAG–DAPK
FLAG–TSC2 (1–1516)
–
–
+
–
+
–
+++
E
FLAG
IP: HA
Actin
TSC2 (1–1516)
TSC2
DAPK
FLAG–TSC2
HA-FLAG–DAPK
FLAG–TSC2 (1–1516)
–
–

+
–
+
–
+++
F
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 antibod-
ies 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.
TSC2 promotes the degradation of DAPK Y Lin et al.
360 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS
with TSC1, we overexpressed FLAG–TSC2 or FLAG–
TSC2 (1–1516) in the presence or absence of FLAG–
TSC1. Consistent with previous reports, TSC1 overex-
pression 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 dis-
ruption 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 deter-
mine 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 anti-
TSC1-specific IgG. Once again, TSC2 and TSC2 (1–
1516) coprecipitated with TSC1 to a similar degree
(Fig. 4C) and endogenous DAPK was also coprecipi-
tated with each of the TSC complexes (Fig. 4C). Con-
sistent with our previous observations, overexpression
of TSC2, but not TSC2 (1–1516), resulted in a reduc-
tion 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.
Death domain binding is required for TSC2 to
reduce DAPK protein levels
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 pro-
tein 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. Immuno-
precipitation 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 coex-
pressed 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 bind-
ing 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
TSC2 (1–1516)
TSC1
TSC2
TSC1
FLAG
FLAG
Actin
FLAG–TSC2
FLAG–TSC1
–
++
+
B

A
IB: FLAG
IB: TSC1
FLAG–TSC2
FLAG–TSC2 (1–1516)
–
+
–
+
TSC2 (1–1516)
TSC2
TSC2 (1–1516)
TSC2
IB: Actin
IB: FLAG
IB: TSC1
IP: TSC1
Lysate
IB: FLAG
IB: TSC1
IB: FLAG
IB: DAPK
IB: DAPK
IB: TSC1
FLAG–TSC2
FLAG–TSC2 (1–1516)
–
+
–+
TSC2 (1–1516)

TSC2
TSC2 (1–1516)
TSC2
IB: Actin
IP: TSC1
Lysate
C
Fig. 4. 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 antibod-
ies 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 pro-
teins 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.
Y Lin et al. TSC2 promotes the degradation of DAPK
FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 361
evaluated DAPK and DAPK (1–1313) protein half-
lives in HEK293 cells treated with the protein synthe-
sis 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 sta-
bility of DAPK via the death domain [18]. Impor-
tantly, however, the stability of the wild-type DAPK
protein is increased in TSC2 () ⁄ )) cells compared
with HEK293 cells, confirming that endogenous
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 overexpres-
sion 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].
A
378 641 835
Kinase

CaM
Ankyrin
Cyto
DD
P-loops
1
1431
378 641 835
1
1313
DAPK
DAPK (1–1313)
B
TSC2
HA
IP: HA
Lysate
HA–DAPK
HA–DAPK (1–1313)
+
–
+
–
TSC2
HA
Actin
C
HA–DAPK (1–1313)
HA
FLAG–TSC2

Actin
HA–DAPK +–
+
–
+–
+
–
FLAG–TSC2 – –
++
0 1 2 3 4 5 6 7 8
0.0
0.2
0.4
0.6
0.8
1.0
DAPK
DAPK (+TSC2)
DAPK 1–1313
DAPK 1–1313 (+TSC2)
*
*
Cycloheximide (h)
Relative DAPK level
F
D
HA–DAPK
Actin
Chx (h)
FLAG–TSC2

02468 02468
E
HA–DAPK (1–1313)
Actin
Chx (h)
0 2 46 8 024 68
FLAG–TSC2
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 antibod-
ies 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, fol-
lowed 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).
TSC2 promotes the degradation of DAPK Y Lin et al.
362 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS
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 endog-

enous 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 indic-
ative of ubiquitination (Fig. 6A). We used p53 as a
control, with levels clearly increased upon proteasomal
A
p53
DAPK
–+MG132
Actin
C
DAPK
Actin
Leupeptin
E-64 d
Chloro
–
+–
–
––
+
–
––
–
+
E
DAPK
Actin
Chloro – + +

MG132 – – +
B
Lysate
His-Ub
pulldown
–+MG132
HA–DAPK
HA–DAP
K
0.0
0.5
1.0
1.5
2.0
Leupeptin
E64D
Chloro
–
+–
–
––
+
–
––
–+
Relative DAPK level
D
0.0
0.5
1.0

1.5
2.0
Chloro
MG132
+
+
+
–
–
–
Relative DAPK level
F
G
3-MA
DAPK
p62
Actin
–
+
LC3-I
LC3-II
H
ATG7
DAPK
p62
Actin
LC3-I
LC3-II
siRNA con
siRNA ATG7

–+
–+
Fig. 6. DAPK is regulated by the lysosome
pathway. (A) HEK293 cells were treated
with 10 l
M MG132 for 6 h. Cell extracts
were prepared and immunoblotted with anti-
bodies 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 l
M) for 6 h. DAPK ubiquitination was
analysed by His–ubiquitin capture on Ni-aga-
rose beads followed by immunoblotting with
HA antibodies. Direct lysates were immu-
noblotted for DAPK with HA antibodies. (C)
HEK293 cells were left untreated as control
or incubated in the presence of leupeptin
(200 l
M), E64D (10 lgÆmL
-1
) or chloroquine
(100 l
M) for 24 h. Cell lysates were immu-
noblotted with antibodies to detect the lev-
els 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 l
M) for 24 h, or combined
chloroquine (100 l
M, 24 h) and MG132
(10 l
M, 6 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 m
M 3-MA for 6 h. Cell lysates were pre-
pared and immunoblotted with antibodies to
detect endogenous DAPK, p62, LC3 and
actin. (H) HEK293 cells were transfected
with 20 l
M 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.
Y Lin et al. TSC2 promotes the degradation of DAPK
FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 363
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 exoge-
nous DAPK was observed (Fig. 6B). We have previ-
ously 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 there-
fore 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–lyso-
some pathway, cells were treated with the phosphati-
dylinositol 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 6 h, cell extracts were prepared and
immunoblotted for endogenous DAPK, p62 ⁄ SQSTM1
(p62; a protein that is destroyed within the autolyso-

some [33]) and the conversion of LC3-I to its mem-
brane-associated lipidated form LC3-II, which can be
used to monitor autophagy levels [34]. 3-MA treat-
ment resulted in an increase in p62 levels and a reduc-
tion 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 inhi-
bition 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 lysosome-
dependent but autophagy-independent regulation.
TSC2 promotes the degradation of DAPK through
the lysosomal pathway
To determine whether TSC2 promotes the degradation
of DAPK through the ubiquitin–proteasome or lyso-
some pathway we compared the effectiveness of pro-
teasome 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 sig-
nificant 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 prote-
asomal 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 chlo-
roquine dramatically increased the levels of TSC2 and
DAPK and was very effective in blocking the degrada-
tion of DAPK promoted by TSC2 (Fig. 7C,D). These
results further establish that DAPK is subject to regu-
lation 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 modu-
lar domains that enable it to function in a diverse
range of signal transduction pathways such as cell sur-
vival, apoptosis and autophagy. Given the size of
DAPK it is not surprising that almost 20 DAPK-bind-
ing 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, uncov-
ered a role for DAPK in the control of growth factor
signalling to mTORC1 and have implicated the lyso-
some 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
TSC2 promotes the degradation of DAPK Y Lin et al.
364 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS
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 bind-
ing of the ring finger containing E3 ligase DIP-1 ⁄ Mib1

to the ankyrin repeats region can promote the polyubiq-
uitination 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 prote-
asomal degradation to control interferon responses [18].
Protein phosphatase 2A has also recently been shown to
negatively regulate DAPK levels by enhancing protea-
some-mediated degradation of the kinase [19]. DIP-
1 ⁄ 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 grow-
ing without stress. At present, it is unclear whether deg-
radation of DAPK by TSC2 can be triggered or indeed
C
HA–DAPK
FLAG–TSC2
Actin

HA–DAPK
FLAG–TSC2
Chloro
+
––
+
++
+
+
–
+
+
–
+
+
––
–
+
A
FLAG–TSC2
HA–DAPK
HA–DAPK
FLAG–TSC2
MG132
+
–
–+
+
+
++

–
++
–
++
–
––+
Actin
p53
B
0.0
0.5
1.0
HA–DAPK
FLAG–TSC2
MG132
+
+
+
+
+
+
+
+
***
–
–
–
–
Relative DAPK level
0.0

0.5
1.0
1.5
2.0
2.5
HA–DAPK
FLAG–TSC2
Chloro
+
+
+
+
+
+
+
+
*
–
–
–
–
Relative DAPK level
D
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 l
M) 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 chloro-
quine (100 l

M) 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).
Y Lin et al. TSC2 promotes the degradation of DAPK
FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 365
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 previ-
ously demonstrated that DAPK protein levels are unaf-
fected by stimulation of cells with epidermal growth
factor, which preferentially activates the RAS–mitogen-
activated protein kinase (MEK)–extracellular signal-
regulated kinase (ERK) pathway, or insulin which pref-
erentially activates the phosphatidylinositol 3-kinase–
AKT signalling pathway [21]. Furthermore, we observed
no change in DAPK levels in response to serum-starva-
tion 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 molecu-
lar 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 sub-
strates 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 auto-
phagy 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 previ-
ously 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
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 growth-
suppressing activity [37]. The death domain of DAPK
regulates its proapoptotic function, in part by interact-
ing with the mitogen-activated protein kinase ERK
[38], which is in turn abrogated by a common poly-
morphism in DAPK at codon 1347 [39]. The death
domain of DAPK also regulates its proapoptotic func-
tion 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 degrada-
tion 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, sug-
gesting 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 degra-
dation 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 recipro-
cal regulation we have identified between DAPK and
TSC2 promotes the degradation of DAPK Y Lin et al.
366 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS
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 patho-
genesis 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). Genera-
tion 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 4 were all gifts from Andrew Tee
(University of Cardiff, UK). For the generation of FLAG-
tagged 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¢.
Cell culture, transfection and immunoblotting
HEK293 and A549 (which express TSC2 and active DAPK)
and TSC2 MEFs (a gift from Andrew Tee, University of Car-
diff, UK) were grown in Dulbecco’s modified Eagle’s med-
ium (Gibco, Rockville, MD, USA) supplemented with 10%
FCS (Gibco) at 37 °C in a 5% CO
2
⁄ H
2
O-saturated atmo-
sphere. Cells for transient transfection were plated out 24 h
before transfection at  1.5 · 10
6
cells per 100 mm dish or
5 · 10
5
cells per 60 mm dish. For Lipofectamine 2000 trans-
fection (Invitrogen, Carlsbad, CA, USA), 2 lL of Lipofecta-
mine was used for every 1 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 NaCl
2
,5mm EDTA, 0.5% NP-40, 5 mm NaF,
1mm sodium vanadate, 1 · protease inhibitor cocktail) for
30 min and centrifuged at 18 894 g for 15 min 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 elec-
trophoresis, 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 ⁄ P
i
–10% nonfat
milk for 30 min, then incubated with primary antibody over-
night at 4 °C in NaCl ⁄ P
i
–5% nonfat milk–0.1% Tween-20.
After washing (3 · 10 min) in NaCl ⁄ P
i
–Tween-20, the blot
was incubated with secondary antibody, either horseradish
peroxidase-conjugated anti-rabbit or anti-mouse IgG (Dako,
Carpinteria, CA, USA; 1 : 5000), for 1 h at room tempera-
ture in NaCl ⁄ P
i
–5% nonfat milk–0.1% Tween-20. After

washing (3 · 10 min) in NaCl ⁄ P
i
–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 Pon-
ceau 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 Lab-
oratories (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 Signal-
ing Technology (Beverly, MA, USA). p62 antibody was
TSC1
mTORC1
S6K
-T389
P
Cell growth
protein synthesis
TSC2
DAPK
Degradation
S6

-S235/236
P
Autophagy
Rheb
Growth
factors
Phosphorylation
Apoptosis
Excessive growth
factor signalling,
IFN-γ, TGF-β,
DNA damage,
Oncogenes,
ER-stress
IFN-γ,
ER-stress
Fig. 8. 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 sig-
naling, 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 activ-
ity. 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 con-
stitutive DAPK inhibitory pathway is coordinated by TSC2 to drive
the lysosome-dependent degradation of DAPK.
Y Lin et al. TSC2 promotes the degradation of DAPK
FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS 367
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 4 °C with con-
stant rotation, together with cell extract ( 1 mg) diluted
to a volume of 500 lL in extraction buffer (50 mm Tris
pH 7.4, 150 mm NaCl
2
,5mm EDTA, 0.5% NP-40, 5 mm
NaF, 1 mm sodium vanadate, 1 · protease inhibitor cock-
tail). The bead pellets were then washed five times in
extraction buffer before being resuspended in 3 · SDS-
loading buffer and analysed by denaturing gel electro-
phoresis and immunoblotting. For immunoprecipitation of
endogenous TSC1 from cells, the same procedure was
followed except TSC1 antibodies were bound to protein G
beads.
siRNA
For the knockdown of gene expression cells were transfect-
ed with TSC2 siRNA (Cell Signaling Technology), ATG7
siRNA (Cell Signaling Technology), Rheb siRNA (Dharm-

acon, 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 NaCl
2
,5mm EDTA, 0.5% NP-40,
5mm NaF, 1 mm sodium vanadate, 1 · protease inhibitor
cocktail) for 30 min and centrifuged at 18 894 g for 15 min
to remove insoluble material. Samples were resolved
by denaturing gel electrophoresis followed by immuno-
blotting.
Drug treatments
The following drugs were added to cell media at the indi-
cated concentrations; MG132 (10 lm; Calbiochem, San
Diego, CA, USA), leupeptin (200 lm; Calbiochem), E-64d
(10 lgÆmL
)1
; Calbiochem), chloroquine (100 lm; Invitro-
gen), 3-MA (10 mm; Calbiochem), tunicamycin (1 lgÆmL
)1
;
Sigma) and cycloheximide (10 lgÆmL
)1
; Supleco, Bellefonte,
PA, USA).
Cell-based ubiquitination assays
HEK293 cells were transfected with the appropriate con-
structs for 24 h, then treated with MG132 (10 lm) for 6 h
prior to harvesting. Twenty per cent of the cell suspension
was used for direct western blot analysis with HA antibod-

ies. For the purification of His–ubiquitinated conjugates the
remainder of the cell suspension was lysed in 5 mL of
His–ubiquitin lysis buffer (guanidinium–HCl 6 m,
Na
2
HPO
4
95 mm, NaH
2
PO
4
5mm, Tris ⁄ HCl 0.01 m
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
for 5 min in each of the following wash buffers: 6 m guanidi-
um–HCl, Na
2
HPO
4
95 mm, NaH
2
PO
4
5mm, Tris ⁄ HCl
0.01 m pH 8.0 (buffer A); 8 m urea, Na
2
HPO
4
95 mm,

NaH
2
PO
4
5mm, Tris ⁄ HCl 0.01 m pH 8.0 and 10 mm b-mer-
catoethanol (buffer B); 8 m urea, Na
2
HPO
4
22.5 mm,
NaH
2
PO
4
77.5 mm, Tris ⁄ HCl 0.01 m pH 6.3 and 10 mm
b-mercatoethanol (buffer C); buffer C with 0.2% Tri-
ton 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 min at
room temp. Eluates were mixed in 1 : 1 ratio with SDS
sample buffer and were analysed by 4–12% NuPAGE ⁄
immunoblot and probed with HA antibody.
RNA extraction and real-time PCR
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 Opti-
con 4 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).
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).
TSC2 promotes the degradation of DAPK Y Lin et al.
368 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS
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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
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from supporting information (other than missing files)
should be addressed to the authors.
TSC2 promotes the degradation of DAPK Y Lin et al.
370 FEBS Journal 278 (2011) 354–370 ª 2010 The Authors Journal compilation ª 2010 FEBS