Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK and actin or FLAG antibodies to detect TSC2.. Following transfection, cell lysates were prepared an
Trang 1death-associated protein kinase-1 (DAPK) through a
lysosome-dependent degradation pathway
Yao Lin1, Paul Henderson1,2, Susanne Pettersson1, Jack Satsangi1, Ted Hupp1and 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
DAPK; degradation; lysosome; mTORC1;
TSC2
Correspondence
C Stevens, University of Edinburgh,
Institute of Genetics and Molecular
Medicine, Edinburgh, EH4 2XR, UK
Fax: +44 131 651 1085
Tel: +44 131 651 1025
E-mail: craig.stevens@ed.ac.uk
(Received 28 July 2010, revised 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 lysolyso-some 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 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-8057213 : TSC1 (uniprotkb: Q92574 ) physically interacts ( MI:0914 ) with DAPK (uni-protkb: P53355 ) and TSC2 (uniprotkb: P49815 ) by anti bait coimmunoprecipitation ( MI:0006 )
l MINT-8057200 : TSC1 (uniprotkb: Q92574 ) physically interacts ( MI:0915 ) with TSC2 (uni-protkb: P49815 ) by anti bait coimmunoprecipitation ( MI:0006 )
Abbreviations
DAPK, death-associated protein kinase-1; GAP, GTPase-activating protein; IFN, interferon; 3-MA, 3-methyladenine; MEF, mouse embryonic fibroblast; mTORC1, mammalian target of rapamycin complex 1; siRNA, short interfering RNA; TNF, tumour necrosis factor; TSC2, tuberous sclerosis-2.
Trang 2Death-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 apoptonecro-sis [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
Trang 3activation 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
Trang 40.25 0.50 0.75 1.0 2.0 0.0
0.2 0.4 0.6 0.8
Concentration of FLAG–TSC2 (µg)
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
G
0.0 0.5 1.0 1.5
NS
Concentration of FLAG–TSC2 (µg)
FLAG
DAPK
Actin TSC2
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.
Trang 5A
DAPK S6K P-T389 S6K S6 P-S235/236 S6
Actin
B
DAPK S6 P-S235/236 S6
Actin
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
– +
–
DAPK HA S6 P-S235/236 S6 P-S235/236 (short exposure)
Actin S6 Serum-starved
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 n M 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 indicattransfect-ed, for 48 h Following transfection, cell lysates were prepartransfect-ed 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.
Trang 6Rheb 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
Trang 7with 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
TSC2
TSC2 (1–1516)
LZ
CC
N1693K
FLAG–TSC2 Actin
FLAG–TSC2 (1–1516) Actin
Chx (h)
FLAG–TSC2 (N1693K) Actin
B
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)
**
***
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.
Trang 8with 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 antibodantibod-ies 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.
Trang 9evaluated 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
P-loops
378 641 835
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 – – + +
0.0 0.2 0.4 0.6 0.8 1.0
DAPK DAPK (+TSC2) DAPK 1–1313 DAPK 1–1313 (+TSC2)
*
*
Cycloheximide (h)
F
D
HA–DAPK Actin Chx (h)
FLAG–TSC2
E
HA–DAPK (1–1313) Actin
Chx (h) 0 2 4 6 8 0 2 4 6 8
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 antibodantibod-ies 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).
Trang 10Moreover, 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
B
Lysate
His-Ub pulldown
MG132
HA–DAPK
HA–DAPK
0.0 0.5 1.0 1.5 2.0
Leupeptin E64D Chloro
D
0.0 0.5 1.0 1.5 2.0
Chloro MG132
+ + + – – –
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.