An alternative transcript from the death-associated
protein kinase 1 locus encoding a small protein selectively
mediates membrane blebbing
Yao Lin
1
, Craig Stevens
1
, Roman Hrstka
2
, Ben Harrison
1
, Argyro Fourtouna
1
, Suresh Pathuri
1
,
Borek Vojtesek
2
and Ted Hupp
1
1 Institute of Genetics and Molecular Medicine, Cell Signalling Unit, CRUK p53 Signal Transduction Group, University of Edinburgh, UK
2 Masaryk Memorial Cancer Institute, Brno, Czech Republic
Death-associated protein kinase 1 (DAPK-1) is a
Ca
2+
⁄ calmodulin-regulated serine ⁄ threonine kinase
composed of multiple functional domains, including a
kinase domain, a calmodulin-binding domain, eight
ankyrin repeats, two P-loop motifs, a cytoskeletal
binding domain, a death domain, and a C-terminal
regulatory tail [1]. It has been shown that DAPK-1

activated protein kinase kinase ⁄ extracellular signal-regulated kinase-depen-
dent apoptotic transfection assay. However, the transfection of s-DAPK-1
was able to mimic full-length DAPK-1 in the induction of membrane bleb-
bing. The 44 kDa protease-resistant mutant s-DAPK-1G296A ⁄ R297A had
very low activity in membrane blebbing, whereas the 40 kDa s-DAPK-
1Dtail protein exhibited the highest levels of membrane blebbing. Deletion
of the tail extension of s-DAPK-1 increased its half-life, shifted the equilib-
rium of the protein from cytoskeletal to soluble cytosolic pools, and altered
green fluorescent protein-tagged s-DAPK-1 protein localization as observed
by confocal microscopy. These data highlight the existence of an alternative
product of the DAPK-1 locus, and suggest that proteolytic removal of the
C-terminal tail of s-DAPK-1 is required to stimulate maximally its mem-
brane-blebbing function.
Abbreviations
GFP, green fluorescent protein; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein
kinase kinase; TM, tail mutant.
2574 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS
including apoptosis, cell survival, and autophagy path-
ways, with each role depending on the cellular context
and the upstream signals [1–5]. No apparent defects
in developmental cell death were observed in DAPK-
1-knockout mice [1], thus providing no obvious
insights into its stress-regulated functions. However,
recent research has found that loss or reduced expres-
sion of DAPK-1 underlies cases of heritable predispo-
sition to chronic lymphocytic leukemia and the
majority of cases of sporadic chronic lymphocytic leu-
kemia [6], suggesting an important role of DAPK-1 in
altering the incidence of certain cancer types. This is
consistent with the ability of DAPK-1 to play a funda-

attenuated in the absence of DAPK-1 [14]. The anky-
rin-repeat region of DAPK-1 is required for its proper
localization to the actin stress fibers [8] and for stable
binding with DAPK-1’s ubiquitin E3 ligase, called
DAPK-1-interacting protein 1 [4]. Recently, it was
shown that the leukocyte common antigen-related
tyrosine phosphatase interacts with the ankyrin-repeat
region of DAPK-1 and dephosphorylates DAPK-1 at
pY491 ⁄ 492 to stimulate its proapoptotic and antimi-
gration activities [15]. There are many regions ⁄ minido-
mains on DAPK-1 without an ascribed function, and
it is likely that further biochemical characterization
will result in a greater understanding of the DAPK-1
gene product in autophagic and apoptotic cell
signaling.
Here we report on an mRNA product of the
DAPK-1 locus that encodes a small miniprotein
(named s-DAPK-1), which shares some domains with
full-length DAPK-1: from part of the ankyrin-repeat
region, through to part of the cytoskeleton binding
domain, and concluding with a unique tail extension
of 42 amino acids that is not present in full-length
DAPK-1. Unlike DAPK-1, s-DAPK-1 cannot induce
apoptosis in response to MEK ⁄ ERK signaling. How-
ever, s-DAPK-1 can mimic full-length DAPK-1’s
ability to promote membrane blebbing. The unique
C-terminal tail of s-DAPK-1 contains an internal pro-
teolytic processing site whose removal stimulates maxi-
mally the membrane-blebbing-promoting effect of
s-DAPK-1. These data together identify a novel func-

amino acid protein are identical to the region of the
Y. Lin et al. Functional transcript expressed by DAPK-1 locus
FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS 2575
Fig. 1. The identification of a small transcript from the DAPK-1 locus. (A) A schematic map of s-DAPK-1 mRNA in relation to the DAPK-1
gene structure. The mRNA of s-DAPK-1 starts in intron 13–14 of the DAPK-1 gene. Its coding region starts from the 10th base pair on
exon 15 of the DAPK-1 gene, and shares the same splicing as full-length DAPK-1 through the rest of exons 15, 16, 17, 18, 19 and 20.
s-DAPK-1’s coding region stops at the 126th base pair of intron 20–21 of the DAPK-1 gene, and the 3¢-UTR extends through the middle of
intron 20–21. (B) Comparison of the protein sequences of DAPK-1 and s-DAPK-1. The first 295 amino acids of s-DAPK-1 are identical to
amino acids 447–743 of full-length DAPK-1; however, the last 42 amino acids comprise a unique tail. (C) Identification of s-DAPK-1 mRNA.
RT-PCR was performed using the Stratagene QPCR Human Reference Total RNA, and the products were subjected to electrophoresis and
staining with ethidium bromide. (D) mRNA level test using SYBR Green real-time PCR. The relative mRNA level is depicted as a ratio of
DAPK-1 ⁄ s-DAPK-1 to actin. (E, F) s-DAPK-1, DAPK-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantification in colon
carcinoma and rectal carcinoma as compared to normal colonic tissue. Colon carcinoma cells, rectal carcinoma cells and their normal healthy
tissue counterparts were harvested (1a, carcinoma cells; 4a, normal tissues), and the relative mRNA was quantified using SYBR Green real-
time PCR as described previously for the DAPK-1 gene [2].
Functional transcript expressed by DAPK-1 locus Y. Lin et al.
2576 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS
DAPK-1 protein from residues 447–743, whereas the
last 42 amino acids are unique for this product
(Fig. 1B). These data suggest that this product is
highly similar to and may be a splice variant of
DAPK-1. Because of its smaller size as compared to
full-length DAPK-1, we have named it s-DAPK-1. The
transcription of s-DAPK-1 was demonstrated further
by RT-PCR using the Stratagene (La Jolla, CA, USA)
QPCR Human Reference Total RNA and the primers
located on both ends of the coding region of s-DAPK-1
mRNA (Fig. 1C).
In order to determine the expression of s-DAPK-1,
we first compared its mRNA expression with that of

fected bands were observed: a 44 kDa upper band,
and a 40 kDa lower band (Fig. 2B). In order to deter-
mine which band corresponded to s-DAPK-1, the
C-terminal Myc tag was deleted (Fig. 2A). Upon trans-
fection, the same lower protein band was observed in
the Flag–s-DAPK-1- and the Flag–s-DAPK-1-Myc-
transfected cells, whereas the upper band in the Flag–
s-DAPK-1 transfection lane was slightly smaller
(Fig. 2C, lane 2 versus lane 1). This suggests that the
depletion of the Myc tag only changes the size of the
upper band, and that therefore the upper band repre-
sents the ‘full-length’ s-DAPK-1.
Two s-DAPK-1 deletion mutants, Flag-AO (Anky-
rin repeat Only) and Flag-TD (Tail Deletion;
s-DAPK-1Dtail) were created (Fig. 2A) to further
investigate why the lower molecular mass protein was
observed. Upon transfection, the Flag-TD vector pro-
duces only one major band (s-DAPK-1Dtail) of lower
molecular mass (Fig. 2D, lane 3) similar to the 40 kDa
lower band produced from the full-length s-DAPK-1
(Fig. 2D, lane 4 versus lane 3). This suggests that the
lower band might be a cleavage product of the full-
length s-DAPK-1, and that the cleavage signal is
within the C-terminal tail extension. This is further
suggested by the in vitro cleavage assay, in which the
purified glutathione S-transferase (GST)–s-DAPK-1
was incubated with HCT116 p53
+ ⁄ +
cell lysates. With
increasing amount of cell lysates, GST–s-DAPK-1 was

third and fourth amino acids are involved in the regu-
lation of this cleavage. This also further fine-maps the
site of cleavage, and indicates that the tail deletion
(s-DAPK-1Dtail) may be used as a mimic of the in vivo
processed form of full-length s-DAPK-1. s-DAPK-
1
H300A
(TM3) surprisingly produced a specific shift in
size under denaturing conditions, suggesting that the
modification of the fifth amino acid on the tail may
alter its secondary structure in denaturing polyacryl-
amide gels or might yield an undefined covalent adduct
(Fig. 3B, lane 4).
Y. Lin et al. Functional transcript expressed by DAPK-1 locus
FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS 2577
Fig. 2. Identification of a proteolytic cleavage within the C-terminal tail of s-DAPK-1 protein. (A) A schematic diagram of the Flag–Myc
vector with the s-DAPK-1 clone and its mutants created by site-directed mutagenesis. The vector encoding the s-DAPK-1Dtail with a 42
amino acid tail deletion is named Flag-TD. (B–D) Transfected s-DAPK-1 and its mutants identified a cleavage within its tail. HCT116
p53
+ ⁄ +
cells were transfected with the respective vectors, as indicated, for 24 h prior to harvesting. Expression of the
ectopically expressed s-DAPK-1 and its mutants was detected using an antibody to Flag (Sigma). (E) In vitro cleavage of purified
GST–s-DAPK-1. Recombinant GST–s-DAPK-1 was purified from Bl21 cells and incubated at 30 °C with increasing amounts of
HCT116 p53
+ ⁄ +
cell lysates (0, 1, 5, 10 and 20 lL) as indicated. The sample mixtures after in vitro cleavage were subjected to immu-
noblotting.
Functional transcript expressed by DAPK-1 locus Y. Lin et al.
2578 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS
The proapoptotic effect in response to MEK ⁄ ERK

full-length s-DAPK-1, s-DAPK-1Dtail and s-DAPK-
1G296AR297A were examined. As compared to
DAPK-1, s-DAPK-1 shows more specific localization
in the cytoplasm (Fig. 5A). s-DAPK-1Dtail predomi-
nantly localizes around the nucleus, and s-DAPK-
1G296AR297A spreads throughout the cytosol and
tends to form some ‘aggregating bodies’ (Fig. 5). More-
over, the half-life of s-DAPK-1Dtail is much longer than
those of s-DAPK-1 and s-DAPK-1G296AR297A
(Fig. 6A–D), suggesting that the increased membrane-
blebbing function of s-DAPK-1Dtail is due to its slower
Fig. 3. Identification of the critical sites for
proteolytic cleavage of the C-terminal tail of
s-DAPK-1. (A) A schematic diagram of the
tail mutants of s-DAPK-1 created by site-
directed mutagenesis. (B) Expression of the
tail mutants of s-DAPK-1. HCT116 p53
+ ⁄ +
cells were transfected with the respective
vectors, as indicated, for 24 h prior to har-
vesting. Expression of the s-DAPK-1 tail
mutants was detected by immunoblotting.
(C) Cleavage of the tail of s-DAPK-1 is not
inhibited by common protease inhibitors.
HCT116 p53
+ ⁄ +
cells were transfected with
the Flag–s-DAPK-1 vector for 24 h and trea-
ted with the indicated protease inhibitors
6 h prior to harvesting. The Flag–s-DAPK-1

sion using real-time PCR, we found a significant corre-
lation in their expression, whether using cancer cell
lines or normal human tissues, suggesting that mRNA
from the locus is coordinately produced. Future work
will be required to understand the regulation of the
translation of these mRNAs and whether stress-regu-
lated signaling pathways regulate these two proteins
differently in cell growth control.
Despite the many functions attributed to DAPK-1,
the two standard cellular assays for defining its func-
tion include proapoptotic pathways and membrane
blebbing. Therefore, we have examined the ability of
the s-DAPK-1 protein to play a role in these two pro-
cesses. We found that although s-DAPK-1 cannot
induce apoptosis in response to the activated
MEK ⁄ ERK signal like DAPK-1, it can mimic DAPK-
1 and induce membrane blebbing. A function was also
attributed to the unique tail of s-DAPK-1: it can regu-
late the localization and half-life of the protein and
Fig. 4. The C-terminal tail of s-DAPK-1 negatively regulates its mem-
brane-blebbing function. (A) s-DAPK-1 does not induce apoptosis in
response to MEK ⁄ ERK signaling. HEK293 cells were transfected
with the respective vectors, as indicated, for 24 h prior to harvest-
ing. PARP and PARP cleavage were detected with a PARP-specific
antibody (Cell Signalling). (B) s-DAPK-1 induces membrane blebbing.
A375 cells were transfected with the respective vectors as indi-
cated, and evaluated for membrane blebbing in transfected cells as
described previously [10]. The top panel (B) shows the normal (1)
and the blebbing (2) morphology. (C) The C-terminal tail modulates
membrane blebbing by s-DAPK-1. A375 cells were transfected with

region deletion mutant of DAPK-1 mislocalized to
focal contacts and lost its ability to induce morphologi-
cal changes [8], indicating a functional role of this
region in DAPK-1’s activity. This might explain the
membrane-blebbing-promoting effect of s-DAPK-1, as
it shares a portion of the ankyrin-repeat region of
DAPK-1. However, the functional significance of the
s-DAPK-1-induced membrane blebs is not clear, as
s-DAPK-1 cannot induce MEK ⁄ ERK-stimulated apop-
totic signals (Fig. 5A). A recent study has provided a
novel insight into membrane blebbing [19]; it was
shown that membrane blebbing is due to the reassembly
of the contractile cortex. Therefore, distinct from the
alternative models showing that membrane blebbing is
linked to autophagic or cell death pathways, membrane
blebbing may also be part of a normal cell division pro-
cesses such as cytokinesis. Considering that ankyrin B
plays an important role in the membrane-blebbing pro-
cess [19], DAPK-1 and s-DAPK-1 may be able to inter-
act with ankyrin B via their ankyrin repeats and thus
promote membrane blebbing. Although these data pro-
vide an explanation for the significance of the ankyrin-
repeat region of DAPK-1 in inducing morphological
changes, they do not necessarily indicate that DAPK-1-
or s-DAPK-1-induced membrane blebbing is part of a
normal cell division cycle. Considering that physiologi-
cal membrane blebbing is a transient process [19], it
also remains possible that DAPK-1 and s-DAPK-1
arrest the cells at the blebbing stage and thus halt the
cell division cycle. Therefore, the actual biological sig-

are available upon request. Prior to transfection, Lipofecta-
mine ( 2 lLÆl g
)1
DNA) was added to Optimum medium
without fetal bovine serum. After a 5 min incubation, the
mixture was added to the DNA constructs, and after a
30 min incubation at room temperature, the whole solution
was added to the cells. The translation inhibitor cyclohexi-
mide from Supleco (Bellefonte, PA, USA) was used at a
concentration of 10 lgÆmL
)1
.
Protein analysis
Proteins were extracted by lysing the cells with lysis buffer
(1% NP40, 0.15 m NaCl, 50 mm Tris, pH 7.5, 1 mm
Fig. 6. The C-terminal tail of s-DAPK-1 regu-
lates its half-life. (A–D) The half-life of
s-DAPK-1 is regulated by its C-terminal tail.
HCT116 p53
+ ⁄ +
cells were transfected with
the respective vectors as indicated for 24 h,
in combination with cycloheximide treat-
ment at the indicated times, prior to har-
vesting. Expression of the Flag-tagged
proteins was evaluated by immunoblotting.
Functional transcript expressed by DAPK-1 locus Y. Lin et al.
2582 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS
dithiothreitol and 1· protease inhibitor mixture), and the
protein concentrations were determined using the Bradford

5¢-GATGGAGCCGCCGATCCACACGG-3¢. The DAPK-
1 primers were as follows: forward, 5¢-CGAGGTGA
TGGTGTATGGTG-3¢, reverse, 5¢-CTGTGCTTTGCTGG
TGGA-3¢. The s-DAPK-1 primers were as follows: for-
ward, 5¢-CGTCTCTCCAGCAGGTGTT-3¢; reverse, 5¢-TA
AGGCCACAGGGTCCAGTA-3¢.
Immunostaining and membrane-blebbing assay
A375 cells were analyzed by immunostaining and mem-
brane blebbing. Twenty-four hours post-transfection, cells
were fixed with 4% paraformaldehyde in NaCl ⁄ P
i
for
10 min, washed, and blocked with antibody dilution buf-
fer (3% BSA in NaCl ⁄ P
i
) for 1 h. For the non-green
fluorescent protein (GFP)-tagged proteins, the transfected
cells were then visualized using HA.11 antibody
(Covance) and antibody to Flag (Sigma). After incuba-
tion with the appropriate primary antibodies for 1 h,
cells were washed with NaCl ⁄ P
i
, stained with mouse
Alexa488-conjugated secondary antibody, and mounted
for observation by immunostaining or by examining
membrane-blebbing morphology using a Leica fluorescent
microscope. For immunostaining, the transfected cells
were incubated with Topro-3 from Invitrogen (1 : 1000 in
NaCl ⁄ P
i

3 Chen CH, Wang WJ, Kuo JC, Tsai HC, Lin JR, Chang
ZF & Chen RH (2005) Bidirectional signals transduced
by DAPK–ERK interaction promote the apoptotic
effect of DAPK. EMBO J 24, 294–304.
4 Jin Y, Blue EK, Dixon S, Shao Z & Gallagher PJ
(2002) A death-associated protein kinase (DAPK)-inter-
acting protein, DIP-1, is an E3 ubiquitin ligase that
promotes tumor necrosis factor-induced apoptosis and
regulates the cellular levels of DAPK. J Biol Chem 277,
46980–46986.
5 Gozuacik D & Kimchi A (2006) DAPk protein family
and cancer. Autophagy 2, 74–79.
6 Raval A, Tanner SM, Byrd JC, Angerman EB, Perko
JD, Chen SS, Hackanson B, Grever MR, Lucas DM,
Matkovic JJ et al. (2007) Downregulation of death-
associated protein kinase 1 (DAPK1) in chronic
lymphocytic leukemia. Cell 129, 879–890.
Y. Lin et al. Functional transcript expressed by DAPK-1 locus
FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS 2583
7 Raveh T, Droguett G, Horwitz MS, DePinho RA &
Kimchi A (2001) DAP kinase activates a p19ARF ⁄
p53-mediated apoptotic checkpoint to suppress onco-
genic transformation. Nat Cell Biol 3, 1–7.
8 Bialik S, Bresnick AR & Kimchi A (2004) DAP-kinase-
mediated morphological changes are localization depen-
dent and involve myosin-II phosphorylation. Cell Death
Differ 11, 631–644.
9 Kuo JC, Wang WJ, Yao CC, Wu PR & Chen RH
(2006) The tumor suppressor DAPK inhibits cell motil-
ity by blocking the integrin-mediated polarity pathway.

Biochemistry 45, 15168–15178.
17 Kuo JC, Lin JR, Staddon JM, Hosoya H & Chen RH
(2003) Uncoordinated regulation of stress fibers and
focal adhesions by DAP kinase. J Cell Sci 116, 4777–
4790.
18 Houle F, Poirier A, Dumaresq J & Huot J (2007) DAP
kinase mediates the phosphorylation of tropomyosin-1
downstream of the ERK pathway, which regulates the
formation of stress fibers in response to oxidative stress.
J Cell Sci 120, 3666–3677.
19 Charras GT, Hu CK, Coughlin M & Mitchison TJ
(2006) Reassembly of contractile actin cortex in cell
blebs. J Cell Biol 175, 477–490.
Functional transcript expressed by DAPK-1 locus Y. Lin et al.
2584 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS