Báo cáo khoa học: DYRK1A phosphorylates caspase 9 at an inhibitory site and is potently inhibited in human cells by harmine pptx

DYRK1A phosphorylates caspase 9 at an inhibitory site
and is potently inhibited in human cells by harmine
Anne Seifert, Lindsey A. Allan and Paul R. Clarke
Biomedical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, UK
DYRK1A is the most extensively characterized mem-
ber of the evolutionarily conserved dual-specificity
tyrosine-phosphorylation-regulated protein kinase
(DYRK) family, which is distantly related to mitogen-
activated protein kinases (MAPKs), cyclin-dependent
protein kinases (CDKs), CDK-like kinases (CLKs)
and glycogen synthase kinase 3 [1]. The DYRK family
comprises several members in mammals, of which
DYRK1A and DYRK1B are predominantly localized
to the nucleus, whereas DYRK2 is cytoplasmic [2,3].
Functional studies in mammals and Drosophila suggest
a conserved regulatory role for DYRK1A in neurogen-
esis. Mutant flies with reduced expression of minibrain
kinase, the Drosophila orthologue of DYRK1A, dis-
play specific reductions in the size of the optic lobes
and central brain hemispheres as well as distinctive
behavioural abnormalities [4]. The human DYRK1A
gene has been implicated as having a role in patho-
genesis due to its location in the ‘Down’s syndrome
critical region’ (DSCR) on chromosome 21 [5,6],
which is present in three copies in Down’s syndrome
individuals.
The molecular mechanisms underlying Down’s syn-
drome and its associated pathologies are likely to be
complex. An important role has been proposed for the
transcription factor nuclear factor of activated T cells
(NFAT), which is dysregulated by increased gene dos-

age of DYRK1A and DSCR1, another DSCR gene
that encodes an inhibitor of the protein phosphatase
calcineurin ⁄ PP-2B [7]. Phosphorylation of NFAT by
DYRKs counteracts its dephosphorylation by calci-
neurin, thereby retaining NFAT in the cytoplasm and
Keywords
apoptosis; caspase; DYRK; harmine;
protein kinase
Correspondence
P. R. Clarke, Biomedical Research Institute,
University of Dundee, Level 5, Ninewells
Hospital and Medical School, Dundee DD1
9SY, UK
Fax: +44 1382 669993
Tel: +44 1382 425580
E-mail:
(Received 28 August 2008, revised 8
October 2008, accepted 21 October 2008)
doi:10.1111/j.1742-4658.2008.06751.x
DYRK1A is a member of the dual-specificity tyrosine-phosphorylation-reg-
ulated protein kinase family and is implicated in Down’s syndrome. Here,
we identify the cysteine aspartyl protease caspase 9, a critical component of
the intrinsic apoptotic pathway, as a substrate of DYRK1A. Depletion of
DYRK1A from human cells by short interfering RNA inhibits the basal
phosphorylation of caspase 9 at an inhibitory site, Thr125. DYRK1A-
dependent phosphorylation of Thr125 is also blocked by harmine, confirm-
ing the use of this b-carboline alkaloid as a potent inhibitor of DYRK1A
in cells. We show that harmine not only inhibits the protein–serine ⁄ threo-
nine kinase activity of mature DYRK1A, but also its autophosphorylation
on tyrosine during translation, indicating that harmine prevents formation

of the active enzyme. When co-expressed in cells, DYRK1A interacts with
caspase 9, strongly induces Thr125 phosphorylation and inhibits caspase 9
auto-processing. Phosphorylation of caspase 9 by DYRK1A involves
co-localization to the nucleus. These results indicate that DYRK1A sets a
threshold for the activation of caspase 9 through basal inhibitory phos-
phorylation of this protease. Regulation of apoptosis through inhibitory
phosphorylation of caspase 9 may play a role in the function of DYRK1A
during development and in pathogenesis.
Abbreviations
CDK, cyclin-dependent kinase; CLK, CDK-like kinase; DYRK, dual-specificity tyrosine phosphorylation-regulated kinase; ERK, extracellular
signal-regulated kinase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; NES, nuclear export signal; NFAT, nuclear
factor of activated T cells; NLS, nuclear localization signal; siRNA, short interfering RNA; TPA, 12-O-tetradecanoylphorbol-13-acetate.
6268 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS
inhibiting its transcriptional activity [8]. However, the
substrates and cellular functions of DYRK1A during
normal development and in pathological conditions
remain to be fully identified.
Here, we identify a novel substrate for DYRK1A,
the cysteine aspartyl protease caspase 9, which is a
critical component of the intrinsic or mitochondrial
apoptotic pathway. Caspase 9 is fully activated by a
variety of apoptotic stimuli that trigger the release of
cytochrome c from mitochondria. Once in the cytosol,
cytochrome c induces the oligomerization of Apaf-1
and subsequent recruitment of procaspase 9 into a
high-molecular-mass multimeric complex, termed the
apoptosome [9]. Apaf-1-induced dimerization of pro-
caspase 9 leads to its activation and autocatalytic pro-
cessing [10]. Active caspase 9 initiates a proteolytic
cascade by processing and activating downstream

effector caspases such as caspase 3 and caspase 7, lead-
ing to the organized disassembly of the cell [11].
Regulation of apoptosome formation is controlled
at the level of cytochrome c release from mitochondria
by pro- and anti-apoptotic proteins of the Bcl-2 family
[12]. In addition, the pathway is controlled down-
stream of cytochrome c release at the level of the
apoptosome [13]. Caspase 9 activation is subject to
modulation by protein kinases activated in signal
transduction pathways initiated by extracellular signals
or cellular stresses [14–16]. We have shown previously
that extracellular signal-regulated kinase (ERK)1 and
ERK2 MAPKs, which are activated in response to sur-
vival signals, restrain caspase 9 activation by direct
phosphorylation on a critical inhibitory site, Thr125
[14,17]. Furthermore, CDK1–cyclin B1 protects mitotic
cells from apoptosis induced by microtubule poisons
by phosphorylating caspase 9 on the same residue [18].
During these studies, we obtained evidence that
Thr125 may be subject to phosphorylation by addi-
tional protein kinase activities, because a basal level of
Thr125 phosphorylation persists when ERK1 ⁄ 2 and
CDK1–cyclin B1 are inhibited [14,18].
Here, we identify DYRK1A as an additional kinase
that targets Thr125 of caspase 9 in cells. These results
suggest a function of DYRK1A in the regulation of
apoptosis that may be relevant to its roles during
development and in pathogenesis. We also present evi-
dence that harmine, a potent and specific inhibitor of
DYRKs in vitro [19], efficiently inhibits DYRK1A

activity towards caspase 9 in cells and also blocks the
co-translational activating tyrosine autophosphoryla-
tion of DYRK1A, showing that this b-carboline alka-
loid can be used to test proposed cellular targets of
DYRK1A and potentially could be used to reverse the
effects of DYRK1A overexpression.
Results
Identification of DYRK1A as a Thr125 kinase
in cells
Previous studies have shown that caspase 9 is phosphor-
ylated on a single major site, Thr125, catalysed by
the proline-directed kinases ERK1 ⁄ 2 MAPKs and
CDK1–cyclin B1 in response to growth factors and
during mitosis, respectively. However, residual phos-
phorylation when ERK1 ⁄ 2 and CDK1 are inhibited
suggests that an additional kinase also targets this site
[14,18]. In serum-starved U2.C9–C287A cells, a U2OS-
derived cell line stably expressing catalytically inactive
caspase 9 [18], the MEK1 inhibitors PD0325901 and
U0126, which block ERK1 ⁄ 2 activation, did not reduce
basal Thr125 phosphorylation (Fig. 1A). By contrast,
both inhibitors blocked ERK1 ⁄ 2-dependent phospho-
rylation of Thr125 induced by the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) (Fig. 1B).
Furthermore, although the CDK1–cyclin B1-dependent
phosphorylation of Thr125 induced by the microtubule
poison nocodazole, which arrests cells in mitosis, was
inhibited by the CDK inhibitors alsterpaullone, pur-
valanol A or roscovitine (Fig. 1C), only roscovitine and
purvalanol A, but not alsterpaullone, caused a reduc-

tion in basal phospho-Thr125 levels (Fig. 1A). These
results suggest that the basal Thr125 phosphorylation in
unstimulated cells involves a novel kinase that is not
dependent on ERK1 ⁄ 2 or CDK activity, but is sensitive
to roscovitine and purvalanol A.
DYRK1 has been reported to be inhibited by rosco-
vitine and purvalanol A, but not by alsterpaullone
[20,21]. Consistent with the idea that DYRK1A might
be a physiologically relevant Thr125 kinase, the amino
acid context of Thr125 fulfils the reported primary
sequence requirements of DYRK kinases (Fig. 1D).
DYRK1A and related DYRKs are proline-directed
kinases which require a proline immediately C-terminal
to the phosphorylated serine or threonine residue.
They also favour the presence of an arginine residue at
the )2or)3 position N-terminal to the phosphory-
lated serine or threonine residue [22,23], although
exceptions exist [24,25]. DYRK1A in particular
favours an additional proline at the )2 position
[22,23], as found in caspase 9 (Fig. 1D). Our initial
results therefore lead us to investigate further the
potential role of DYRK1A as a Thr125 kinase.
To test the role of DYRK1A independently of
chemical inhibitors, we depleted DYRK1A from
U2.C9–C287A cells using RNA interference. Transfec-
tion of two distinct synthetic short interfering RNA
(siRNA) duplexes targeting DYRK1A mRNA resulted
A. Seifert et al. Phosphorylation of caspase 9 by DYRK1A
FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6269
in a strong decrease of endogenous DYRK1A protein

levels (Fig. 2A). These siRNA duplexes had no effect
on the expression of the most closely related family
member DYRK1B (85% identical amino acids in the
catalytic domain) [3] when this kinase was expressed as
a green fluorescent protein (GFP) fusion protein, indi-
AB
CD
–
p-Casp9 (T125)
Casp9
Actin
p-ERK1/2
ERK1/2
PD
U0
Alst
PA
Rosc
Casp9
p-ERK1/2
ERK1/2
Actin
–
PD
U0
TPA
–
p-Casp9 (T125)
Actin
Casp9

p-Casp9 (T125)
Nocodazole
–
Alst
PA
Rosc
–
H.sapiens
M.musculus
R.norvegicus
Fig. 1. Small-molecule inhibitors suggest DYRK1A as a potential kinase that phosphorylates caspase 9 on Thr125. (A) U2.C9–C287A cells
stably expressing caspase 9(C287A) were serum-starved and treated with protein kinase inhibitors for 30 min as indicated. (B) U2.C9–C287A
cells were serum-starved and preincubated with inhibitors 30 min prior to addition of TPA for 15 min or (C) incubated with nocodazole for
16 h before addition of inhibitors for 15 min. Inhibitors used were PD0325901 (PD; 0.1 l
M), U0126 (U0; 10 lM), alsterpaullone (Alst; 10 lM),
purvalanol A (PA; 10 l
M) or roscovitine (Rosc; 20 lM). Cell lysates were probed on blots with antibodies against the specified proteins.
(D) Comparison of the amino acid sequence surrounding Thr125 of human, mouse and rat caspase 9 with the reported DYRK and DYRK1A
consensus phosphorylation sequences [22,23]. Phosphorylated residues are highlighted in bold.
AB
CD
Luc
1A #1
1A #2
Actin
DYRK1A
siRNA
Luc
1A #1
1A #2

1B
GFP-DYRK1B-p69
Actin
siRNA
siRNA
Luc
1A #1
1A #2
1B
Actin
Casp9
p-ERK1/2
p-Casp9 (T125)
ERK1/2
Actin
Casp9
p-ERK1/2
p-Casp9 (T125)
ERK1/2
Luc
1A #1
1A #2
1B
siRNA
Fig. 2. DYRK1A depletion inhibits phosphorylation of caspase 9 on Thr125 in cells. (A) Transfection with two DYRK1A-targeting siRNA
duplexes ablates endogenous DYRK1A protein. U2.C9–C287A cells were transfected with siRNA duplexes targeting DYRK1A (1A#1 and
1A#2) or luciferase (Luc) as control, serum-starved for 48 h after transfection and lysed 24 h later. Endogenous DYRK1A was precipitated
from cell lysates using Ni
2+
-NTA agarose. (B) Transfection of U2.C9–C287A cells with DYRK1A targeting siRNA duplexes (1A#1 and 1A#2)

has no effect on protein levels of overexpressed GFP–DYRK1B–p69, whereas transfection with DYRK1B targeting siRNA (1B) decreases
GFP–DYRK1B–p69 protein levels. siRNA transfections were carried out 24 h prior to DNA transfections. Cells were lysed 72 h after siRNA
transfections. (C) Depletion of DYRK1A decreases Thr125 phosphorylation in serum-starved U2.C9–C287A cells. Transfections were carried
out as in (A). (D) Depletion of DYRK1A decreases Thr125 phosphorylation in U2.C9–C287A cells in the presence of serum. Transfections
were carried out as in (A). In all cases, proteins were detected on immunoblots probed with the indicated antibodies.
Phosphorylation of caspase 9 by DYRK1A A. Seifert et al.
6270 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS
cating their specificity for DYRK1A (Fig. 2B). Deple-
tion of DYRK1A by siRNA significantly inhibited
basal Thr125 phosphorylation in cells both in the
absence (Fig. 2C) and in the presence (Fig. 2D) of
serum, demonstrating a role for DYRK1A in the phos-
phorylation of caspase 9 in cells. DYRK1A depletion
did not impinge on ERK1 ⁄ 2 phosphorylation
(Fig. 2C,D) nor did it prevent the phosphorylation of
caspase 9 at Thr125 induced by the phorbol ester TPA
(data not shown), showing that DYRK1A is not
required for the ERK1 ⁄ 2-dependent phosphorylation of
caspase 9. In contrast to the effect of depleting
DYRK1A, a DYRK1B-specific siRNA (see Fig. 2B for
validation of knockdown efficiency) had no effect on
Thr125 phosphorylation (Fig. 2C,D). Therefore, we
conclude that DYRK1A is required for basal phosphor-
ylation of caspase 9 at Thr125 and we can exclude
DYRK1B as a significant Thr125 kinase in U2OS cells.
Direct phosphorylation of caspase 9 by DYRK1A
To establish whether DYRK1A can phosphorylate cas-
pase 9 directly, we carried out an in vitro kinase assay
using [
32

P]ATP[cP] and catalytically inactive His
6
–
caspase 9(C287A) as a substrate. Active DYRK1A
produced in bacteria catalysed the incorporation of
radiolabelled phosphate into caspase 9, whereas a cas-
pase 9 mutant in which Thr125 was mutated to alanine
(T125A) was not phosphorylated (Fig. 3A). Thus,
DYRK1A phosphorylates caspase 9 directly in vitro
AB
CD
Casp9
FLAG
IP: -FLAG
DYRK WT
DYRK K188R
C9
C9 + DYRK WT
C9 + DYRK K188R
C9 T125A + DYRK WT
C9 T125A
Casp9
FLAG
Actin
Input
p-Casp9 (T125)
Casp9
p-ERK1/2
ERK1/2
Actin

FLAG
WT
K188R
Y321F
FLAG-DYRK1A
EV
p-Casp9 (T125)
Casp9
p-ERK1/2
ERK1/2
Actin
GFP
GFP-DYRK1A
GFP-DYRK1B
EV
Autorad
Coomassie
His-C9
His-C9
His-C9 (T125A)
+DYRK1A
Fig. 3. DYRK1A interacts with caspase 9 and induces its phosphorylation on Thr125 in cells. (A) DYRK1A directly phosphorylates caspase 9
at Thr125. Recombinant His
6
–caspase 9 (His–C9) or His
6
–caspase 9(T125A) (both containing the catalytically inactivating C287A mutation)
was incubated with recombinant DYRK1A in the presence of [
32
P]ATP[cP] as indicated. Samples were analysed by SDS ⁄ PAGE, followed by

autoradiography. (B) Expression of DYRK1A causes Thr125 phosphorylation in cells. U2.C9–C287A cells were transiently transfected with
empty vector (EV) or wild-type (WT), K188R or Y321F mutant DYRK1A in pcDNA3–FLAG. (C) Expression of DYRK1B also induces Thr125
phosphorylation. U2.C9–C287A cells were transiently transfected with empty vector (EV), pEGFP–DYRK1A or pEGFP–DYRK1B-p69.
(D) DYRK1A interacts with caspase 9 in cells. U2OS cells were co-transfected with empty vector, wild-type (WT) or K188R DYRK1A
in pcDNA3–FLAG and caspase 9(C287A) (C9) or caspase 9(T125A ⁄ C287A) in pcDNA3. A portion of each cell lysate was retained as an input
sample and FLAG-immunoprecipitations were performed on the remainder. *Indicates bands resulting from IgG; arrows indicate bands of
interest. In (B–D), cell lysates were prepared 24 h after transfection and proteins were detected on immunoblots probed with the indicated
antibodies.
A. Seifert et al. Phosphorylation of caspase 9 by DYRK1A
FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6271
and that Thr125 is the sole phosphorylation site on
caspase 9 targeted by DYRK1A.
Expression of exogenous FLAG-tagged DYRK1A
in U2.C9–C287A cells caused a strong increase in the
phosphorylation of caspase 9 on Thr125 (Fig. 3B),
confirming the ability of DYRK1A to target caspase 9
in cells. The FLAG–DYRK1A mutants K188R or
Y321F, the catalytic activity of which is reduced due
to a mutation in the ATP-binding site or the activa-
tion-loop, respectively [26,27], did not result in strong
Thr125 phosphorylation (Fig. 3B), confirming that the
protein kinase activity of DYRK1A is required.
Expression of DYRK1B as a GFP fusion protein
caused strong Thr125 phosphorylation like DYRK1A
(Fig. 3C), showing that DYRK1B is capable of cataly-
sing Thr125 phosphorylation even though it is not
responsible for basal Thr125 phosphorylation in U2OS
cells.
Many protein kinases interact with their substrates
in complexes that can be co-precipitated. For example,

caspase 9 associates with CDK1–cyclin B1 in cells dur-
ing G2 and mitosis [18]. To test for the interaction
between caspase 9 and DYRK1A, U2OS cells were
transiently co-transfected with expression vectors
encoding caspase 9 and FLAG–DYRK1A or FLAG–
DYRK1A(K188R). Both wild-type and K188R mutant
FLAG–DYRK1A were able to precipitate caspase 9,
indicating caspase 9 and DYRK1A do indeed asso-
ciate in cells, but DYRK1A kinase activity is not
required. Furthermore, caspase 9(C287A ⁄ T125A) lack-
ing Thr125 still precipitated with FLAG–DYRK1A
(Fig. 3D), showing that Thr125 and its phosphoryla-
tion are dispensable for the interaction. Taken together
with the ability of DYRK1A to catalyse the phospho-
rylation of caspase 9 at Thr125 in vitro, these results
strongly indicate that DYRK1A targets caspase 9
directly in cells.
Harmine is a potent inhibitor of DYRK1A in cells
The b-carboline alkaloid harmine has recently been
reported as a specific DYRK inhibitor in vitro by Bain
et al. [19]. Harmine inhibits purified DYRK1A in the
nanomolar range, with DYRK2 and DYRK3 inhibited
 10-fold less potently, and little or no inhibition by
1 lm harmine of a panel of 67 other protein kinases
[19]. We found that the basal phosphorylation of
Thr125 in U2.C9–C287A cells that is due to DYRK1A
was potently inhibited by harmine, with a partial inhi-
bition even at a concentration of 0.01 lm and almost
complete inhibition at 1 lm (Fig. 4A). We did not
observe any impairment of the basal phosphorylation

of ERK1 ⁄ 2 by harmine, excluding the possibility that
the reduction of Thr125 phosphorylation is due to
defective ERK1⁄ 2 activation (Fig. 4A). In agreement,
we also found no effect of 1 lm harmine on TPA-
induced ERK1 ⁄ 2 activation and the subsequent phos-
phorylation of Thr125 (Fig. 4B).
A previous study identified harmine as an inhibitor
of CDKs at micromolar concentrations, with an
IC
50
=17lm for CDK1–cyclin B [28]. Bain et al.
[19], however, demonstrated no significant inhibitory
activity against another cyclin-dependent kinase,
CDK2–cyclin A, at 1 lm harmine. Consistent with the
latter study, we observed that 1 lm harmine had no
effect on CDK1–cyclin B1-dependent Thr125 phos-
phorylation induced by nocodazole (Fig. 4C). These
results confirm that harmine selectively inhibits the
basal phosphorylation of caspase 9 at Thr125 that is
due to DYRK1A, but not the ERK1 ⁄ 2-dependent
phosphorylation induced by mitogens or the CDK1–
cyclin B1-dependent phosphorylation induced by
mitotic arrest. When the phosphorylation of Thr125 in
caspase 9 was strongly induced by the expression of
FLAG–DYRK1A, this activity was also completely
inhibited by harmine, although higher concentrations
of the inhibitor were required, presumably because of
the elevated levels of DYRK1A in the cells (Fig. 4D).
We also analysed the effect of 1 lm harmine on the
phosphorylation of Thr125 on endogenous caspase 9

immunoprecipitated from HeLa cells. Immunoblots
showed a reduction of Thr125 phosphorylation
in response to harmine, confirming that endogenous
caspase 9 is also phosphorylated in a DYRK1A-depen-
dent manner (Fig. 4E).
DYRKs are unusual dual-specificity kinases that
require autophosphorylation of an essential Tyr–Xaa–
Tyr motif in the activation loop to form a mature
kinase that has specificity towards serine and threonine
residues in substrate proteins [21,26,29]. Therefore, we
were interested in studying whether harmine, like pur-
valanol A [21], inhibits the autophosphorylation of
DYRK1A on tyrosine in addition to the phosphoryla-
tion of exogenous substrates. Using an assay developed
by Lochhead et al. [21], we translated FLAG–
DYRK1A in rabbit reticulocyte lysate in the absence
or presence of harmine and found that harmine also
blocks the tyrosine autophosphorylation of DYRK1A,
with almost complete inhibition at 1 lm (Fig. 4F).
Inhibition of caspase 9 auto-processing
by DYRK1A
Previous work has shown an inhibitory effect of
Thr125 phosphorylation on caspase 9 activation,
thereby blocking downstream caspase 3 activation and
Phosphorylation of caspase 9 by DYRK1A A. Seifert et al.
6272 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS
apoptosis [14,18]. Although proteolytic processing of
caspase 9 is not required for its activation, generation
of a processed p35 form is dependent on the catalytic
activity of the enzyme [30]. When we expressed catalyt-

ically active caspase 9 in U2OS cells, generation of the
p35 auto-processed form was significantly reduced by
co-expression of DYRK1A (Fig. 5). Auto-processing
of caspase 9 was not antagonized by DYRK1A if the
Thr125 residue of caspase 9 was converted to alanine
(Fig. 5A). Furthermore, inhibition of caspase 9 auto-
processing required the kinase activity of DYRK1A,
because co-expression of the DYRK1A–K188R
mutant did not block caspase 9 auto-processing
(Fig. 5B). Thus, DYRK1A inhibits the auto-processing
of caspase 9 in cells through the phosphorylation of
Thr125 on caspase 9. This result indicates that phos-
phorylation of Thr125 by DYRK1A inhibits caspase 9
activation, consistent with previous studies on the
effects of phosphorylation of this site by other kinases
[14,18].
DYRK1A phosphorylates caspase 9 in the nucleus
DYRK1A has been reported as a predominantly
nuclear kinase [2]. We therefore wished to determine
whether DYRK1A and caspase 9 co-localize. As anti-
cipated, DYRK1A expressed as a fusion protein with
GFP was found to be predominantly nuclear by con-
focal microscopy (Fig. 6A). When caspase 9 was
expressed as the catalytically inactive mutant C287A in
AB
CD
EF
p-Casp9(T125)
Actin
Harmine(

M) –
0.1
0.01
1
0.001
Casp9
p-ERK1/2
ERK1/2
FLAG-DYRK1AEV
p-Casp9(T125)
Actin
Flag
Casp9
100
50
10
1
0.1
p-Tyr
FLAG-DYRK1A
EV
FLAG-DYRK1A
Harmine PA
1
10
5
100
0.1
Inhibitor ( M)
Actin

0.1
0.01
1
0.001
Nocodazole
Casp9
p-Casp9(T125)
p-Casp9(T125)
Actin
Harmine(
M) –
0.1
0.01
1
0.001
TPA
Casp9
p-ERK1/2
ERK1/2
p-Casp9(T125)
Casp9
Casp9
Actin
IP
Input
Mock
-caspase-9
1
–
Harmine(

M) –
–
Harmine(
M) –
–
Harmine(
M)
––
––
–
Fig. 4. Harmine inhibits DYRK1A-dependent phosphorylation of caspase 9 on Thr125 in cells. (A) Harmine inhibits the basal phosphorylation
of Th125. Serum-starved U2.C9–C287A cells were treated with indicated concentrations of harmine for 30 min. (B) Harmine does not inhibit
ERK1 ⁄ 2 activation or TPA-induced Thr125 phosphorylation. Serum-starved U2.C9–C287A cells were incubated with harmine for 15 min prior
to addition of TPA for further 15 min. (C) Harmine does not inhibit mitotic phosphorylation of Thr125. U2.C9–C287A cells were incubated in
the presence of nocodazole for 16 h prior to addition of harmine for 30 min. (D) Harmine inhibits Thr125 phosphorylation caused by DYRK1A
overexpression in cells. U2OS cells were transfected with pcDNA3–caspase 9(C287A) and pcDNA3–FLAG–DYRK1A or empty vector (EV).
Twenty hours after transfection, cells were incubated with indicated concentrations of harmine for 30 min. (E) HeLa cells were incubated in
the presence of 1 l
M harmine where indicated for 30 min, followed by immunoprecipitation of endogenous caspase 9 using a caspase 9-spe-
cific antibody. Mock immunoprecipitations were carried out using an anti-Myc IgG. (F) FLAG–DYRK1A was in vitro translated in rabbit reticulo-
cyte lysate in the absence or presence of indicated concentrations of harmine or purvalanol A (PA), followed by immunoprecipitation using
anti-(FLAG agarose). EV indicates empty vector. In all cases, proteins were detected on immunoblots probed with the indicated antibodies.
A. Seifert et al. Phosphorylation of caspase 9 by DYRK1A
FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6273
U2OS cells, it localized to both the cytoplasm and the
nucleus. Nuclear speckle-like foci were detected by
the pThr125 antibody in the absence of transfected
caspase 9 (Fig. 6A). These speckles were not removed
by siRNA-mediated depletion of endogenous caspase 9
(Fig. S1); therefore the speckles are not likely to corre-

spond to phosphorylated caspase 9 and probably origi-
nate from another epitope. However, in cells in which
caspase 9 was co-expressed with DYRK1A, the signal
detected by the pThr125 antibody was strongly
increased in the nucleus, and pThr125 epitopes also
appeared in the cytoplasm. The increased signal gener-
ated by DYRK1A expression was due entirely to the
phosphorylation of caspase 9 at Thr125, because no
increased signal was detected when cells were co-trans-
fected with a non-phosphorylatable mutant of cas-
pase 9(T125A) (Fig. 6A). The increase in Thr125
phosphorylation also required the kinase activity of
DYRK1A, because it was not induced by the ATP-
binding site mutant K188R (data not shown).
To test if caspase 9 phosphorylation takes place in
the nucleus where DYRK1A is localized, we engi-
neered GFP–caspase 9 fusion constructs tagged with
either a nuclear localization signal (NLS) or a nuclear
export signal (NES). When expressed in cells, the
NLS–GFP–caspase 9 and NES–GFP–caspase 9 pro-
teins localized to the nucleus and cytoplasm, respec-
tively (Fig. 6B). When co-expressed with DYRK1A,
GFP–caspase 9 and NLS–GFP–caspase 9 exhibited a
higher level of Thr125 phosphorylation than NES–
GFP–caspase 9 (Fig. 6C). In agreement with this
result, we also found that the endogenous basal
Thr125 kinase had a stronger preference for nuclear
caspase 9 than for cytoplasmic caspase 9 (Fig. 6D).
This nuclear kinase activity towards caspase 9 was sen-
sitive to harmine and thus due to DYRK1A (Fig. S2).

Together, these results demonstrate that DYRK1A
and caspase 9 co-localize to the nucleus and that
DYRK1A phosphorylates caspase 9 in the nuclear
compartment.
Discussion
The dual-specificity tyrosine phosphorylation-regulated
protein kinase DYRK1A plays important roles during
development and in human pathologies. However, lit-
tle is currently known about the substrates through
which it exerts these effects. Here, we have identified
the apoptotic protease caspase 9 as a substrate for
phosphorylation by DYRK1A at a critical site,
Thr125. Previously we have shown that phosphoryla-
tion of this site inhibits the activation of caspase 9 and
restrains apoptosis in human cells [14,18]. We propose
that basal phosphorylation of Thr125 in caspase 9 by
DYRK1A sets a threshold in the response to apoptotic
stimuli that is augmented in proliferating cells through
the activities of ERK1 ⁄ 2 and CDK1–cyclin B1 kinases
[14,18].
Although DYRK1A appears to be synthesized as a
constitutively active enzyme, work on the cytoplasmic
Caenorhabditis elegans DYRK orthologue MBK-2 has
shown a cell-cycle and developmental stimulus-depen-
dent regulation of DYRK activity [31], and DYRK1A
may also be regulated through alterations of its expres-
sion during development and the cell cycle in mamma-
lian cells [32,33]. Thus, the level of phosphorylation of
caspase 9 catalysed by DYRK1A and its significance
for cell survival is likely to be modulated by changes

in DYRK1A expression in vivo.
DYRK1A has been mapped to the Down’s syndrome
critical region on chromosome 21 that is present as an
additional copy in Down’s syndrome individuals [5,6].
DYRK1A is overexpressed in Down’s syndrome brains
p-Casp9(T125)
Casp9
Actin
WT
EV
EV
DYRK1A
DYRK1A
FLAG
Procaspase9
Processed Casp9
Procaspase9
Processed Casp9
T125A
A
B
p-Casp9(T125)
Casp9
Actin
DYRK1A
DYRK1A WT
DYRK1A K188R
EV
Procaspase9
Processed Casp9

Procaspase9
Processed Casp9
Fig. 5. Phosphorylation of Thr125 by DYRK1A inhibits caspase 9
activation. (A) U2OS cells were transfected with pcDNA3 vectors
encoding catalytically active caspase 9 (wild-type; WT) or catalyti-
cally active caspase 9(T125A) and FLAG–DYRK1A or empty vector
(EV). (B) U2OS cells were transfected with pcDNA3 vectors encod-
ing catalytically active caspase 9 (wild-type) and wild-type (WT) or
K188R FLAG–DYRK1A or empty vector (EV). Cell lysates were
prepared 7 h after transfection and blotted with the indicated anti-
bodies.
Phosphorylation of caspase 9 by DYRK1A A. Seifert et al.
6274 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS
A
BC
D
Casp9/
GFP-DYRK1A
Casp9/
GFP
Casp9(T125A)/
GFP
Casp9(T125A)/
GFP-DYRK1A
Casp9
Casp9
Casp9
Casp9
p-Casp9(T125)
p-Casp9(T125)

p-Casp9(T125)
p-Casp9(T125)
GFP-DYRK1A
GFP
GFP-DYRK1A
GFP
DNA
DNA
DNA
DNA
NES-GFP-Casp9
NLS-GFP-Casp9
GFP-Casp9
GFP DNA
p-Casp9 (T125)
GFP-Casp9
Actin
FLAG
FLAG-
DYRK1A
Casp9
NLS-Casp9
NES-Casp9
Casp9
NLS-Casp9
NES-Casp9
EV
Casp9
NES-Casp9
NLS-Casp9

p-Casp9(T125)
GFP-Casp9
Actin
Fig. 6. Phosphorylation of caspase 9 on Thr125 by DYRK1A in the nucleus. (A) Immunofluorescence staining of U2OS cells transiently trans-
fected with vectors encoding caspase 9(C287A), caspase 9(T125A ⁄ C287A), GFP or GFP–DYRK1A. Cells were fixed 20–24 h after transfec-
tion and stained with antibodies directed against total caspase 9 and caspase 9 phosphorylated on Thr125. DNA was DAPI-stained and cells
were analysed by confocal microscopy. Scale bars, 10 lm. (B) Localization of GFP–caspase 9(C287A), NES–GFP–caspase 9(C287A) and
NLS–GFP–caspase 9(C287A) in U2OS cells. Cells were transiently transfected with pEGFP vectors encoding the respective fusion proteins,
followed by fixation after 8–9 h. Scale bars, 10 lm. (C) Overexpressed DYRK1A predominantly phosphorylates nuclear caspase 9. U2OS
cells were transfected as in (B), but in combination with empty vector (EV) or DYRK1A in pcDNA3–FLAG and lysed 8–9 h after transfection.
(D) Endogenous Thr125 kinase(s) preferentially phosphorylate(s) nuclear caspase 9. U2OS cells were transfected as in (B). In (C, D), cell
lysates were blotted with antibodies against the specified proteins. Note that samples in (C) were harvested 8–9 h after transfection,
whereas cells in (D) were lysed 20–24 h after transfection. This difference in expression time accounts for the absence of a p-Casp-9(T125)
signal in lanes 1–3 in (C).
A. Seifert et al. Phosphorylation of caspase 9 by DYRK1A
FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6275
[34], suggesting a role in neurogenesis like its Drosophila
orthologue, minibrain (mnb) [4]. Studies in both mouse
and Drosophila have found an important role for
DYRK1A ⁄ minibrain kinase in determining the number
of neurons during post-embryonic neurogenesis: muta-
tion of minibrain causes reduction of the size of the
optic lobes and central brain hemispheres [4], whereas
mice lacking one copy of the DYRK1A gene exhibit
region-specific reductions in brain size [35]. Although
the molecular mechanisms underlying this phenotype
are not understood, regulation of apoptosis might be
involved. This idea is particularly appealing because
caspase 9 also has an essential function in mouse brain
development [36,37].

DYRK1A is a predominantly nuclear kinase that is
localized to intranuclear splicing speckles, which are
sites of mRNA processing [38]. Our pThr125 antibody
also detects these speckles (Fig. 6; data not shown),
although the phosphoepitope is probably not due to
caspase 9 (Fig. S1). This study shows that caspase 9 is
partially localized to the nucleus and its phosphoryla-
tion by DYRK1A is promoted by nuclear targeting
and diminished by cytoplasmic targeting. Previously,
although caspase 9 was reported to be mainly localized
to mitochondria and cytosol when analysed by subcel-
lular fractionation of Jurkat cells [39], GFP–caspase 9
was found partially localized to nuclei in Jurkat and
HEK293 cells [40]. Nuclear caspase 9 has also been
observed in mammary epithelial cells [41]. Our results
show that caspase 9 is distributed in both the nucleus
and cytoplasm in U2OS cells. Caspase 9 would
encounter DYRK1A in the nucleus and become phos-
phorylated, before being redistributed to the cyto-
plasm. In this way, a nuclear kinase, DYRK1A, can
regulate the cytoplasmic activity of caspase 9 as an ini-
tiator of apoptosis. It does, however, remain possible
that caspase 9 has a distinct function within the
nucleus that is controlled by DYRK1A.
Identification of caspase 9 as a bona fide substrate
for DYRK1A in cells has enabled us to confirm the
b-carboline alkaloid harmine as an intracellular inhibi-
tor of DYRK1A, as suggested by its identification as a
potent and selective inhibitor of DYRKs in vitro [19].
b-Carbolines are present in Peganum harmala and

other plants which have been used as medicinal pre-
parations as well as hallucinogens in traditional rituals.
Harmine has a long history of use as a chemothera-
peutic drug for a number of diseases, including malar-
ial infection and Parkinson’s disease [42]. Harmine and
related b-carbolines have cytotoxic activity towards
human tumour cell lines in culture [43], suggesting a
possible use in anti-cancer therapy. Several putative
molecular targets for harmine have been identified
[28,44], but inhibition of DYRK1A at low (sub-micro-
molar) concentrations in cells strongly suggests that
inhibition of this kinase is involved in the biological
activity of harmine in vivo. Interestingly, our results
show that harmine not only inhibits the protein–ser-
ine ⁄ threonine kinase activity of the mature enzyme,
but also the tyrosine autophosphorylation that is
required for maturation of the active enzyme [21]. This
indicates that harmine also inhibits formation of active
DYRK1A in cells. Identification of harmine as a cell-
permeable DYRK1A inhibitor is anticipated to facili-
tate the identification of further DYRK1A substrates
in vivo and also suggests its potential use to reverse the
pathological effects of DYRK1A overexpression.
Experimental procedures
Plasmids and recombinant proteins
Caspase 9 cDNA in pcDNA3 (Invitrogen, Carlsbad, CA,
USA) or pET28a (Novagen, Madison, WI, USA) has been
described previously [14]. To generate an expression con-
struct for GFP–caspase 9 fusion protein, caspase 9 cDNA
was subcloned into pEGFP(C2). Vectors encoding NLS–

GFP–caspase 9 and NES–GFP–caspase 9 were constructed
by insertion of the SV40T NLS or the NES from the pro-
tein kinase A inhibitor between the initiating ATG and
the second codon of EGFP using the QuikChangeÔ site-
directed mutagenesis kit. Wild-type and K188R mutant
pEGFP(C1)–DYRK1A (rat) as well as pEGFP(C1)–
DYRK1B-p69 (human) were kind gifts from W. Becker
(Aachen, Germany). An expression construct for FLAG–
DYRK1A was generated by subcloning DYRK1A cDNA
into pcDNA3–FLAG (kindly provided by D. Meek,
University of Dundee, UK). Expression of recombinant
His
6
–caspase 9(C287A) and His
6
–caspase 9(T125A ⁄ C287A)
proteins was carried out as described previously [14]. All
expression constructs encoding proteins bearing amino acid
substitutions were generated by site-directed mutagenesis
using the QuikChangeÔ kit (Stratagene, Cedar Creek, TX,
USA) according to the manufacturer’s instructions. Recom-
binant DYRK1A, expressed as a fusion protein with gluta-
thione S-transferase (GST–DYRK1A) in Escherichia coli,
was purchased from Millipore (Watford, UK).
Antibodies and reagents
The following antibodies were used for western blotting
and immunological staining according to standard proto-
cols: caspase 9 mAb (Chemicon, Temecula, CA, USA),
phospho-ERK1 ⁄ 2, phospho-Tyr-100 (both Cell Signalling
Technology, Beverly, MA, USA), ERK1 ⁄ 2 (Millipore),

GFP, DYRK1A G-19 (both Santa Cruz Biotechnology,
Santa Cruz, CA), Actin, FLAG-M2 (both Sigma-Aldrich,
Phosphorylation of caspase 9 by DYRK1A A. Seifert et al.
6276 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS
St Louis, MO, USA), myc-9E10 (Cancer Research UK,
London, UK). Generation and characterization of rabbit
anti-[phospho-caspase 9(T125)] IgG was described previ-
ously [14]. Reagents used were: nocodazole (Sigma), TPA
and protein kinase inhibitors (Calbiochem, San Diego, CA,
USA), harmine (Sigma-Aldrich) and PD0325901 (kindly
provided by P. Cohen, University of Dundee, UK).
DYRK1A kinase assay
Recombinant His
6
–caspase 9 (1.5 lg) was added to a total
reaction volume of 15 lL kinase assay buffer (50 mm Tris
pH 7.5, 10 mm MgCl
2
, 100 lm ATP, 1 mm dithiothreitol)
containing 1.5 lCi [
32
P]ATP[cP] (from a 10 mCiÆmL
)1
stock with specific activity 3000 CiÆmmol
)1
). The kinase
assay was initiated by adding 30 ng of active recombinant
GST–DYRK1A. Reaction mixtures were incubated at
30 °C for 30 min. Reactions were stopped by boiling in
SDS ⁄ PAGE sample buffer and half the volume of a reac-

tion was analysed by SDS ⁄ PAGE, followed by autoradio-
graphy.
Cell culture, DNA transfections and treatments
HeLa and U2OS cells (obtained from Cancer Research UK
Cell Services) were maintained in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal bovine
serum, 50 UÆmL
)1
penicillin, 50 lgÆmL
)1
streptomycin and
2mml-glutamine (Invitrogen, Carlsbad, CA). For U2.C9–
C287A cells [18], which stably express catalytically inactive
caspase 9(C287A), the growth medium was supplemented
with G418 sulfate (400 ngÆmL
)1
, Calbiochem). Where indi-
cated, serum starvation was performed by culturing cells in
Dulbecco’s modified Eagle’s medium containing 0% fetal
bovine serum for 20–24 h. DNA transfections were carried
out using CsCl-purified plasmid DNA and Superfect (Qia-
gen, Valencia, CA, USA) according to manufacturer’s pro-
tocol. To arrest cells in mitosis, cells were treated with
100 ngÆmL
)1
nocodazole for 16 h. To activate ERK1 ⁄ 2
MAPK signalling, cells were incubated with 1 lm TPA for
15 min. The protein kinase inhibitors PD0325901 (0.1 lm),
U0126 (10 lm), alsterpaullone (10 lm), purvalanol A
(10 lm), roscovitine (20 lm) or harmine (routinely, 1 lm)

were added as indicated. The specificity of these inhibitors
towards a panel of purified protein kinases is reported by
Bain et al. [19]. For analysis by immunoblotting, cells were
lysed in SDS ⁄ PAGE sample buffer.
RNA interference
For siRNA transfections, cells were transfected with
100 nm siRNA duplex and Lipofectamine 2000 following
the manufacturer’s instructions (Invitrogen). Then, cells
were trypsinised and cultured for 72 h before analysis. The
following siRNA duplexes were used to deplete DYRK1A
(sense strands): 5¢-UAAGGAUGCUUGAUUAUGAdTdT-3¢
(DYRK1A#1), 5¢-AAACUCGAAUUCAACCUUAdTdT-3¢
(DYRK1A#2). Other siRNAs used were 5¢-CGUACG
CGGAAUACUUCGAdTdT-3¢ (Luciferase), 5¢-CGACCU
GACUGCCAAGAAAdTdT-3¢ (Caspase 9) and DYRK1B
SmartPool (Dharmacon) comprising four different duplexes:
5¢-GAAAUUGACUCGCUCAUUGrUrU-3¢,5¢-ACACGG
AGAUGAAGUACUArUrU-3¢,5¢-GCCAGAGGAUCUA
CCAGUArUrU-3¢,5¢-GCACAUCAAUGAGGUAUACr
UrU-3¢. Single siRNA duplexes were synthesized by MWG
(Martinsried, Germany).
Caspase 9 and FLAG immunoprecipitations
Cells were lysed in IP buffer (20 mm Tris pH 7.6, 137 mm
NaCl, 2 m m EDTA, 1 mm Na
3
VO
4
,50mm NaF, 5 mm
b-glycerophosphate, 1% Triton X-100, 1 lm okadaic acid,
1mm phenylmenthanesulfonyl fluoride, 1 lgÆmL

)1
each
aprotinin, leupeptin and pepstatin A). Immunoprecipitation
of endogenous caspase 9 from HeLa cells was carried out
as described previously [14]. For co-immunoprecipitation of
FLAG–DYRK1A and caspase 9 from U2OS cells, cell
lysate (0.5 mg) was incubated with 15 lL anti-(FLAG aga-
rose) (Sigma) for 1 h at 4 °C. Beads were washed three
times in IP buffer and boiled in SDS ⁄ PAGE sample buffer.
Samples were analysed by western blotting.
Ni
2+
-pulldown of endogenous DYRK1A from cells
DYRK1A contains an internal stretch of 13 consecutive
histidine residues, enabling the endogenous DYRK1A pro-
tein to bind Ni
2+
-NTA agarose [45]. U2.C9–C287A cells
were lysed in buffer A (6 m guanidine–HCl, 10 mm Tris,
0.1 m phosphate buffer, pH 8.0) supplemented with 5 m m
imidazole for 5 min, sonicated and incubated with 30 lL
Ni-NTA–agarose (Qiagen) for 4–5 h. Beads were pelleted
and washed once in buffer A, followed by one wash in buf-
fer B (8 m urea, 10 mm Tris, 0.1 m phosphate buffer,
pH 8.0), one wash in buffer C (8 m urea, 10 mm Tris, 0.1 m
phosphate buffer pH 6.5, 0.2% Triton X-100) and one
wash in buffer D (buffer C supplemented with 0.1% Tri-
ton X-100). For elution, beads were boiled in SDS ⁄ PAGE
sample buffer. Supernatant was analysed by western blot-
ting and endogenous DYRK1A was detected with a

DYRK1A-specific antibody.
In vitro translation and immunoprecipitation of
FLAG–DYRK1A from rabbit reticulocyte lysate
In vitro translation of FLAG–DYRK1A was performed in
absence or presence of inhibitors using the TNT Quick cou-
pled transcription ⁄ translation protocol (Promega, Madison,
WI, USA) and pcDNA3–FLAG–DYRK1A as template.
A. Seifert et al. Phosphorylation of caspase 9 by DYRK1A
FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6277
Following incubation of the reaction mixture at 30 °C for
1 h, IP buffer containing 2 mm Na
3
VO
4
and washed anti-
(FLAG–M2) agarose beads (Sigma) were added. Immuno-
precipitation was carried out as outlined below. Protein
was eluted in SDS ⁄ PAGE sample buffer containing 2 mm
Na
3
VO
4
at 37 °C.
Immunofluorescence
Cells were transfected on coverslips and fixed with 3% para-
formaldehyde, permeabilised with 0.2% Triton X-100 in
Tris-buffered saline and incubated in blocking buffer (5%
fetal bovine serum in Tris-buffered saline) for 1 h at room
temperature. Samples were incubated overnight at 4 °C with
anti-(caspase 9) (1 : 100) and anti-[phospho-caspase 9(T125)]

(1 : 50) purified sera, followed by incubation with AlexaFlu-
or647-conjugated anti-mouse (1 : 100; Invitrogen) and
TRITC-conjugated anti-rabbit (1 : 20; DAKO) IgG for
45 min at room temperature. DNA was stained with DAPI.
Microscopy was carried out using a LSM 510 confocal
microscope (Zeiss) with a 63· Plan Apochromat objective.
Fluorescent signals of EGFP (excitation 488 nm using an
argon laser), TRITC (excitation 543 nm using a HeNe1 laser)
and AlexaFluor-647 (excitation 633 nm using a HeNe3 laser)
were detected using band-pass 505–530 nm, 560–615 nm and
long-pass 650 nm emission filters, respectively.
Acknowledgements
We thank P. Cohen (Dundee, UK) for reagents and
helpful discussion of this work. We are also grateful to
W. Becker (Aachen, Germany) for providing GFP–
DYRK1A and GFP–DYRK1B DNA constructs, and
to D. Meek (Dundee, UK) for the pcDNA3–FLAG
vector. We thank the members of the Clarke labora-
tory, A. Cole, M. Soutar, C. Sutherland and S. Keyse
for their help. This work was supported by a Cancer
Research UK Studentship (AS), the Association for
International Cancer Research (LAA) and a Royal
Society–Wolfson Research Merit Award (PRC).
References
1 Becker W & Joost HG (1999) Structural and functional
characteristics of Dyrk, a novel subfamily of protein
kinases with dual specificity. Prog Nucleic Acid Res Mol
Biol 62, 1–17.
2 Becker W, Weber Y, Wetzel K, Eirmbter K, Tejedor FJ
& Joost HG (1998) Sequence characteristics, subcellular

localization, and substrate specificity of DYRK-related
kinases, a novel family of dual specificity protein kinases.
J Biol Chem 273, 25893–25902.
3 Leder S, Weber Y, Altafaj X, Estivill X, Joost HG &
Becker W (1999) Cloning and characterization of
DYRK1B, a novel member of the DYRK family of
protein kinases. Biochem Biophys Res Commun 254,
474–479.
4 Tejedor F, Zhu XR, Kaltenbach E, Ackermann A,
Baumann A, Canal I, Heisenberg M, Fischbach KF &
Pongs O (1995) Minibrain: a new protein kinase family
involved in postembryonic neurogenesis in Drosophila.
Neuron 14, 287–301.
5 Song WJ, Sternberg LR, Kasten-Sportes C, Keuren
ML, Chung SH, Slack AC, Miller DE, Glover TW,
Chiang PW, Lou L et al. (1996) Isolation of human
and murine homologues of the Drosophila minibrain
gene: human homologue maps to 21q22.2 in the Down
syndrome ‘critical region’. Genomics 38, 331–339.
6 Guimera J, Casas C, Pucharcos C, Solans A, Domenech
A, Planas AM, Ashley J, Lovett M, Estivill X & Prit-
chard MA (1996) A human homologue of Drosophila
minibrain (MNB) is expressed in the neuronal regions
affected in Down syndrome and maps to the critical
region. Hum Mol Genet 5, 1305–1310.
7 Arron JR, Winslow MM, Polleri A, Chang CP, Wu H,
Gao X, Neilson JR, Chen L, Heit JJ, Kim SK et al.
(2006) NFAT dysregulation by increased dosage of
DSCR1 and DYRK1A on chromosome 21. Nature 441,
595–600.

8 Gwack Y, Sharma S, Nardone J, Tanasa B, Iuga A,
Srikanth S, Okamura H, Bolton D, Feske S, Hogan PG
et al. (2006) A genome-wide Drosophila RNAi screen
identifies DYRK-family kinases as regulators of NFAT.
Nature 441, 646–650.
9 Cain K, Bratton SB & Cohen GM (2002) The Apaf-1
apoptosome: a large caspase-activating complex.
Biochimie 84, 203–214.
10 Pop C, Timmer J, Sperandio S & Salvesen GS (2006)
The apoptosome activates caspase-9 by dimerization.
Mol Cell 22, 269–275.
11 Budihardjo I, Oliver H, Lutter M, Luo X & Wang X
(1999) Biochemical pathways of caspase activation
during apoptosis. Annu Rev Cell Dev Biol 15, 269–290.
12 Youle RJ & Strasser A (2008) The BCL-2 protein
family: opposing activities that mediate cell death. Nat
Rev Mol Cell Biol 9, 47–59.
13 Schafer ZT & Kornbluth S (2006) The apoptosome:
physiological, developmental, and pathological modes
of regulation. Dev Cell 10, 549–561.
14 Allan LA, Morrice N, Brady S, Magee G, Pathak S
& Clarke PR (2003) Inhibition of caspase-9 through
phosphorylation at Thr 125 by ERK MAPK. Nat Cell
Biol 5, 647–654.
15 Brady SC, Allan LA & Clarke PR (2005) Regulation of
caspase 9 through phosphorylation by protein kinase C
zeta in response to hyperosmotic stress. Mol Cell Biol
25, 10543–10555.
16 Martin MC, Allan LA, Lickrish M, Sampson C, Mor-
rice N & Clarke PR (2005) Protein kinase A regulates

Phosphorylation of caspase 9 by DYRK1A A. Seifert et al.
6278 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS
caspase-9 activation by Apaf-1 downstream of cyto-
chrome c. J Biol Chem 280, 15449–15455.
17 Martin MC, Allan LA, Mancini EJ & Clarke PR (2008)
The docking interaction of caspase-9 with ERK2
provides a mechanism for the selective inhibitory
phosphorylation of caspase-9 at threonine 125. J Biol
Chem 283, 3854–3865.
18 Allan LA & Clarke PR (2007) Phosphorylation of
caspase-9 by CDK1 ⁄ cyclin B1 protects mitotic cells
against apoptosis. Mol Cell 26, 301–310.
19 Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ,
McLauchlan H, Klevernic I, Arthur JS, Alessi DR &
Cohen P (2007) The selectivity of protein kinase
inhibitors: a further update. Biochem J 408, 297–315.
20 Bain J, McLauchlan H, Elliott M & Cohen P (2003)
The specificities of protein kinase inhibitors: an update.
Biochem J 371, 199–204.
21 Lochhead PA, Sibbet G, Morrice N & Cleghon V
(2005) Activation-loop autophosphorylation is mediated
by a novel transitional intermediate form of DYRKs.
Cell 121, 925–936.
22 Himpel S, Tegge W, Frank R, Leder S, Joost HG &
Becker W (2000) Specificity determinants of substrate
recognition by the protein kinase DYRK1A. J Biol
Chem 275, 2431–2438.
23 Campbell LE & Proud CG (2002) Differing substrate
specificities of members of the DYRK family of argi-
nine-directed protein kinases. FEBS Lett 510, 31–36.

24 de Graaf K, Czajkowska H, Rottmann S, Packman LC,
Lilischkis R, Luscher B & Becker W (2006) The protein
kinase DYRK1A phosphorylates the splicing factor
SF3b1 ⁄ SAP155 at Thr434, a novel in vivo phosphoryla-
tion site. BMC Biochem 7,7.
25 de Graaf K, Hekerman P, Spelten O, Herrmann A,
Packman LC, Bussow K, Muller-Newen G & Becker W
(2004) Characterization of cyclin L2, a novel cyclin with
an arginine ⁄ serine-rich domain: phosphorylation by
DYRK1A and colocalization with splicing factors.
J Biol Chem 279, 4612–4624.
26 Kentrup H, Becker W, Heukelbach J, Wilmes A,
Schurmann A, Huppertz C, Kainulainen H & Joost HG
(1996) Dyrk, a dual specificity protein kinase with
unique structural features whose activity is dependent
on tyrosine residues between subdomains VII and VIII.
J Biol Chem 271, 3488–3495.
27 Wiechmann S, Czajkowska H, de Graaf K, Grotzinger
J, Joost HG & Becker W (2003) Unusual function of
the activation loop in the protein kinase DYRK1A.
Biochem Biophys Res Commun 302, 403–408.
28 Song Y, Kesuma D, Wang J, Deng Y, Duan J, Wang
JH & Qi RZ (2004) Specific inhibition of cyclin-depen-
dent kinases and cell proliferation by harmine. Biochem
Biophys Res Commun 317, 128–132.
29 Himpel S, Panzer P, Eirmbter K, Czajkowska H, Sayed
M, Packman LC, Blundell T, Kentrup H, Grotzinger J,
Joost HG et al. (2001) Identification of the autophos-
phorylation sites and characterization of their effects in
the protein kinase DYRK1A. Biochem J 359, 497–505.

30 Stennicke HR, Deveraux QL, Humke EW, Reed JC,
Dixit VM & Salvesen GS (1999) Caspase-9 can be acti-
vated without proteolytic processing. J Biol Chem 274,
8359–8362.
31 Stitzel ML, Cheng KC & Seydoux G (2007) Regulation
of MBK-2 ⁄ Dyrk kinase by dynamic cortical anchoring
during the oocyte-to-zygote transition. Curr Biol
17,
1545–1554.
32 Okui M, Ide T, Morita K, Funakoshi E, Ito F, Ogita
K, Yoneda Y, Kudoh J & Shimizu N (1999) High-level
expression of the Mnb ⁄ Dyrk1A gene in brain and heart
during rat early development. Genomics 62, 165–171.
33 Maenz B, Hekerman P, Vela EM, Galceran J & Becker
W (2008) Characterization of the human DYRK1A
promoter and its regulation by the transcription factor
E2F1. BMC Mol Biol 9, 30.
34 Guimera J, Casas C, Estivill X & Pritchard M (1999)
Human minibrain homologue (MNBH ⁄ DYRK1):
characterization, alternative splicing, differential tissue
expression, and overexpression in Down syndrome.
Genomics 57, 407–418.
35 Fotaki V, Dierssen M, Alcantara S, Martinez S, Marti
E, Casas C, Visa J, Soriano E, Estivill X & Arbones
ML (2002) Dyrk1A haploinsufficiency affects viability
and causes developmental delay and abnormal brain
morphology in mice. Mol Cell Biol 22, 6636–6647.
36 Kuida K, Haydar TF, Kuan CY, Gu Y, Taya C,
Karasuyama H, Su MS, Rakic P & Flavell RA (1998)
Reduced apoptosis and cytochrome c-mediated caspase

activation in mice lacking caspase 9. Cell 94, 325–337.
37 Hakem R, Hakem A, Duncan GS, Henderson JT,
Woo M, Soengas MS, Elia A, de la Pompa JL, Kagi D,
Khoo W et al. (1998) Differential requirement for cas-
pase 9 in apoptotic pathways in vivo. Cell 94, 339–352.
38 Alvarez M, Estivill X & de la Luna S (2003) DYRK1A
accumulates in splicing speckles through a novel target-
ing signal and induces speckle disassembly. J Cell Sci
116, 3099–3107.
39 Zhivotovsky B, Samali A, Gahm A & Orrenius S (1999)
Caspases: their intracellular localization and transloca-
tion during apoptosis. Cell Death Differ 6, 644–651.
40 Shikama Y, U M, Miyashita T & Yamada M (2001)
Comprehensive studies on subcellular localizations and
cell death-inducing activities of eight GFP-tagged apop-
tosis-related caspases. Exp Cell Res 264, 315–325.
41 Ritter PM, Marti A, Blanc C, Baltzer A, Krajewski S,
Reed JC & Jaggi R (2000) Nuclear localization of
procaspase-9 and processing by a caspase-3-like activity
in mammary epithelial cells. Eur J Cell Biol 79, 358–364.
42 Coulthard CE, Levene HH & Pyman FL (1933) The
chemotherapy of derivatives of harmine and harmaline.
I. Biochem J 27, 727–739.
A. Seifert et al. Phosphorylation of caspase 9 by DYRK1A
FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6279
43 Ishida J, Wang HK, Bastow KF, Hu CQ & Lee KH
(1999) Antitumor agents 201. Cytotoxicity of harmine
and beta-carboline analogs. Bioorg Med Chem Lett 9,
3319–3324.
44 Waki H, Park KW, Mitro N, Pei L, Damoiseaux R,

Wilpitz DC, Reue K, Saez E & Tontonoz P (2007) The
small molecule harmine is an antidiabetic cell-type-spe-
cific regulator of PPARgamma expression. Cell Metab
5, 357–370.
45 Woods YL, Rena G, Morrice N, Barthel A, Becker W,
Guo S, Unterman TG & Cohen P (2001) The kinase
DYRK1A phosphorylates the transcription factor
FKHR at Ser329 in vitro, a novel in vivo phosphoryla-
tion site. Biochem J 355, 597–607.
Supporting information
The following supplementary material is available:
Fig. S1. Deletion of caspase 9 by siRNA does not
reduce nuclear speckle staining caused by pThr125
antibody.
Fig. S2. Harmine inhibits Thr125 phosphorylation of
nuclear caspase 9.
This supplementary material can be found in the
online version of this article.
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sponding author for the article.
Phosphorylation of caspase 9 by DYRK1A A. Seifert et al.
6280 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS