Báo cáo khoa học: Activation, regulation, and inhibition of DYRK1A Walter Becker1 and Wolfgang Sippl2 pptx

MINIREVIEW
Activation, regulation, and inhibition of DYRK1A
Walter Becker
1
and Wolfgang Sippl
2
1 Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Germany
2 Institute of Pharmacy, Martin-Luther-Universita
¨
t Halle-Wittenberg, Germany
Introduction
Dual-specificity tyrosine phosphorylation-regulated
kinases (DYRKs) are a conserved family of eukaryotic
kinases that are related to the cyclin-dependent kinases
(CDKs), mitogen-activated protein kinases (MAPKs),
glycogen synthase kinases (GSKs) and CDK-like kin-
ases (CLKs), which are collectively termed the CMGC
group. Two distinctive features of DYRK1A have
originally stimulated the cloning and characterization
of this founding member of the mammalian DYRK
family. First, DYRK1A is a dual-specificity protein
kinase that catalyses the phosphorylation of serine and
threonine residues in its substrates as well as the auto-
phosphorylation on a tyrosine residue in the activation
loop [1,2]. Second, the human DYRK1A gene was
identified as a Down syndrome candidate gene,
because of its localization in the Down syndrome criti-
cal region on human chromosome 21 [3] and due
to the previous observation that the orthologous
Drosophila kinase, minibrain (MNB), has an essential
role in postembryonic neurogenesis [4]. Since then,

multiple evidence has been obtained for a function of
DYRK1A in neurodevelopment, supporting the
hypothesis that DYRK1A contributes to the aberrant
brain development underlying mental retardation
Keywords
CMGC kinases; dual-specificity; DYRK1A;
harmine; INDY; kinase inhibitor; structural
model; tyrosine autophosphorylation
Correspondence
W. Becker, Institute of Pharmacology and
Toxicology, RWTH Aachen University,
Wendlingweg 2, 52074 Aachen, Germany
Fax: +49 241 80 82433
Tel: +49 241 80 89124
E-mail:
Database
Structural data is available at the Protein
Data Bank under the accession numbers
2VX3 and 3KVW
(Received 15 July 2010, revised 26 August
2010, accepted 2 September 2010)
doi:10.1111/j.1742-4658.2010.07956.x
Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A)
is a protein kinase with diverse functions in neuronal development and
adult brain physiology. Higher than normal levels of DYRK1A are associ-
ated with the pathology of neurodegenerative diseases and have been impli-
cated in some neurobiological alterations of Down syndrome, such as
mental retardation. It is therefore important to understand the molecular
mechanisms that control the activity of DYRK1A. Here we review the cur-
rent knowledge about the initial self-activation of DYRK1A by tyrosine

autophosphorylation and propose that this mechanism presents an ances-
tral feature of the CMGC group of kinases. However, tyrosine phosphory-
lation does not appear to regulate the enzymatic activity of DYRK1A.
Control of DYRK1A may take place on the level of gene expression, inter-
action with regulatory proteins and regulated nuclear translocation.
Finally, we compare the properties of small molecule inhibitors that target
DYRK1A and evaluate their potential application and limitations. The
b-carboline alkaloid harmine is currently the most selective and potent
inhibitor of DYRK1A and has proven very useful in cellular assays.
Abbreviations
CDK, cyclin-dependent kinase; CLK, CDK-like kinase; DMAT, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DYRK1A, dual-specificity
tyrosine phosphorylation-regulated kinase 1A; EGCG, epigallocatechin-gallate; GSK, glycogen synthase kinase; MAPK, mitogen-activated
protein kinase; MNB, protein kinase encoded by the minibrain gene from Drosophila; NFAT, nuclear factor of activated T-cells; SPRED,
sprouty-related protein with an EVH1 domain; TBB, 4,5,6,7-tetrabromo-1H-benzotriazole.
246 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS
in Down syndrome [5,6]. More recently, overexpres-
sion of DYRK1A has also been associated with
neurodegenerative diseases [7]. The function of
DYRK1A in neuronal development and in neurode-
generation is covered by the two accompanying
reviews within this minireview series [8,9]. Because the
level of activity of DYRK1A is of key importance for
its physiological and pathological effects, this review
focuses on the molecular mechanisms of activation and
regulation of DYRK1A. In addition, we provide an
overview of the small molecule drugs that inhibit the
catalytic activity of DYRK1A.
Activation
Most protein kinases function as molecular switches
that can adopt distinct active and inactive conforma-

tions. Conversion between these states is in many cases
regulated by reversible phosphorylation of discrete ser-
ine, threonine or tyrosine residues in the centrally
located so-called ‘activation loop’. Phosphorylation of
the activation loop stabilizes a conformation with an
appropriately positioned substrate binding site [10].
Typically, this conformational change is driven by the
electrostatic interactions between the phosphoresidue
and a positively charged binding site named the RD
pocket. Main contributors to the basic nature of this
pocket are a conserved arginine (R) immediately pre-
ceding the invariant aspartate (D) in the catalytic loop
and a basic residue in the b9 strand [11] (Fig. 1).
Kinases of the DYRK family depend on the phos-
phorylation of an absolutely conserved tyrosine residue
in the activation loop to achieve full activity [1,2]. This
tyrosine (Y321 in DYRK1A) corresponds to Y185 in
the doubly phosphorylated TEY motif of ERK2, a res-
idue classified as a ‘secondary activation loop phos-
phorylation site’ because it does not interact with the
RD pocket [10]. Instead, the phosphorylated tyrosine
forms salt bridges with two arginines in the P + 1
loop (R189 and R192 in ERK2). Many kinases of the
CMGC group contain a tyrosine at the same position,
either in combination with a threonine as the ‘primary
phosphorylation site’ in a TxY motif, as in the MAP-
Ks, or in the absence of a primary site, as in the
DYRKs and GSK3a ⁄ b (Fig. 1). Although PRP4 and
HIPK1-3 harbour phosphorylatable serine or threonine
AB

Fig. 1. Tyrosine phosphorylation in the activation loop of CMGC kinases. (A) CMGC kinases with a tyrosine phosphorylation site in the acti-
vation loop belong to different branches of the CMGC group. Human Kinome provided courtesy of Cell Signaling Technology (http://
www.cellsignal.com). (B) Sequence motifs directly involved in the activation mechanism. Basic residues that interact with the primary phos-
phorylation site (RD pocket, formed by the residue preceding the catalytic aspartic acid and the b9 strand) and the basic residues in the
P + 1 loop that interact with the phosphotyrosine are shown in blue. In GSK3, the RD pocket binds a phosphorylated residue in the sub-
strate. Two conserved cysteines in the DYRK family that may form a reversible disulfide bond are highlighted in yellow. The mechanism of
tyrosine phosphorylation is indicated by symbols as indicated. For HIPK2, tyrosine phosphorylation was shown in a kinase-negative point
mutant, but so far there is no evidence for an upstream kinase [80]. The TEY motif was shown to be essential for full activity of MOK, but
as yet there is no direct evidence of tyrosine phosphorylation [81]. CLK1-4, ERK3-4, SRPK ⁄ MSSK1 and the CDK subfamily do not contain a
tyrosine in the respective position of the activation loop. Evidence for tyrosine phosphorylation in the activation loop was extracted from the
indicated references [82–86] or the PhosphoSitePlus database (P) ().
W. Becker and W. Sippl Activation, regulation, and inhibition of DYRK1A
FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS 247
residues at the primary phosphorylation site, they
lack the RD pocket, and phosphoproteomic studies
() only provide strong evi-
dence for the phosphorylation of the tyrosine in these
kinases (Table 1). In DYRK1A, pY321 is engaged in
the same interactions with the two arginines as pY185
in ERK2 (R325 and R328 in DYRK1A) (Fig. 2).
Whereas the dual-phosphorylation of the MAP
kinases is a classical paradigm for the on ⁄ off regulation
by upstream protein kinases, tyrosine phosphorylation
of DYRKs and GSK3 occurs by autophosphorylation
and appears to be constitutive [2,13]. This one-off
autoactivation takes place during or immediately after
translation by an intramolecular reaction [14,15]. In
Drosophila DYRK2 (dDYRK2), tyrosine autophos-
phorylation depends on the presence of an N-terminal
autophosphorylation accessory region. The N-terminal

autophosphorylation accessory region is conserved in a
subgroup of the DYRK family (class 2 DYRKs), but
is not required for tyrosine autophosphorylation of
MNB [16]. We have shown for mammalian DYRK1A
that tyrosine autophosphorylation is an intrinsic
capacity of the catalytic domain and does not depend
on other domains or any cofactor [17]. Notably, a few
kinases with the doubly phosphorylated TxY motif
also activate by autophosphorylation (ERK7, CDKL5,
Table 1), and some degree of activation loop auto-
phosphorylation has historically even been observed in
ERK1 and ERK2 [18,19]. Thus, a sometimes latent
capacity of tyrosine autophosphorylation appears to
be a characteristic of many CMGC group protein kin-
ases and may be an evolutionarily ancestral feature.
A very interesting intermediate mechanism exists in the
closely related kinases ICK and MAK, which auto-
phosphorylate the tyrosine in the TDY motif, but are
fully active only after threonine phosphorylation by an
upstream kinase [20,21]. Another variation of the
theme is realized in the stress-activated kinase p38a,
which can be activated either by upstream kinases or
by regulated tyrosine autophosphorylation [22]. Fur-
ther work is required to dissect the full spectrum of
activation modes of the different CMGC group kinas-
es. However, so far there is no evidence that the cata-
lytic activity of any member of the DYRK family is
reversibly regulated by phosphorylation ⁄ dephosphory-
lation of the activation loop tyrosine.
Mature DYRKs phosphorylate their substrates only

on serine or threonine residues and cannot rephospho-
rylate on tyrosine after phosphatase treatment [14,23].
How can a serine ⁄ threonine-specific protein kinase
autophosphorylate on tyrosine? Lochhead et al. [14]
proposed that only a translational folding intermediate
of DYRKs with biochemical properties different from
the mature kinase is capable of tyrosine phosphoryla-
tion. This idea is supported by their finding that the
tyrosine phosphorylation of newly synthesized Dro-
sophila dDYRK2 and substrate phosphorylation by
the mature kinase exhibit differential sensitivity
towards kinase inhibitors. This result suggests that the
‘dual-specificity’ of DYRKs comes together with a
‘dual-sensitivity’ to kinase inhibitors [14].
There are several open questions concerning the acti-
vation mechanism of DYRK1A. The substitution of
the activation loop tyrosine by phenylalanine markedly
reduces (> 80%) the catalytic activity of DYRK1A
[1,2,24]. Surprisingly, dephosphorylation does not inac-
tivate mature DYRK1A [23] and reduces the activity
of Drosophila dDYRK2 and MNB by only  50%
Table 1. DYRK1A inhibitors. List of small molecule drugs that have been used as DYRK1A inhibitors.
Inhibitor IC
50
for DYRK1A
a
IC
50
for other kinases
a

Comments References
Purvalanol 300 n
M 100 nM CDK2 Inhibits tyrosine autophosphorylation
of dDYRK2
[11,58]
DMAT 120 n
M 150 nM CK2 [75]
TBB 4360 n
M 150 nM CK2
990 n
M DYRK2
Does not inhibit tyrosine autophosphorylation
of dDYRK2
[11,75,76]
Pyrazolidin-diones
18 and 21
600 n
M ND IC
50
was determined in an autophosphorylation
assay
[55]
TG003 12 n
M
b
930 lM
19 nM CLK1
130 n
M DYRK1B
Less potent inhibitor of DYRK1A than harmine

in HeLa cells
c
[77,78]
[12]
INDY 240 n
M 230 nM DYRK1B Structurally related with TG003 [12]
EGCG 330 n
M 1000 nM PRAK Non-ATP-competitive inhibitor [58,79]
Harmine 80 n
M
b
33 nM
150 nM DYRK1B
900 n
M DYRK2
800 n
M DYRK3
IC
50
= 1900 nM for tyrosine auto-phosphorylation
of DYRK1A
[17,59]
a
IC
50
values were determined in in vitro kinase assays at variable reaction conditions.
b
Deviating results obtained by different assays.
c
M. Rahbari & W. Becker, unpublished results.

Activation, regulation, and inhibition of DYRK1A W. Becker and W. Sippl
248 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS
[14]. It is conceivable that tyrosine phosphorylation is
only required for the switch into the active conformation
but not for maintaining this state. Alternatively, the
stabilizing effect of the salt bridges between the phosp-
hotyrosine with the two arginines in the P + 1 may be
critical in living cells, but less so in in vitro assays. For
comparison, it is interesting to consider the role of the
phosphotyrosine (pY216) in GSK3b, which resembles
the DYRKs in its autoactivation mechanism [13,15].
Analysis of crystal structures revealed that unphos-
phorylated GSK3b, in stark contrast to unphosphory-
lated ERK2, can acquire a catalytically active
conformation [25,26]. However, dephosphorylation de-
stabilizes the active conformation and leads to a loss
of activity over time [13].
Regulation
The available evidence suggests that DYRK1A is
always catalytically active when isolated from tissues
or cells. However, the role of protein kinases as cellu-
lar regulators implies that their own activity is some-
how regulated. Given that the activation loop
phosphorylation is apparently constitutive, how then is
the activity of DYRK1A modulated? Genetic evidence
indicates that small changes in expression levels of
DYRK1A have severe phenotypic consequences, as
exemplified by patients with homozygous deficiency of
DYRK1A [27] and transgenic animal models [28].
Thus, the function of DYRK1A may be controlled by

more subtle changes in activity than the paradigmatic
on ⁄ off switches as known from MAPKs, cAMP-
dependent protein kinase, receptor tyrosine kinases
and many other kinases [11]. Although our present
understanding of the regulation of DYRK1A function
is only rudimentary, we will discuss here potential
mechanisms by which the function of DYRK1A may
be controlled, including changes in expression levels,
association with regulatory proteins, changes in subcel-
lular localization.
There is evidence for a regulation of DYRK1A on
the level of gene expression and protein abundance. A
recent study demonstrated circadian changes in
DYRK1A levels in the mouse liver and identified
DYRK1A as a novel component of the molecular
clock [29]. Numerous microarray studies have revealed
striking changes in DYRK1A mRNA levels in various
systems of cellular differentiation and proliferation (for
references see [30]). However, very little is known
about the transcriptional regulation of DYRK1A gene
expression. Activator protein 4 was described as a neg-
ative regulator of DYRK1A expression in non-neural
cells [31]. In the neuroblastoma cell line SH-SY5Y, an
increase in DYRK1A mRNA was induced by the
b-amyloid peptide, but nothing is known about the
potential mechanism of regulation [32]. The transcrip-
tion factor E2F1 increases DYRK1A mRNA levels by
enhancing promoter activity [30]. In a recent report,
nuclear factor of activated T-cells (NFATc1) was
shown to upregulate DYRK1A levels in bone marrow

macrophages [33]. This control mechanism forms part
of a negative feedback loop, because NFATs are nega-
tively regulated by DYRK1A [34]. The presence of
potentially destabilizing AUUUA elements in the
3¢-UTR of the DYRK1A mRNA and the PEST region
in the DYRK1A protein [1] supports the hypothesis
that DYRK1A levels are subject to rapid changes.
PEST sequences are rich in the amino acids proline
(P), glutamate (E), serine (S) and threonine (T) and
correlate with rapid protein turnover in eukaryotic
cells, often by acting as degradation tags that direct
proteins for proteasome-mediated destruction [35].
However, data on the half-life of DYRK1A or on the
regulation of its degradation have not been reported.
Several studies suggest a regulation of DYRK1A by
interacting proteins. Binding of 14-3-3b to an autop-
hosphorylated serine residue in the C-terminal domain
of DYRK1A (Ser529, numbered Ser520 in the short
splicing variant of DYRK1A) stimulates the catalytic
activity of DYRK1A up to a factor of 2 [36]. Binding
Fig. 2. Potential intramolecular disulfide bridge in DYRK1A observed
in the X-ray structure of DYRK1A (PDB acc. 2VX3). The distance
between the thiol groups of the two conserved cysteines (coloured
green, C286 and C312 in DYRK1A) is  4.3 A
˚
, indicating the
probability of forming a disulfide bridge in the oxidized form. In
addition, arginines R325 and R328 interacting with the phos-
phorylated Y321 are shown (hydrogen bonds are shown as dashed
lines).

W. Becker and W. Sippl Activation, regulation, and inhibition of DYRK1A
FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS 249
of RanBPM (=RanBP9) to a neighbouring region in
the C-terminal domain (550–563) negatively modulates
DYRK1A activity [37]. The WD40 repeat protein,
WDR68 (also called HAN11, official human gene sym-
bol DCAF7), has repeatedly been found to be associ-
ated with DYRK1A and ⁄ or DYRK1B and may
function as a regulatory subunit [38–41]. Overexpres-
sion of WDR68 inhibited the DYRK1A-mediated stim-
ulation of GLI1-dependent reporter gene activity [39].
Recently, SPRED1 and SPRED2 (sprouty-related pro-
tein with an EVH1 domain) were found to directly
interact with the catalytic domain of DYRK1A and to
inhibit the phosphorylation of the substrate proteins,
Tau and STAT3 [42]. Interestingly, the inhibitory effect
of the SPRED proteins appears to be due to competi-
tion with the substrate proteins and not due to a gen-
eral inhibition of catalytic activity. Further research is
clearly required to elucidate the role of these interacting
proteins in the regulation of DYRK1A.
A novel regulatory mechanism was recently revealed
for the DYRK2 orthologous kinase in Caenorhabditis
elegans. In this case, the catalytically inactive ‘pseudo-
phosphatases’ EGG-4 and EGG-5 inhibit substrate
phosphorylation by MBK-2 by binding to the phosp-
hotyrosine motif in the activation loop of the kinase
[43,44]. Catalytically inactive tyrosine phosphatases
also exist in mammals, and it will be exciting to see
whether this mechanism applies to other members of

the DYRK family, including DYRK1A.
Regulated nuclear translocation has been reported
for mammalian DYRK2 and the DYRK in budding
yeast, Yak1p [45,46]. DYRK1A harbours a functional
nuclear localization sequence in its N-terminal domain
and has been found in both the nucleus and in the
cytoplasm (see also the accompanying minireview by
Wegiel et al. [9]), possibly depending on the cell type.
Furthermore, DYRK1A phosphorylates both nuclear
and cytoplasmic proteins [8,47]. DYRK1A controls
nuclear import (GLI1) or export (FOXO1, NFAT) of
several transcription factors [34,48,49], but the ques-
tion whether DYRK1A itself undergoes cytoplasmic–
nuclear shuttling has not yet been directly addressed.
Interestingly, dynamic changes in the intracellular
localization of the chicken DYRK1A orthologue,
MNB, have been deduced from immunofluorescence
analyses of developing neurons during brain develop-
ment [5].
Finally, we want to propose a new potential mode
of regulation of DYRKs that has not yet been experi-
mentally tested. As we and others have previously
noted [50,51], two cysteine residues are located in those
positions that correspond to the RD pocket in kinases
regulated by the primary phosphorylation site in the
activation loop (RD kinases, Fig. 1). This unique
structural peculiarity is conserved in DYRK family
kinases from fungi, plants and animals, but cannot
easily be explained by the catalytic mechanism or
structural constraints and thus must have another

essential function. In the three-dimensional structures
of DYRK1A and DYRK2 (PDB acc. 2VX3; 2WO6;
3KVW; 3K2L), the thiol moieties of these cysteines are
sufficiently close to each other ( 4.3 A
˚
in DYRK1A
and DYRK2) to allow the formation of a disulfide
bridge (Fig. 2). It is tempting to speculate that redox
regulation of DYRK1A can occur through changes in
the redox state of these cysteines. Redox modification
at C286 and C312 in DYRK1A (-SH reduced to -S–S-
oxidized state) could result in a conformational
change, which could then somehow influence the cata-
lytic activity of DYRK kinases.
DYRK1A inhibitors
Small cell-permeant inhibitors of protein kinases are
important tools for studying intracellular signal trans-
duction pathways. Although genetic techniques such as
RNA interference offer an alternative to study kinase
function, small-molecule inhibitors provide more rapid
temporal control. Moreover, comparisons of genetic
kinase knockouts with effects of inhibitors have often
revealed major differences in the resulting phenotype
[52]. The hypothesis that the elevated activity of
DYRK1A contributes to the cognitive deficits in
Down syndrome and the development of Alzheimer’s
disease has stimulated interest in DYRK1A as a
potential target for therapeutic inhibitors [53,54].
Synthetic inhibitors
The first targeted approach to develop an inhibitor of

DYRK1A resulted in the identification and optimiza-
tion of pyrazolidinedione compounds that inhibit
DYRK1A autophosphorylation with IC
50
values from
0.6–2.5 lm. [53,55]. These inhibitors remain to be fur-
ther characterized for their specificity against a broad
panel of kinases and their effects in cells or in experi-
mental animals. Ogawa et al. recently reported the
characterisation of a new benzothiazol inhibitor of
DYRK1A, designated INDY, as well as the three-
dimensional structure of the DYRK1A/INDY complex
[PDB accession 3ANQ]. Furthermore, the authors
demonstrate that harmine resembles TG003 and INDY
in its capacity to inhibit CLKs at a level comparable
with the inhibition of DYRK1A [12].
Several compounds originally designed to target other
protein kinases were uncovered as fairly efficient inhibi-
Activation, regulation, and inhibition of DYRK1A W. Becker and W. Sippl
250 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS
tors of DYRK1A (Table 1, Fig. 3). Purvalanol A had
been developed as an inhibitor of CDKs, 2-dimethyla-
mino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT)
and 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) as CK2
inhibitors, and TG003 as an inhibitor of CLKs. Obvi-
ously, the promiscuous behaviour of these inhibitors
limits their value as tools for the analysis of signalling
pathways. Nevertheless, use of purvalanol A has guided
the identification of DYRK1A as the kinase responsible
for the phosphorylation of specific sites in MAP1B and

caspase 9 [56,57]. A prodrug of INDY was successfully
used to reverse malformations of Xenopus embryos
induced by DYRK1A overexpression [12]. Furthermore,
cross-reactivity with other kinases is not an issue in
mechanistic studies with purified enzymes, such as the
analysis of the autoactivation mechanism of dDYRK2
and MNB [14].
Natural compounds
Two plant compounds, epigallocatechin-gallate
(EGCG) and harmine, have been identified as
DYRK1A inhibitors in selectivity profiling studies
[58,59] (Table 1). EGCG, the major polyphenolic com-
pound of green tea, inhibited DYRK1A rather specifi-
cally among 29 kinases tested [58]. However, multiple
and heterogeneous effects of EGCG on signalling path-
ways have been described (e.g. [60–62]). Furthermore,
EGCG exhibits complex pharmacokinetic properties
and poor bioavailability [63], which limit its usefulness
in cell and animal experiments. EGCG was used in cell
culture studies to confirm the presumed role of
DYRK1A in signalling events [56,64–66] and to rescue
brain defects of DYRK1A-overexpressing mice [67].
Harmine is a b-carboline alkaloid that has long been
known as a potent inhibitor of monoamine oxidase A
(IC
50
=5nm) [68]. Harmine is produced by divergent
plant species, including the South American vine Ban-
isteriopsis caapi and the mideastern shrub Peganum
harmala (Syrian rue). Banisteriopsis is a component of

hoasca (also called ayahuasca or yage
´
), an hallucino-
genic brew of plant extracts used in shamanic rituals
and South American sects for its visionary effects. The
monoamine oxidase-inhibiting activity of harmine
blocks the first pass metabolism of dimethyltryptamine
and thereby allows the oral ingestion of this natural
hallucinogenic. It is interesting to note that plasma lev-
els of harmine in hoasca users are within a range
( 0.5 lm) [69] expected to cause a substantial inhibi-
tion of DYRK1A.
As a kinase inhibitor, harmine displays excellent
specificity for DYRK1A among 69 protein kinases
[59]. Obviously, side-effects on kinases not included in
the screen cannot be excluded. Development of an
optimized DYRK1A inhibitor on the basis of harmine
as a lead structure appears feasible, because harmine is
a rather small molecule (212 Da). The crystal struc-
tures of DYRK1A complexed with an indazol inhibi-
tor and DYRK2 complexed with an indirubin
inhibitor have been solved recently [70]. Based on the
analysis of the two highly similar 3D structures, only
three amino acid residues in the binding pocket have
been found to be different. These include V222, M240
and V306, which are substituted by the more spacious
residues I212, L230 and I294 in DYRK2. Docking
studies showed that harmine interacts with the residues
of the ATP binding pocket and is involved in two
hydrogen bonds – one to the hinge region (backbone

NH of M240) and one to the conserved K188. Based
on the model of the DYRK1A ⁄ harmine complex, it is
suggested that the accessible volume of the ATP bind-
ing pocket can accommodate substituents at the
b-carboline structure (Fig. 4A). Interestingly, due to
the substitution of L230 for M240 and I212 for V222,
the binding pocket in DYRK2 is more restricted
(Fig. 4B), resulting in a slightly different orientation of
the docked harmine. For DYRK2 the observed bind-
ing mode is less favourable, showing longer hydrogen
bond distances between inhibitor and kinase (not
shown).
The closest relative of DYRK1A, DYRK1B, is
inhibited at somewhat higher concentrations by har-
mine (Table 1), but this difference is not sufficient
to discriminate these kinases pharmacologically [17].
Harmine inhibits DYRK1A in cultured cells with simi-
Fig. 3. Chemical structures of known DYRK1A inhibitors.
W. Becker and W. Sippl Activation, regulation, and inhibition of DYRK1A
FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS 251
lar potency as in vitro at concentrations where little
toxicity is observed [17]. Effects of harmine on many
other targets have been described, but generally require
much higher concentrations than the inhibition of
DYRK1A. The antidiabetic effect of harmine as an
inducer of Id2 and PPARc expression in preadipocytes
also required concentrations greater than 1 lm [70,71].
The molecular target responsible for these effects is
not yet known. Harmine is arguably the most useful
inhibitor of DYRK1A presently available and has

been used in several studies to support a presumed cel-
lular function of DYRK1A [57,72–74]. However, the
inhibitory effect on monoamine oxidase clearly limits
its use in animals and in experimental systems suscepti-
ble to the influence of monoamine oxidase (e.g. brain
slices).
Specific inhibition of tyrosine
autophosphorylation?
As noted above, Lochhead et al. [14] provided
evidence that the ‘dual-specificity’ of the Drosophila
DYRKs is associated with a differential inhibitor
sensitivity of the transitory folding intermediate and
the mature conformation of the kinase. We have
recently shown that harmine inhibits tyrosine auto-
phosphorylation of mammalian DYRK1A much less
potently than the phosphorylation of exogenous sub-
strates [17]. However, it must be noted that these
reactions are kinetically very different. It is possible
that a much lower degree of catalytic activity is
required for the intramolecular autophosphorylation
(a first-order reaction) than the trans-phosphorylation
of other substrates in a second-order reaction. The
identification of an inhibitor that specifically or
preferentially inhibits tyrosine autophosphorylation of
DYRK1A versus serine ⁄ threonine phosphorylation of
substrates could prove the concept of dual inhibitor
sensitivity. This is important not only from a
biochemical point of view, because the one-off auto-
activation mechanism of the DYRKs suggests that
inhibitors of tyrosine autophosphorylation should act

irreversibly. However, this hypothesis remains to be
experimentally substantiated.
Acknowledgements
We thank German Erlenkamp for excellent technical
assistance. Financial support of our research on
DYRK1A by the Deutsche Forschungsgemeinschaft
and the Jerome Lejeune Foundation is gratefully
acknowledged (WB).
References
1 Kentrup H, Becker W, Heukelbach J, Wilmes A, Schu
¨
r-
mann A, Huppertz C, Kainulainen H & Joost HG
(1996) Dyrk, a dual specificity protein kinase with
Fig. 4. Structure of the ATP-binding pocket in DYRK1A and DYRK2. (A) Close-up of DYRK1A ATP binding pocket with docked harmine. Only
the interacting amino acid residues and the residues that are different between DYRK1A and DYRK2 are displayed for clarity. Harmine (col-
oured pink) is involved in two hydrogen bonds to M240 and K188 (shown as dashed lines). In addition, the gatekeeper residue F238 is
shown. The model suggests that the accessible volume of the ATP binding pocket can accommodate substituents at the ß-carboline struc-
ture. After submission of this article, the proposed orientation of harmine in our model was confirmed by crystallography of the
DYRK1A ⁄ harmine complex [12]. (B) Local superposition between DYRK1A and DYRK2 (PDB acc. 2VX3 and 3KVW), illustrating differences in
the ATP binding pocket.
Activation, regulation, and inhibition of DYRK1A W. Becker and W. Sippl
252 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS
unique structural features whose activity is dependent
on tyrosine residues between subdomains VII and VIII.
J Biol Chem 271, 3488–3495.
2 Himpel S, Panzer P, Eirmbter K, Czajkowska H,
Sayed M, Packman LC, Blundell T, Kentrup H,
Gro
¨

tzinger J, Joost HG et al. (2001) Identification of
the autophosphorylation sites and characterization of
their effects in the protein kinase DYRK1A. Biochem
J 359, 497–505.
3 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.
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.
5Ha
¨
mmerle B, Elizalde C, Galceran J, Becker W &
Tejedor FJ (2003) The MNB ⁄ DYRK1A protein
kinase: neurobiological functions and Down
syndrome implications. J Neural Transm Suppl 67,
129–137.
6 Park J, Song WJ & Chung KC (2009) Function and
regulation of Dyrk1A: towards understanding Down
syndrome. Cell Mol Life Sci 66, 3235–3240.
7 Ferrer I, Barrachina M, Puig B, Martinez de LM,
Marti E, Avila J & Dierssen M (2005) Constitutive
Dyrk1A is abnormally expressed in Alzheimer disease,
Down syndrome, Pick disease, and related transgenic
models. Neurobiol Dis 20, 392–400.

8Ha
¨
mmerle B & Tejedor FJ (2010) MNB ⁄ DYRK1A: a
multiple regulator of neuronal development. FEBS J
278, 223–235.
9 Wegiel J, Gong CX & Hwang YW (2010) DYRK1A:
the role in neurodegenerative diseases. FEBS J 278,
236–245.
10 Nolen B, Taylor S & Ghosh G (2004) Regulation of
protein kinases; controlling activity through activation
segment conformation. Mol Cell 15, 661–675.
11 Johnson LN, Noble ME & Owen DJ (1996) Active and
inactive protein kinases: structural basis for regulation.
Cell 85, 149–158.
12 Ogawa Y, Nonaka Y, Goto T, Ohnishi E, Hiramatsu
T, Kii I, Yoshida M, Ikura T, Onogi H, Shibuya H
et al. (2010) Development of a novel selective inhibitor
of the Down syndrome-related kinase Dyrk1A. Nat
Commun 1, 1–9.
13 Cole A, Frame S & Cohen P (2004) Further
evidence that the tyrosine phosphorylation of glycogen
synthase kinase-3 (GSK3) in mammalian cells is an
autophosphorylation event. Biochem J 377, 249–
255.
14 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.
15 Lochhead PA, Kinstrie R, Sibbet G, Rawjee T, Morrice
N & Cleghon V (2006) A chaperone-dependent

GSK3beta transitional intermediate mediates activation-
loop autophosphorylation. Mol Cell 24, 627–633.
16 Kinstrie R, Luebbering N, Miranda-Saavedra D, Sibbet
G, Han J, Lochhead PA & Cleghon V (2010) Charac-
terization of a domain that transiently converts class 2
DYRKs into intramolecular tyrosine kinases. Sci Signal
3, ra16.
17 Go
¨
ckler N, Jofre G, Papadopoulos C, Soppa U, Teje-
dor FJ & Becker W (2009) Harmine specifically inhibits
protein kinase DYRK1A and interferes with neurite
formation. FEBS J 276, 6324–6337.
18 Crews CM, Alessandrini AA & Erikson RL (1991)
Mouse Erk-1 gene product is a serine ⁄ threonine protein
kinase that has the potential to phosphorylate tyrosine.
Proc Natl Acad Sci USA 88, 8845–8849.
19 Seger R, Ahn NG, Boulton TG, Yancopoulos GD,
Panayotatos N, Radziejewska E, Ericsson L, Bratlien
RL, Cobb MH & Krebs EG (1991) Microtubule-associ-
ated protein 2 kinases, ERK1 and ERK2, undergo
autophosphorylation on both tyrosine and threonine
residues: implications for their mechanism of activation.
Proc Natl Acad Sci USA 88, 6142–6146.
20 Fu Z, Schroeder MJ, Shabanowitz J, Kaldis P, Togawa
K, Rustgi AK, Hunt DF & Sturgill TW (2005) Activa-
tion of a nuclear Cdc2-related kinase within a mitogen-
activated protein kinase-like TDY motif by autophos-
phorylation and cyclin-dependent protein kinase-activat-
ing kinase. Mol Cell Biol 25, 6047–6064.

21 Fu Z, Larson KA, Chitta RK, Parker SA, Turk BE,
Lawrence MW, Kaldis P, Galaktionov K, Cohn SM,
Shabanowitz J et al. (2006) Identification of yin-yang
regulators and a phosphorylation consensus for male
germ cell-associated kinase (MAK)-related kinase. Mol
Cell Biol 26, 8639–8654.
22 Ge B, Gram H, Di Padova F, Huang B, New L, Ulevitch
RJ, Luo Y & Han J (2002) MAPKK-independent activa-
tion of p38alpha mediated by TAB 1-dependent auto-
phosphorylation of p38alpha. Science 295, 1291–1294.
23 Adayev T, Chen-Hwang MC, Murakami N, Lee E, Bol-
ton DC & Hwang YW (2007) Dual-specificity tyrosine
phosphorylation-regulated kinase 1A does not require
tyrosine phosphorylation for activity in vitro. Biochemis-
try 46, 7614–7624.
24 Wiechmann S, Czajkowska H, de Graaf K, Gro
¨
tzinger
J, Joost HG & Becker W (2003) Unusual function of
the activation loop in the protein kinase DYRK1A.
Biochem Biophys Res Commun 302, 403–408.
25 Dajani R, Fraser E, Roe SM, Young N, Good V, Dale
TC & Pearl LH (2001) Crystal structure of glycogen
W. Becker and W. Sippl Activation, regulation, and inhibition of DYRK1A
FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS 253
synthase kinase 3 beta: structural basis for phosphate-
primed substrate specificity and autoinhibition. Cell
105, 721–732.
26 Dajani R, Fraser E, Roe SM, Yeo M, Good VM,
Thompson V, Dale TC & Pearl LH (2003) Structural

basis for recruitment of glycogen synthase kinase 3beta
to the axin-APC scaffold complex. EMBO J 22, 494–
501.
27 Møller RS, Ku
¨
bart S, Hoeltzenbein M, Heye B, Vogel
I, Hansen CP, Menzel C, Ullmann R, Tommerup N,
Ropers HH et al. (2008) Truncation of the Down syn-
drome candidate gene DYRK1A in two unrelated
patients with microcephaly. Am J Hum Genet 82, 1165–
1170.
28 Dierssen M & de Lagran MM (2006) DYRK1A (dual-
specificity tyrosine-phosphorylated and -regulated
kinase 1A): a gene with dosage effect during develop-
ment and neurogenesis. Sci World J 6 , 1911–1922.
29 Kurabayashi N, Hirota T, Sakai M, Sanada K &
Fukada Y (2010) DYRK1A and glycogen synthase
kinase 3beta, a dual-kinase mechanism directing
proteasomal degradation of CRY2 for circadian
timekeeping. Mol Cell Biol 30, 1757–1768.
30 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.
31 Kim MY, Jeong BC, Lee JH, Kee HJ, Kook H,
Kim NS, Kim YH, Kim JK, Ahn KY & Kim KK
(2006) A repressor complex, AP4 transcription factor
and geminin, negatively regulates expression of target
genes in nonneuronal cells. Proc Natl Acad Sci USA
103, 13074–13079.

32 Kimura R, Kamino K, Yamamoto M, Nuripa A, Kida
T, Kazui H, Hashimoto R, Tanaka T, Kudo T, Yamag-
ata H et al. (2007) The DYRK1A gene, encoded in
chromosome 21 Down syndrome critical region, bridges
between beta-amyloid production and tau phosphoryla-
tion in Alzheimer disease. Hum Mol Genet 16, 15–23.
33 Lee Y, Ha J, Kim HJ, Kim YS, Chang EJ, Song WJ
& Kim HH (2009) Negative feedback inhibition of
NFATc1 by DYRK1A regulates bone homeostasis.
J Biol Chem 284, 33343–33351.
34 Arron JR, Winslow MM, Polleri A, Chang CP, Wu H,
Gao X, Neilson JR, Chen L, Heit JJ, Yamasaki N et al.
(2006) NFAT dysregulation by increased dosage of
DSCR1 and DYRK1A on chromosome 21. Nature 441,
595–601.
35 Rechsteiner M & Rogers SW (1996) PEST sequences
and regulation by proteolysis. Trends Biochem Sci 21,
267–271.
36 Alvarez M, Altafaj X, Aranda S & de la Luna S (2007)
DYRK1A autophosphorylation on serine residue 520
modulates its kinase activity via 14-3-3 binding. Mol
Biol Cell 18, 1167–1178.
37 Zou Y, Lim S, Lee K, Deng X & Friedman E (2003)
Serine ⁄ threonine kinase Mirk ⁄ Dyrk1B is an inhibitor of
epithelial cell migration and is negatively regulated by
the Met adaptor Ran-binding protein M. J Biol Chem
278, 49573–49581.
38 Skurat AV & Dietrich AD (2004) Phosphorylation of
Ser640 in muscle glycogen synthase by DYRK family
protein kinases. J Biol Chem 279, 2490–2498.

39 Morita K, Lo Celso C, Spencer-Dene B, Zouboulis CC
& Watt FM (2006) HAN11 binds mDia1 and controls
GLI1 transcriptional activity. J Dermatol Sci 44 , 11–20.
40 Mazmanian G, Kovshilovsky M, Yen D, Mohanty A,
Mohanty S, Nee A & Nissen RM (2010) The zebrafish
dyrk1b gene is important for endoderm formation.
Genesis 48, 20–30.
41 Komorek J, Kuppuswamy M, Subramanian T,
Vijayalingam S, Lomonosova E, Zhao LJ, Mymryk JS,
Schmitt K & Chinnadurai G (2010) Adenovirus type 5
E1A and E6 proteins of low-risk cutaneous beta-human
papillomaviruses suppress cell transformation through
interaction with FOXK1 ⁄ K2 transcription factors.
J Virol 84, 2719–2731.
42 Li D, Jackson RA, Yusoff P & Guy GR (2010) The
direct association of
sprouty-related protein with an
EVH1 domain (SPRED) 1 or SPRED2 with DYRK1A
modifies substrate kinase interactions. J Biol Chem 285,
35374–35385.
43 Cheng KC, Klancer R, Singson A & Seydoux G (2009)
Regulation of MBK-2 ⁄ DYRK by CDK-1 and the
pseudophosphatases EGG-4 and EGG-5 during the
oocyte-to-embryo transition. Cell 139, 560–572.
44 Parry JM, Velarde NV, Lefkovith AJ, Zegarek MH,
Hang JS, Ohm J, Klancer R, Maruyama R, Druzhinina
MK, Grant BD et al. (2009) EGG-4 and EGG-5 link
events of the oocyte-to-embryo transition with meiotic
progression in C. elegans. Curr Biol 19, 1752–1757.
45 Taira N, Nihira K, Yamaguchi T, Miki Y & Yoshida

K (2007) DYRK2 is targeted to the nucleus and
controls p53 via Ser46 phosphorylation in the apoptotic
response to DNA damage. Mol Cell 25, 725–738.
46 Moriya H, Shimizu-Yoshida Y, Omori A, Iwashita S,
Katoh M & Sakai A (2001) Yak1p, a DYRK family
kinase, translocates to the nucleus and phosphorylates
yeast Pop2p in response to a glucose signal. Genes Dev
15, 1217–1228.
47 Galcera
´
n J, de Graaf K, Tejedor FJ & Becker W (2003)
The MNB ⁄ DYRK1A protein kinase: genetic and
biochemical properties. J Neural Transm Suppl 67 ,
139–148.
48 Mao J, Maye P, Kogerman P, Tejedor FJ, Toftgard R,
Xie W, Wu G & Wu D (2002) Regulation of Gli1 tran-
scriptional activity in the nucleus by Dyrk1. J Biol
Chem 277, 35156–35161.
49 Woods YL, Rena G, Morrice N, Barthel A, Becker W,
Guo S, Unterman TG & Cohen P (2001) The kinase
Activation, regulation, and inhibition of DYRK1A W. Becker and W. Sippl
254 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS
DYRK1A phosphorylates the transcription factor
FKHR at Ser329 in vitro, a novel in vivo phosphoryla-
tion site. Biochem J 355, 597–607.
50 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.
51 Kannan N & Neuwald AF (2004) Evolutionary con-

straints associated with functional specificity of the
CMGC protein kinases MAPK, CDK, GSK, SRPK,
DYRK, and CK2alpha. Protein Sci 13, 2059–2077.
52 Knight ZA & Shokat KM (2007) Chemical genetics:
where genetics and pharmacology meet. Cell 128, 425–
430.
53 Kim ND, Yoon J, Kim JH, Lee JT, Chon YS, Hwang
MK, Ha I & Song WJ (2006) Putative therapeutic
agents for the learning and memory deficits of people
with Down syndrome. Bioorg Med Chem Lett 16, 3772–
3776.
54 Savage MJ & Gingrich DE (2009) Advances in the
development of kinase inhibitor therapeutics for Alzhei-
mer’s disease. Drug Dev Res 70, 125–144.
55 Koo KA, Kim ND, Chon YS, Jung MS, Lee BJ,
Kim JH & Song WJ (2009) QSAR analysis of
pyrazolidine-3,5-diones derivatives as Dyrk1A
inhibitors. Bioorg Med Chem Lett 19, 2324–2328.
56 Scales TM, Lin S, Kraus M, Goold RG & Gordon-
Weeks PR (2009) Nonprimed and DYRK1A-primed
GSK3 beta-phosphorylation sites on MAP1B regulate
microtubule dynamics in growing axons. J Cell Sci 122,
2424–2435.
57 Seifert A, Allan LA & Clarke PR (2008) DYRK1A
phosphorylates caspase 9 at an inhibitory site and is
potently inhibited in human cells by harmine. FEBS J
275, 6268–6280.
58 Bain J, McLauchlan H, Elliott M & Cohen P (2003)
The specificities of protein kinase inhibitors: an update.
Biochem J 371, 199–204.

59 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.
60 Tachibana H, Koga K, Fujimura Y & Yamada K
(2004) A receptor for green tea polyphenol EGCG. Nat
Struct Mol Biol 11, 380–381.
61 Khan N, Afaq F, Saleem M, Ahmad N & Mukhtar H
(2006) Targeting multiple signaling pathways by green
tea polyphenol (-)-epigallocatechin-3-gallate. Cancer Res
66, 2500–2505.
62 Bode AM & Dong Z (2006) Molecular and cellular
targets. Mol Carcinog 45, 422–430.
63 Lambert JD, Sang S & Yang CS (2007) Biotransfor-
mation of green tea polyphenols and the biological
activities of those metabolites. Mol Pharm 4, 819–
825.
64 Canzonetta C, Mulligan C, Deutsch S, Ruf S, O’Doher-
ty A, Lyle R, Borel C, Lin-Marq N, Delom F, Groet J
et al. (2008) DYRK1A-dosage imbalance perturbs
NRSF ⁄ REST levels, deregulating pluripotency and
embryonic stem cell fate in Down syndrome. Am J
Hum Genet 83
, 388–400.
65 Murakami N, Xie W, Lu RC, Chen-Hwang MC, Wie-
raszko A & Hwang YW (2006) Phosphorylation of am-
phiphysin I by minibrain kinase ⁄ dual-specificity tyrosine
phosphorylation-regulated kinase, a kinase implicated
in Down syndrome. J Biol Chem 281, 23712–23724.
66 Shi J, Zhang T, Zhou C, Chohan MO, Gu X, Wegiel J,

Zhou J, Hwang YW, Iqbal K, Grundke-Iqbal I et al.
(2008) Increased dosage of Dyrk1A alters alternative
splicing factor (ASF)-regulated alternative splicing of
tau in Down syndrome. J Biol Chem 283 , 28660–28669.
67 Guedj F, Se
´
brie
´
C, Rivals I, Ledru A, Paly E, Bizot JC,
Smith D, Rubin E, Gillet B, Arbones M et al. (2009)
Green tea polyphenols rescue of brain defects induced
by overexpression of DYRK1A. PLoS ONE 4, e4606.
68 Kim H, Sablin SO & Ramsay RR (1997) Inhibition of
monoamine oxidase A by beta-carboline derivatives.
Arch Biochem Biophys, 337, 137–142.
69 Callaway JC, McKenna DJ, Grob CS, Brito GS, Ray-
mon LP, Poland RE, Andrade EN, Andrade EO &
Mash DC (1999) Pharmacokinetics of Hoasca alkaloids
in healthy humans. J Ethnopharmacol 65, 243–256.
70 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.
71 Park KW, Waki H, Villanueva CJ, Monticelli LA,
Hong C, Kang S, MacDougald OA, Goldrath AW &
Tontonoz P (2008) Inhibitor of DNA binding 2 is a
small molecule-inducible modulator of peroxisome pro-
liferator-activated receptor-gamma expression and adi-
pocyte differentiation. Mol Endocrinol 22, 2038–2048.

72 Laguna A, Aranda S, Barallobre MJ, Barhoum R,
Fernandez E, Fotaki V, Delabar JM, de la LS, de la
Villa P & Arbones ML (2008) The protein kinase
DYRK1A regulates caspase-9-mediated apoptosis
during retina development. Dev Cell 15, 841–853.
73 Sitz JH, Baumga
¨
rtel K, Ha
¨
mmerle B, Papadopoulos C,
Hekerman P, Tejedor FJ, Becker W & Lutz B (2008)
The Down syndrome candidate dual-specificity tyrosine
phosphorylation-regulated kinase 1A phosphorylates
the neurodegeneration-related septin 4. Neuroscience
157, 596–605.
74 Kuhn C, Frank D, Will R, Jaschinski C, Frauen R,
Katus HA & Frey N (2009) DYRK1A is a novel
negative regulator of cardiomyocyte hypertrophy. J Biol
Chem 284, 17320–17327.
75 Pagano MA, Bain J, Kazimierczuk Z, Sarno S, Ruzzene
M, Di Maira G, Elliott M, Orzeszko A, Cozza G,
W. Becker and W. Sippl Activation, regulation, and inhibition of DYRK1A
FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS 255
Meggio F et al. (2008) The selectivity of inhibitors of
protein kinase CK2: an update. Biochem J 415, 353–365.
76 Sarno S, Reddy H, Meggio F, Ruzzene M, Davies SP,
Donella Deana A, Shugar D & Pinna LA (2001)
Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP
site-directed inhibitor of protein kinase CK2 (‘casein
kinase-2’). FEBS Lett 496, 44–48.

77 Muraki M, Ohkawara B, Hosoya T, Onogi H, Koizumi
J, Koizumi T, Sumi K, Yomoda J, Murray MV, Kim-
ura H et al. (2004) Manipulation of alternative splicing
by a newly developed inhibitor of Clks. J Biol Chem
279, 24246–24254.
78 Mott BT, Tanega C, Shen M, Maloney DJ, Shinn P,
Leister W, Marugan JJ, Inglese J, Austin CP, Misteli T
et al. (2009) Evaluation of substituted 6-arylquinazolin-
4-amines as potent and selective inhibitors of cdc2-like
kinases (Clk). Bioorg Med Chem Lett 19, 6700–6705.
79 Adayev T, Chen-Hwang MC, Murakami N, Wegiel J &
Hwang YW (2006) Kinetic properties of a
MNB ⁄ DYRK1A mutant suitable for the elucidation of
biochemical pathways. Biochemistry 45, 12011–12019.
80 Pierantoni GM, Fedele M, Pentimalli F, Benvenuto G,
Pero R, Viglietto G, Santoro M, Chiariotti L & Fusco
A (2001) High mobility group I (Y) proteins bind
HIPK2, a serine-threonine kinase protein which inhibits
cell growth. Oncogene 20, 6132–6141.
81 Miyata Y, Akashi M & Nishida E (1999) Molecular
cloning and characterization of a novel member of the
MAP kinase superfamily. Genes Cells 4, 299–309.
82 Hughes K, Nikolakaki E, Plyte SE, Totty NF &
Woodgett JR (1993) Modulation of the glycogen syn-
thase kinase-3 family by tyrosine phosphorylation.
EMBO J 12, 803–808.
83 Bertani I, Rusconi L, Bolognese F, Forlani G, Conca
B, De Monte L, Badaracco G, Landsberger N & Kilst-
rup-Nielsen C (2006) Functional consequences of muta-
tions in CDKL5, an X-linked gene involved in infantile

spasms and mental retardation. J Biol Chem 281,
32048–32056.
84 Abe MK, Kahle KT, Saelzler MP, Orth K, Dixon JE &
Rosner MR (2001) ERK7 is an autoactivated member
of the MAPK family. J Biol Chem 276, 21272–21279.
85 Klevernic IV, Stafford MJ, Morrice N, Peggie M, Mor-
ton S & Cohen P (2006) Characterization of the revers-
ible phosphorylation and activation of ERK8. Biochem
J 394, 365–373.
86 Chang L & Karin M (2001) Mammalian MAP kinase
signalling cascades. Nature 410, 37–40.
Activation, regulation, and inhibition of DYRK1A W. Becker and W. Sippl
256 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS