Activation, regulation, and inhibition of DYRK1AWalter Becker1 and Wolfgang Sippl2 1 Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Germany 2 In
Trang 1Activation, regulation, and inhibition of DYRK1A
Walter Becker1 and Wolfgang Sippl2
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: wbecker@ukaachen.de
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.
Trang 2in 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
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) (http://www.phosphosite.org).
Trang 3residues at the primary phosphorylation site, they
lack the RD pocket, and phosphoproteomic studies
(http://www.phosphosite.org) 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.
of dDYRK2
[11,58]
990 n M DYRK2
Does not inhibit tyrosine autophosphorylation
of dDYRK2
[11,75,76] Pyrazolidin-diones
18 and 21
assay
[55]
930 l M
19 n M CLK1
130 n M DYRK1B
Less potent inhibitor of DYRK1A than harmine
in HeLa cellsc
[77,78] [12]
33 n M
150 n M DYRK1B
900 n M DYRK2
800 n M DYRK3
IC 50 = 1900 n M for tyrosine auto-phosphorylation
of DYRK1A
[17,59]
a IC50values 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.
Trang 4[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).
Trang 5of 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 IC50 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
Trang 6inhibi-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 (IC50= 5 nm) [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, showbind-ing 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.
Trang 7lar 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)
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