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J Neuropathol Exp Neurol. Author manuscript; available in PMC 2013 December 01.
Published in final edited form as:
J Neuropathol Exp Neurol. 2012 December ; 71(12): 11001112. doi:10.1097/NEN.0b013e31827733c8.

Gene-Dosage-Dependent Association of DYRK1A with the

Cytoskeleton in the Brain and Lymphocytes of Down Syndrome
Patients
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Karol Dowjat, PhD, Tatyana Adayev, PhD, Wojciech KaczmarskiPhD, Jerzy Wegiel, PhD,
and Yu-Wen Hwang, PhD
Department of Developmental Neurobiology (KD, WK, JW), Department of Molecular Biology (TA,
Y-WH), NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY

Abstract
The triplication of the DYRK1A gene encoding proline-directed serine/threonine kinase and
located in the critical region of Down syndrome (DS) has been implicated in cognitive deficits and
intellectual disability of individuals with DS. We investigated the effect of abnormal levels of this
kinase on the cytoskeleton in brain and peripheral tissues of DS subjects. In DS tissues, the
predictable 1.5-fold enhancement of the levels of DYRK1A protein was demonstrated. An
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association of DYRK1A with all 3 major cytoskeleton networks was identified using
immunoprecipitation. We concentrated on the actin cytoskeleton because its association with
DYRK1A was the most affected by the enzyme levels. As measured by co-immunoprecipitation in
DS tissues, but not in fragile X lymphocytes, actin association with DYRK1A was reduced. This
reduced association was dependent on the state of phosphorylation of cytoskeletal proteins and
was present only in cells overproducing DYRK1A kinase; therefore, the effect was attributable to
the DYRK1A gene dosage. Alterations of DYRK1A-actin assemblies were detected in newborn
and infant groups, thereby linking DYRK1A overexpression with abnormal brain development of
DS children. The identification of the actin cytoskeleton as one of cellular targets of DYRK1A
action provides new insights into a gene-dosage-sensitive mechanism by which DYRK1A could
contribute to the pathogenesis of DS. In addition, the presence of this DS-specific cytoskeleton
anomaly in lymphocytes attests to the systemic nature of some features of DS. To our knowledge
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this is the first study conducted in human tissue that shows DYRK1A association with the
cytoskeleton.

Keywords
Actin cytoskeleton; Brain; Co-immunoprecipitation; Down syndrome; DYRK1A kinase;
Lymphocytes

INTRODUCTION
DYRK1A gene, the homolog of Drosophila minibrain gene, encodes a proline-directed
serine/threonine kinase (1, 2). The gene is located on chromosome 21 in the Down
syndrome (DS) critical region containing a subset of genes implicated in DS (3). It is
assumed that many of the phenotypic features of DS stem from enhanced expression of
these genes (4, 5). Accordingly, DYRK1A levels in the brain tissue of DS subjects are
increased in a gene-dosage-dependent manner (6), and its abnormal expression has been

Send correspondence and reprint requests to: Karol Dowjat PhD, NYS Institute for Basic Research in Developmental Disabilities,
1050 Forest Hill Road, Staten Island, NY 10314. Tel.: +1 718 494 5321; fax: +1 718 494 4856; kdowjat@gmail.com.
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linked to brain anomalies and deficiencies in complex neuronal networks, both in the
developing (710) and the mature (11, 12) CNS. This expression may lead to the formation
of defective neuronal circuits and contribute to the pathogenesis of mental retardation in DS
(13). Both DYRK1A triallelic (1417) and monoallelic (10) mice showed morphogenesis
defects of the brain and learning and memory deficits. Importantly, in humans, microcephaly
and mental retardation were found to be associated with deletion (18) and truncation of
DYRK1A (19). Moreover, similar phenotypes have been described for monosomy 21-
associated mental retardation (2023). These observations point to the crucial role of
maintaining a dosage balance of this gene for normal development and CNS function.
Changes in phenotypes of neuronal cells with abnormal expression of DYRK1A, such as
reduced size, impeded neurite outgrowth, and lowered density of dendritic branching and
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spines, have also been reported (16, 2427). These results further support the postulated role
of DYRK1A in mental retardation and cognitive deficits in DS individuals.

Because of its ability to bind and phosphorylate numerous proteins (28, 29), multiple
functions have been attributed to DYRK1A. DYRK1A contains the nuclear localization
sequence (30); however, the majority of the endogenous kinase is found in the Triton-
insoluble post-nuclear fraction (31), presumably a fraction containing cytoskeletal matrices.
Protein-protein interactions have been widely employed to position a kinase in the proximity
of its targets, and ultimately to determine its function in a defined cellular context. In this
study, we use the immunoprecipitation (IP) technique with anti-DYRK1A antibodies to
investigate the interaction of DYRK1A with cytoskeletal proteins in brain tissue and in
immortalized B-lymphocytes of control and DS subjects. Our findings show that DYRK1A
is associated with the cytoskeleton and this association, particularly with actin, is
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abnormally affected by the gene-dosage-sensitive mechanism, both in the brain and

peripheral tissue of DS individuals.

MATERIALS AND METHODS

Brain Tissue
Brain tissue was obtained from 2 sources: the Brain and Tissue Bank for Developmental
Disorders of the NICHD, University of Maryland at Baltimore and the Brain and Tissue
Bank for Developmental Disabilities and Aging of the Institute for Basic Research (IBR
Brain Bank). All groups represented subjects of both sexes and all ethnicities because more
stringent selection of the cases was limited by the availability of the tissue. The cases used
are listed in Table 1. All procedures involving human tissue were performed in accordance
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with the Declaration of Helsinki. Experimental protocols were approved by the Institutional
Review Board of the NYS Institute for Basic Research in Developmental Disabilities. All
specimens were kept frozen at 70C until used.

Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (LCLs) of control donors were obtained from the Corriell Cell
Repositories (Camden, NJ). All DS and 2 fragile-X cell lines were established at the
Department of Human Genetics of our Institute. The full list of LCLs used is presented in
Table 2. Lymphocytes cultures were maintained in RPMI 1640 medium supplemented with
10% fetal bovine serum and a mixture of penicillin and streptomycin. All culture media and
supplements were purchased from Mediatech, Inc. (Manassas, VA). Cells after harvest were
washed 3 times with phosphate-buffered saline (PBS) and stored at 20C until used. In
some experiments freshly harvested cells without prior freezing were used for analysis.

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Antibodies
Anti-DYRK1A antibody 8D9 is an in-house produced monoclonal antibody raised against
peptides containing the first 160 residues of rat DYRK1A (31), with the epitope located
between residues 142147 (isoform 1 numbering) containing non-phosphorylated Tyr-145
(32). Mouse monoclonal anti-DYRK1A antibody 7F3, with the epitope located between
residues 7478, was raised similarly as 8D9 (33). R420 rabbit polyclonal antibody aimed at
the detection of isoform 2 of DYRK1A was produced by immunization with the peptide, the
sequence of which matched the alternative junction of exon 5 and 6b (34). All antibodies
used in the study are listed in Table 3.

Preparation of Brain and Lymphocytes Lysates, Immunoprecipitation and Immunoblot

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Analysis
Frozen dissected frontal cortex tissue was mortar-ground in liquid N2. Pools were made by
mixing 0.1 g of powdered tissue from each subject assigned to a given group solely based on
age; 100 mg of pooled tissue was lysed in 1 ml of ice-cold RIPA buffer (1x PBS, pH 7.4,
1% NP-40, 0.1% sodium dodecyl sulfate (SDS), and 0.5% sodium deoxycholate) containing
Complete protease inhibitor tablets (Roche Diagnostic, Indianapolis, IN) and, when
necessary, phosphatase inhibitors (2 mM NaF, 1 mM Na3VO4, 4 M cyclosporine, and 200
nM okadaic acid), followed by a short-pulse sonication. Lysates were clarified by microfuge
centrifugation at 14 k rpm for 10 minutes; protein concentrations were determined by
bicinchoninic acid assay (Thermo Scientific, Rockford, IL). To assure the specificity of
binding, lysates were pre-cleared by incubation with Dynabeads Protein G (Invitrogen
Dynal AS, Oslo, Norway). For immunoprecipitation, 1 mg of total protein of each brain
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lysate was mixed with Dynabeads primary antibody conjugate, which was prepared by
binding 2 g of the antibody to 40 l suspensions of prewashed beads. The volume of each
sample was made 0.5 ml with complete RIPA buffer and the mixture was incubated with
tilting and rotation for 1 hour at 4C. The complexes were washed 3 times in 1 ml of PBST
using a magnet and eluted by boiling for 5 minutes in 50 l of 1x tricine sample buffer (Bio-
Rad Laboratories, Hercules, CA). Ten l (20% of total) of each eluate were separated
electrophoretically on 8% tricine SDS-polyacrylamide gel and electro-transferred onto
PVDF membranes (Bio-Rad). The membranes were blocked in 5% non-fat milk and
incubated overnight at 4C with the appropriate dilution of primary antibody. The
immunoreactive bands were visualized by using species-specific anti-IgG alkaline
phosphatase-conjugated secondary antibody (Thermo Scientific) and CDP-Star
chemiluminescence reagent (New England Biolabs, Ipswich, MA). After
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chemiluminescence detection, blots were further developed by color reaction using BCIP/
NBT substrate (Sigma-Aldrich, St. Louis, MO). Preparation of lymphocytes lysates was
essentially the same as brain lysates; 15 106 cells were lysed in 1 ml of ice-cold complete
RIPA buffer. Lysates were clarified by centrifugation and the equivalent of 400 g of total
protein was taken for immunoprecipitation and immunoblot analysis, as described for the
brain tissue. Cytochalasin D (CytD) and nocodazole (Noc) treatments were performed by
incubating cell lysate at 30C for 45 minutes in the presence of 25 M CytD or 10 M Noc
(both from Sigma-Aldrich), and then processed for immunoprecipitation and
immunoblotting.

Immunocytochemistry and Confocal Microscopy

Paraffin-embedded sections of white matter from DS fetal (20-week gestation) were stained
by immunohistochemistry with monoclonal 7F3 anti-DYRK1A antibody, as described (33).
For immunofluorescence staining, mouse Neuro2a (N2a) neuroblastoma cells grown in
MEM with 10% fetal bovine serum on 8-well glass chamber slides (Nalge Nunc, Naperville,
IL) were rinsed in PBS and fixed in 80% methanol for 20 minutes. After blocking for 1 hour
in 10% fetal bovine serum in PBS, cells were incubated overnight at 4C with primary

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antibodies diluted in blocking buffer containing 0.1% saponin, which was included in all
subsequent steps of the procedure. Slides were developed by 1-hour incubation with FITC-
or/and Cy 3-conjugated species-specific secondary antibodies (Jackson ImmunoResearch,
West Grove, PA), mounted in the Vectashield mounting medium (Vector Laboratories,
Burlingame, CA) and examined with a Nikon C1 three-laser confocal system mounted on a
Nikon Eclipse 90i microscope (Nikon, Melville, NY).

Transfection of NIH3T3 Cells

NIH3T3 mouse fibroblasts were obtained from American Type Culture Collection
(Manassas, VA) and propagated in DMEM medium supplemented with 10% fetal bovine
serum. pCMV-Script-based DYRK1A expression vectors used for transfection were as
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previously described (35). For transfections, NIH3T3 cells were pre-plated for 24 hours onto
a 6-well plate with cell density of 5 105 cells per well in antibiotic-free DMEM medium
and cultured under normal growth conditions. Next, cells were transfected with 2 g of
plasmid DNA (pCMV-Script, pCMV-MnbWT, and pCMV-MnbK188R) and Lipofectamine
2000 (Life Technologies, Carlsbad, CA), according to the manufacturers protocol followed
by media change 5 hours afterwards. All experimental procedures were performed 48 hours
post-transfection, according to standard protocols, as described for brain tissue and LCL
cultures.

In Vitro Phosphorylation
After immunoprecipitation with anti-DYRK1A antibody-coated Dynabeads, 40 l of the
beads suspension were washed twice and resuspended in 100 l of kinase buffer (25 mM
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Tris-HCl, pH 7.4, 100 mM NaCl, and 5 mM MgCl2 supplemented with protease and
phosphatase inhibitors). Phosphorylation was initiated by adding 100 M ATP alone or with
1 g of C-terminal truncated GST-DYRK1A 497 (35), and allowed to proceed at 30C for
20 minutes with occasional gentle shaking. Immunoprecipitated complexes were then
washed twice with PBST, eluted by boiling in the sample buffer and subjected to
immunoblotting under standard conditions of this work.

Data Analysis
The densitometric quantification of immunoreactive bands was carried out with the 1D Scan
EX 3.1 software program (Scanalytic Corp., Rockville, MD). The results were evaluated for
statistical significance with the two-tailed Student t-test for independent samples using
GraphPad Prism version 4.0 software program (GraphPad Software Inc. San Diego, CA),
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and considered significant at p < 0.05. Figures are representative of at least 3 independent
experiments with similar results.

RESULTS
In our studies of DYRK1A protein expression in the brain of control and DS subjects co-
fractionation of actin with DYRK1A was consistently observed (6). This prompted us to
investigate the association of DYRK1A with actin and other cytoskeletal proteins.
Detergent-soluble fractions of human brain tissue were immunoprecipitated with
commercial (H143 and G19) and in-house produced (R420) anti-DYRK1A antibodies, and
analyzed by immunoblotting with antibodies against major cytoskeleton networks. All 3
anti-DYRK1A antibodies produced very similar immunoprecipitation profiles and
substantial enrichment of DYRK1A was evident in immunoprecipitates. Data for H143
antibody are presented in Figure 1. In the frontal cortex of control brain, neurofilament
heavy subunit (NF-H), -tubulin and -actin were found to co-IP with DYRK1A. The
association of DYRK1A with cytoskeleton was further confirmed by immunocytochemical
staining of brain tissue and confocal microscopy of Neuro-2A (N2a) cells. There was strong

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staining of structures resembling the omnipresent cytoskeleton filamentous network in axons

in fetal white matter (Fig. 1). In an earlier study of DYRK1A in adult brains, it was also
found to be present in neuronal processes (33). In cultured mouse Neuro-2A cells doubly
labeled with anti-DYRK1A and either NF-H, actin or tubulin antibodies, DYRK1A
colocalized with all 3 main cytoskeletal networks, which is in accordance with the co-IP
data. NF-H and tubulin colocalization was restricted to neurites and sites of neurite
formation on the cell periphery. The strongest colocalization was with actin. Although
concentrated in neurites, particularly in their endings, it was also present in the cell body.

We next investigated whether the overexpression of DYRK1A in DS subjects could affect

the association of DYRK1A with the neuronal cytoskeleton. The study included 6 controls
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and 12 DS subjects (Table 1). To reduce the individual variation among the cases (6), we
created pools consisting of frontal cortex of control and DS subjects. Although DYRK1A
was found to be part of the larger complex consisting of 3 main cytoskeleton networks, we
focused on the actin cytoskeleton because its association with DYRK1A was the most
prominent. Accordingly, using IP and quantification by immunoblotting, levels of DYRK1A
and -actin in immunoprecipitated complexes of control and DS subjects were measured
and compared (Fig. 2). The specificity of the IP assay was tested using Dynabeads alone or
beads coated with unrelated rabbit antibody (Fig. 2A). No signal with DYRK1A and only
very weak signal with actin were detected in those samples, ruling out the possibility of non-
specific binding. Figure 2B depicts a representative Western blot analysis of DYRK1A and
actin immunoprecipitates together with plotted results of densitometric quantification. In
accordance with our previous report (6), levels of DYRK1A in crude DS lysate were
increased by 50% (149 8.4%, p < 0.05). The levels of actin were similar in both pools
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asserting that the levels of actin were not altered in DS. In immunoprecipitates, DYRK1A
was elevated by 37% (137% 6.5%, p < 0.05) in DS; however, the levels of actin were
reduced by 43% (57% 4.6%, p < 0.05). Thus, overproduction of DYRK1A protein in DS
can be observed at the level of its association with the actin cytoskeleton resulting in
lowering the yield of actin, as measured in the IP assay.

The association of DYRK1A with the actin cytoskeleton was confirmed by reverse
immunoprecipitation with the actin antibody. In this case, the DS group, compared to
control, showed both less actin and DYRK1A in precipitated materials. Actin was reduced
by 70% and DYRK1A by 30% (Fig. 2B). However, the ratio of DYRK1A to actin in actin
IP were quite similar to that observed in DYRK1A IP. In the DS pool there was 1.7-fold
dominance of DYRK1A over actin compared to 1.6-fold in DYRK1A IP. Denaturing of DS
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lysates by boiling before IP (DS 100o in Fig. 2B) restored the yield of both DYRK1A and
actin to the levels of the control pool; this indicates the importance of preserving the native
conformation of actin-DYRK1A assemblies for displaying characteristic for DS patterns in
the IP assay. As shown in Figure 2C, only the middle band of the normally seen triplet of
DYRK1A-immunoreactive bands was recovered from the actin IP. Apparently, the middle
band represents the only species with specific affinity toward the actin cytoskeleton.

These results establish the association of DYRK1A with the neuronal cytoskeleton and
reveal altered DYRK1A complexes with the cytoskeleton in pathological brains of DS
subjects. Because DS is a developmental disease it is important to examine it at different
stages of brain development. This was tested in the pools grouping brain tissue of newborn
and infants. A group of adults was included for comparison. The number of cases in each
group and their age is listed in Table 1 and the results are presented in Figure 3. In lysates of
newborn and infants groups there was no elevation of DYRK1A, as is observed in the brains
of adult DS subjects (6). In the immunoprecipitates, however, DS subjects in all 3 groups
showed an increase in DYRK1A: in the newborn group by 1.3-fold, infant group by 1.7-fold
and adults by 1.5-fold in contrast to the DYRK1A measurements in the crude lysates.

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Presumably, this apparent discrepancy could be the reflection of overall lower numbers of
cells expressing DYRK1A in DS during the early stages of brain development creating the
dilution effect in the lysates. In DS pools of all age groups the actin yields were reduced by
50% (Fig. 3), resembling the results obtained for adult brains (Fig. 2). The overall yield of
actin in the developmental groups, particularly in infants, was much above the levels
recorded for adults (Fig. 3B). This could be the reflection of gross differences in the
intensity of neuronal plasticity between the developing and mature brains. This also
indicates DYRK1A involvement in the build-up of neuronal connectivity and its negative
gene-dosage effect during a critical time of development.

The availability of LCLs established from DS subjects prompted us to investigate whether

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observations from the brain tissue could be reproduced in peripheral cells. Figure 4A shows
the results of immunoblot testing of 3 control and 5 DS LCLs lines. As in the brain tissue,
all DS lines displayed elevated levels of DYRK1A and also showed some variability. This
variability was particularly evident in the control group in which line from the 68 year old
was much higher than the other 2 of this group and almost as high as the lowest line in DS
group. Because of this variability, the study was conducted on pooled cases from each group
(Fig. 4B). DYRK1A levels in DS crude lysates were enhanced by 39% (139% 8.5%, p <
0.05), thus close to the values obtained for the brain tissue (Fig. 2). No enhancement in
DYRK1A expression was observed in fragile X cases. In DYRK1A immunoprecipitates,
these proportions were retained with 37% (137% 22%, p < 0.05) enhancement over
control and; however, the actin levels were reduced by 40% (6%0 19%, *p < 0.05), as
compared to the control and FraX pools. Thus, the changes of DYRK1A-actin-
immunoprecipitable complexes found in the DS brain tissue were fully reproduced in DS
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LCLs, indicating the systemic nature of the underlying mechanism. Those changes appear to
be specific for DS LCLs as they were not observed in 2 LCLs from donors with fragile X.

Findings in LCLs create an opportunity to study a gene-dosage effect of DYRK1A in easily

obtainable blood-derived cells. We further explored this opportunity to test the effects of
low temperature, microfilament (CytD) and microtubule (Noc) disrupting agents, and
conditions favoring phosphorylation on DYRK1A-actin complexes. All of those conditions
are known to affect the integrity of cytoskeleton filamentous networks. We first tested effect
of freezing cells before lysis. In the IP of lysates of freshly harvested cells, the yields of
actin in control and DS cells were comparable (Fig. 5A). This was not the case in the IP of
frozen cells lysates in which the DS sample had less actin. Although the 1.5 ratio of DS to
control for DYRK1A was preserved in both conditions, the overall yield in IP samples of
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frozen cells was significantly lower. Thus, freezing may affect the stability of DYRK1A-
actin complexes by disrupting actin filaments. That the actin polymerization process is
essential for formation of complexes can be deduced from experiments with CytD treatment,
a potent inhibitor of actin polymerization. CytD treatment of lysates before IP, markedly
prevented formation of the complex, as judged from 80% reduction of the actin yield in
DYRK1A co-IP (Fig. 5B). Importantly, Noc, which causes depolymerization of
microtubules, had no effect. The polymerization and depolymerization of actin filaments is
tightly regulated by phosphorylation and the elevated activity of DYRK1A kinase in DS
may change the dynamic of those processes, thus affecting DYRK1A association with the
actin cytoskeleton. If this were the case, lysates prepared without phosphatase inhibitors
would be expected to affect control and DS samples to different degrees. Originally we used
a phosphatase inhibitors cocktail, but later found that okadaic acid alone, a potent inhibitor
of PP1 and PP2A Ser/Thr phosphatases, was enough for exerting the effect. Accordingly,
lysates of lymphocytes were prepared with or without okadaic acid and processed for IP
with DYRK1A antibody. By omitting okadaic acid in the lysis buffer, the markedly reduced
actin yield that co-IP with DYRK1A in DS lymphocytes, was restored to the levels of
control (Fig. 5C). This effect of okadaic acid on DYRK1A-actin complexes in DS suggests

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that their assembly and disassembly depends on the balanced phosphorylation of

cytoskeletal proteins, possibly by DYRK1A kinase. To investigate phosphorylation
dependency of the process more directly, we tested the effect of conditions favoring
phosphorylation on already formed complexes. DYRK1A-actin complexes bound to
Dynabeads coated with DYRK1A antibody were incubated in kinase buffer with or without
ATP and then analyzed for their stability by immunoblot quantification. For comparison
between control and DS, the experiments were conducted on freshly harvested cells without
prior freezing to ensure that we started with comparable levels of actin (Fig. 5A). In both
control and DS samples, incubation with ATP promoted disassociation of actin from the
complexes, as judged from the decrease of actin, but not antibody-bound DYRK1A (Fig.
5D). The effect was much more pronounced in the DS sample in which the reduction of
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actin in samples incubated with ATP was by 78% vs. 31% in the control. These results
reaffirm that the association of DYRK1A with the actin cytoskeleton is regulated by
phosphorylation and the higher rate of actin dissociation in DS may stem from trisomy-
driven overexpression of the DYRK1A gene.

Frozen brain tissue does not allow direct studies of gene-dosage effect; however, the use of
transfected cell lines abrogates this limitation. Originally, we used mouse neuroblastoma
N2a cells for these experiments, but due to the low and variable efficiency of transfection
we switched to NIH3T3 fibroblasts, which can be easily transfected and also share the same
developmental origin as LCLs. As shown in panels Figure 6A and B, the transfection of
DYRK1A clone resulted in an average of 1.68-fold enhancements over the vector-
transfected controls in lysates, and importantly, a similar increase was also recorded in the
IP samples. The level of total s- actin was not affected by DYRK1A transfection; therefore,
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the level of actin can be used as readout for the analysis without complications. Similarly to
what was observed in lymphocytes (Fig. 5), the yield of actin complexed with DYRK1A in
the wild-type (WT) DYRK1A and vector-transfected cells was differentially affected by
phosphatase inhibitors. Again, the levels of actin co-IP were reduced (0.61-fold decrease, p
< 0.05) only when lysates of DYRK1A transfectants were prepared in the presence of
phosphatase inhibitors. Tubulin was also found to co-IP with DYRK1A, but its yield was the
same in all samples (Fig. 6A, B), indicating that microtubules association with DYRK1A
does not depend on phosphorylation. That the kinase activity of DYRK1A is required for
observing the effect can be deduced from experiments where cells were transfected with
kinase-deficient K188R mutant and subjected to immunoprecipitation with DYRK1A
antibody. Despite a similar elevation of DYRK1A in the WT and mutant transfected cells,
the actin levels were decreased (0.61-fold, *p < 0.05) only in the WT transfectants (Fig. 6C).
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Because the only variable in this particular experimental system is kinase activity of
DYRK1A, we conclude that the reduction in actin co-IP is a direct consequence of
DYRK1A elevation. We subsequently analyzed the effects of DYRK1A on dissociating pre-
formed DYRK1A-actin complexes. The experimental approach was the same as the one
applied to lymphocytes (Fig. 5D), except the inclusion of exogenous recombinant WT
DYRK1A in the sample during incubation. Accordingly, DYRK1A co-IP complexes were
similarly prepared, incubated with ATP or ATP plus recombinant DYRK1A in kinase
buffer, and then DYRK1A and actin retained on Dynabeads were eluted and analyzed by
immunoblotting. Figure 6D shows that incubating complexes along with ATP can
significantly reduce the level of bound actin and it can be further reduced if DYRK1A is
present. These results reaffirm the conclusion that the assembly of DYRK1A-actin
complexes depends upon phosphorylation, driven in part by DYRK1A kinase itself. Thus,
its abnormal levels in DS may lead to derangement of this association. They further
demonstrate that this effect of DYRK1A is independent of the cell type.

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DISCUSSION
Using immunoprecipitation with anti-DYRK1A antibodies, we have identified an
association of DYRK1A with the cytoskeleton. In detergent-soluble fractions of human
brain and cultured lymphocyte homogenates, DYRK1A was found to co-IP with actin,
tubulin and NF-H proteins, the structural components of 3 main cytoskeleton networks
(Figs. 1, 3, 6). The subcellular distribution of DYRK1A and cytoskeletal proteins was
subsequently examined by confocal microscopy (Fig. 1) supporting results of IP
experiments. Because its association with DYRK1A was the most affected by the enzyme
levels, the actin cytoskeleton seemed to be the key target for DYRK1A kinase activity.
Thus, we focused on the interaction of DYRK1A and actins, primarily -actin in this study;
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however, the extension of this study to other cytoskeletal systems is currently under way.

The tissue and cell lysates for IP were prepared in RIPA buffer containing SDS and
deoxycholate, with the intention of improving protein extraction and minimizing non-
specific binding background. Co-IP of various cytoskeletal proteins with DYRK1A under
such harsh conditions not only argues for the strength of the interaction but also allowed
detection of subtle but consistent alterations in DYRK1A-actin complexes in samples
prepared from DS subjects (Figs. 2, 4), in which the level of DYRK1A is elevated due to
triplication of the gene. This phenomenon was also seen in mouse fibroblasts overexpressing
the wild-type but not kinase-deficient mutant of DYRK1A (Fig. 6). Importantly, conditions
favoring phosphorylation, i.e. the presence of phosphatase inhibitors (Fig. 5), are required
for a display of altered properties of DYRK1A-actin complexes in DS samples. The
importance of phosphorylation is also evident from experiments in which precipitated
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DYRK1A-actin complexes were exposed to ATP or active recombinant kinase (Figs. 5D,
6C). All those conditions promoted actin disassociation from the complex. Taken together,
our data support a model that DYRK1A-actin complexes are maintained by a delicate
balance of the kinase-phosphatase equilibrium. An ~50% increase in DYRK1A level due to
gene-dosage elevation in DS subjects or transfection in NIH3T3 cells is sufficient to disturb
this balance resulting in derangement of those complexes. To the best of our knowledge this
is the first documentation of formation and gene-dosage regulation of the DYRK1A-actin
complexes.

Actin depolymerization by CytD treatment and freezing cells before lysis could drastically
reduce the yield of actin that co-IP with DYRK1A. It is conceivable that the effect of
DYRK1A elevation may also be mediated through controlling the stability of actin
filaments. Alterations in actin dynamic through increased stability of actin filaments was
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also observed in DYRK1A trisomic TgDyrk1A mice where DYRK1A is overexpressed to

the levels observed in DS (27). The reduction in actin co-IP was already present in DS
brains of newborn and infants (Fig. 3), linking the DYRK1A interaction with the
cytoskeleton to well-documented deficiencies and delays in brain maturation associated with
DS (36). The presence of this anomaly in newborn subjects contrasts with earlier studies on
DS brain morphometry at the time of birth (3740). It should also be stressed that alterations
of the association of DYRK1A and actin in DS are systemic, as they were seen in both brain
tissue and peripheral cells. The elevation of DYRK1A expression as well as the cytoskeleton
abnormality seen in DS were not observed in fragile X cases, further implicating DYRK1A
imbalance as a factor contributing to the altered stability of DYRK1A-actin complexes.

Actin filament dynamics are involved in shaping dendritic branching complexity and
modulating spine/synapse densities. Altered DYRK1A gene dosage, as in DS brains (13)
and triallelic (16, 27) or monoallelic (10, 24) mice, results in marked changes in spine/
synapse densities and abnormalities in the dendritic arbors. DYRK1A expression has been
associated with neurite formation in cultured neurons (26, 27, 41, 42), and its Drosophila

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 Dowjat et al. Page 9

orthologue-minibrain kinase (MNB) was also found to regulate actin-based protrusions,

specifically in CNS-derived cell lines (43). Administration of tea extract containing
DYRK1A inhibitor, epigallocatechin gallate, to transgenic hYACtgDYRK1A mice
overexpressing DYRK1A nearly completely rescued brain defects in the mice (44). The
downregulation of DYRK1A due to intragenic deletion (18) and truncation (19) of
DYRK1A gene or partial monosomy of 21 containing locus of DYRK1A produced disease
phenotypes very similar to those of DS patients (2023). These studies point to a DYRK1A
gene-dosage-sensitive mechanism as the chief contributor to mental retardation and
cognitive impairment of DS individuals, but the key mechanism underlying its involvement
is yet to be revealed. Our study identifying the actin cytoskeleton as a target of DYRK1A
action provides new insight for better understanding of how this function of DYRK1A can
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be executed.

It is important to emphasize that DYRK1A is only a component of a common machinery of

phosphorylation that involves many kinases and cytoskeletal proteins that modulates
cytoskeleton plasticity. The kinases involved act in a highly regulated interplay inducing
small variations in the phosphorylated states of cytoskeleton proteins, which are important
for dynamic properties and integrated function of the entire cytoskeleton network. Abnormal
activity of DYRK1A kinase may disturb this fine-tuning, causing subtle perturbations in the
smooth operation of this complex machinery. The enormous complexity of this machinery
makes identification of the putative target(s) of DYRK1A enzymatic action a daunting task.
Purified actin itself is not phosphorylated to an appreciable level by recombinant DYRK1A,
nor does actin bind directly to the kinase (Adayev, unpublished observation). This result
suggests that DYRK1A complexing with actin needs adapter molecule(s). Actin interacts
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with a plethora of binding proteins (45). Likewise, more than 2 dozen putative substrates
and interacting proteins of DYRK1A have been identified (28, 29). Unfortunately, a cross
check of both lists did not immediately reveal any potential adapter candidate(s). DYRK1A
has recently been found to phosphorylate N-WASP protein (46), which is involved in
regulating actin polymerization and dynamics. Although a protein such as N-WASP could
be a potential adaptor molecule, the co-IP and colocalization of tubulin and neurofilament
proteins with DYRK1A (Figs. 1, 3) suggest that the adaptor molecules should also function
as linkers of actin filaments with other cytoskeleton networks. The association of actin with
DYRK1A has been found in every cell type examined so far; therefore, the distribution of
adapter(s) should be ubiquitous. Other potential adapters could be heat shock protein 90,
which we have found in DYRK1A and actin immunoprecipitates (Dowjat, unpublished
observation). This molecular chaperone protein is known to be associated with all 3
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cytoskeleton networks, apparently protecting their filamentous structure, and its biological
function is regulated by numerous kinases (47). A systemic approach to characterize the
adapter(s) is currently under way.

Of special interest are our findings in DS lymphocytes attesting to the systemic nature of
some features of DS phenotype. One of those features could be Alzheimer disease (AD)-
type dementia, a highly prevailing condition in aging DS patients. Thus, it will be interesting
to explore whether a similar abnormality in the association of DYRK1A with actin exists in
AD and whether it is required for the onset of clinical AD. Such a connection has been
proposed in an previous publication from this laboratory (48), and fits the well-documented
disruption of cytoskeleton in AD. If this is the case, then lymphocytes from DS and AD
donors may serve as unique cellular model of the disease, and possibly, as diagnostic tool
for predicting or confirming AD.

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 Dowjat et al. Page 10

Acknowledgments
Supported in parts by funds from the New York State Office for People with Developmental Disabilities and grants
R01 HD43960 from the National Institute of Health (to JW) and from Jerome Lejeune Foundation (to Y-WH).

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Figure 1.
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Association of DYRK1A with the cytoskeleton as revealed by immunoprecipitation and

immunostaining of brain tissue and cultured mouse neuroblastoma neuro-2a (N2a) cells.
Left panels: Frozen frontal cortex tissue of a control subject was homogenized in RIPA
buffer and subjected to immunoprecipitation (IP) with anti-DYRK1A (H143) antibody.
Immunoprecipitates were analyzed by Western blotting (WB) with antibodies against the
indicated cytoskeletal proteins. All major structural cytoskeletal proteins were detected in
the DYRK1A IP. Right panels: High magnification image of fetal (20-week gestation)
Down syndrome (DS) white matter immunostained with anti-DYRK1A antibody (7F3).
Merge images are of double immunofluorescent labeling of N2a cells with DYRK1A
(H143) and either NF-H (N52), -tubulin (B-5-1-2) or -actin (AC-15) antibodies.
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Figure 2.
Quantification of DYRK1A and -actin in immunoprecipitates of pooled frontal cortex
tissue of control and Down syndrome (DS) subjects. (A) Specificity of DYRK1A and actin
co-immunoprecipitation (IP). NIH3T3 cell lysate was used for IP with uncoated (Beads),
anti-glutathione S-transferase (GST) antibody coated, and anti-DYRK1A R420 antibody
(DYRK1A) coated Dynabeads. The levels of DYRK1A and -actin in crude lysate and
precipitates were detected by immunoblotting. (B) Analysis of DYRK1A and -actin co-IP.
Pools were made by mixing 0.1 g of tissue powdered in liquid N2 (n = 6 for control and n =
12 for DS) of each brain. Parallel samples of brain lysates were immunoprecipitated with the
rabbit polyclonal anti-DYRK1A antibody directed against the C-terminus (H143) of

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 Dowjat et al. Page 15

DYRK1A polypeptide and monoclonal -actin antibody. IP and Western blot (WB) analysis
of lysates and immunoprecipitates were conducted as described in Materials and Methods.
20 g of each lysate and 10 l of IP sample were taken for analysis. Blots were probed with
1:5000 dilutions of DYRK1A 8D9 and -actin monoclonal antibodies. In the DS sample, IP
with actin antibody was also done with lysate denatured by boiling. Representative Western
blots and the results of quantification are shown. The results are expressed as percent of
values recorded for the control pool. Each bar represents the mean value of 4 IP experiments
conducted on 2 independently prepared pools. Error bars indicate SEM. CTR: control lysate;
DS: Down syndrome lysate; DS 100: boiled DS lysate. *Denotes the differences with
statistical significance at the level of p < 0.05. (C) Patterns of DYRK1A-immunoreactive
bands that co-IP with actin. DYRK1A and actin was independently IP with each
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respective antibody from control lysate and then compared by Western blotting. Running
time for SDS-PAGE was extended to permit the separation of multiple DYRK1A bands
approximately 90 kDa.
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 Dowjat et al. Page 16
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Figure 3.
Immunoblotting profiles of DYRK1A immunoprecipitates in developing brains of control
and Down syndrome (DS) subjects. Pools of frontal cortex tissue of newborns (n = 4 for
control and n = 3 for DS), infants (n = 9 for control and n = 8 for DS), and adults (n = 7 for
control and n = 14 for DS) were subjected to immunoprecipitation with R420 rabbit anti-
DYRK1A antibody, and analyzed by immunoblotting with DYRK1A (8D9) and -actin
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antibodies. Others details are as described in the legend to Figure 2. (A, B) Representative
immunoblots of crude lysates (A) and immunoprecipitates (B) are shown. Results of
densitometric measurements are expressed as a percent of immunoreactivity recorded for
controls in each group. Each bar represents mean value SEM of 4 experiments conducted
on 2 independently prepared pools. The differences between control and DS for all age
groups were significant at *p < 0.05.
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 Dowjat et al. Page 17
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Figure 4.
Quantification of DYRK1A and -actin in immunoprecipitates of pooled lymphoblastoid
cell lines (LCLs) of healthy (CTR), Down syndrome (DS) and fragile X (FraX) subjects. (A)
Levels of DYRK1A and actin in crude lysates. RIPA lysates were prepared from LCLs
established from 3 control and 4 DS donors Samples of each lysate were immunoblotted
with DYRK1A 8D9 and s- actin antibodies. (B) Immunoblot quantification of DYRK1A and
-actin in DYRK1A IP (R420). Preparation of pooled control (5 cases), DS (6 cases) and
FraX (2 cases) LCLs, DYRK1A IP, and analysis of immunoprecipitates were as described in
Materials and Methods. Samples were immunoblotted with DYRK1A 8D9 and -actin
antibodies. Plots of densitometric measurements are expressed as a percent of
immunoreactivity recorded for the control pool. Each bar represents mean value SEM of 4
experiments conducted on 2 independently prepared pools. *Denotes the differences with
statistical significance at the level of p < 0.05

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 Dowjat et al. Page 18
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Figure 5.
Conditions affecting DYRK1A and actin co-immunoprecipitation (IP) in control and
Down syndrome (DS) lymphoblastoid cell lines (LCLs). (A) Effect of freezing. DYRK1A
IP was performed with lysates prepared from freshly harvested cells of control AG09387
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and DS405 and cells after freezing for 2 hours at -20C. DYRK1A and actin complexes in
IP were measured by immunoblotting. (B) Effect of cytochalasin D (CytD) or nocodazole
(Noc) treatment on DYRK1A and actin co-IP. Equal aliquots of control (AG09387) cells
lysate were incubated at 30C with CytD or Noc before IP with DYRK1A antibody
followed by immunoblot analysis for DYRK1A and -actin. Load. crude lysate,() IP from
lysate without treatment, CytD: IP from lysate treated with CytD, and Noc: IP from lysate
treated with Noc. The results were quantified and shown as a bar graph. (C) Effect of
phosphatase inhibitor okadaic acid (OKA). Lysates of control (GM07045) and DS (DS206)
LCLs were prepared with or without 200 nM OKA; and then IP with anti-DYRK1A (R420)
antibody. The representative immunoblot is shown together with the plot of densitometric
measurements where each bar represents values for DS expressed as percent of
corresponding untreated (- OKA) or OKA-treated (+ OKA) control. (D) Effect of incubating
with ATP. DYRK1A IP was performed with lysates of control and DS LCLs as described.
DYRK1A-actin complexes bound to Dynabeads were subsequently incubated for 30 min at
30C with or without ATP in kinase buffer. After extensive washing, DYRK1A and actin
retained on Dynabeads were then analyzed by immunoblotting. KB: incubated only with
kinase buffer, ATP: incubated with ATP in kinase buffer.

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Figure 6.
Effect of exogenous DYRK1A on the formation and stability of DYRK1A-actin complexes
in NIH 3T3 fibroblasts. (A) Effect of phosphatase inhibitors (PPIs) in DYRK1A over-
expressing cells. NIH 3T3 fibroblasts were transfected with the wild-type (WT) DYRK1A
or the cloning vector (VEC) for 48 hours before harvesting. Cells were frozen at -20C for 2
hours, lysed in RIPA with (+) or without (-) PPIs, and processed for IP. Aliquots of cell
lysates containing 400 g of total protein were taken for IP with R420 DYRK1A antibody
and immunoprecipitates were probed with DYRK1A (8D9), s-tubulin, and -actin
antibodies. As a control, crude lysates without IP (lysates) were also probed with DYRK1A
(8D9) and -actin antibodies. The intensity of immunoreactive bands was quantified and
reported as independent values in arbitrary units. Representative Western blots and plots of
densitometric quantification of DYRK1A and actin are shown. Each bar represents mean
value SEM of 3 independent experiments. *Denotes the differences with statistical
significance at the level of p < 0.05. (B) Effect of over-expression of kinase-deficient
mutant. Cells were transfected with the cloning vector (VEC), wild-type (WT) or mutant
(K188R) DYRK1A and processed with PPIs for IP and subsequent Western blotting as in

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(A). Quantification and analysis of the intensity of immunoreactive bands were also
performed as described above. (C) Effect of exogenous recombinant DYRK1A on
Dynabeads immobilized DYRK1A-actin complexes. Complexes bound to DYRK1A
antibody-coated Dynabeads were prepared from untransfected NIH 3T3 lysate as described
earlier. Beads were then incubated in kinase buffer for 30 minutes at 30C, with or without
ATP or recombinant truncated wild-type DYRK1A. The coated beads were then extensively
washed with PBST and DYRK1A and actin that remained on the beads were quantified by
immunoblotting. KB: incubated with kinase buffer only, ATP: incubated with ATP, and
WT: incubated with ATP and DYRK1A.
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Table 1
Cases Studied by Age
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Age group Control (Age range) Down Syndrome (Age range)

Newborns n = 4 (1435 days) n = 3 2130 days
Infants n = 9 (340 months) n = 8 (636 months)
Adults n = 7 (3185 years) n = 14 (3165 years)
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Table 2
Lymphoblastoid Cell Lines

Controls (n = 6) Down Syndrome (n = 6) Fragile X (n = 2)

Dowjat et al.

Age (years) Age (years) Age (years)

* AG08702 30 ** DS405 48 ** FraX3966 19
* GM07041 38 ** DS68 55 ** FraX3537 18

* GM07045 39 ** DS206 57

* AG09387 43 ** DS971 59

* AG08378 60 ** DS211 60

* GM03657 68 ** DS433 60

*
Cell lines obtained from Coriell Cell Repositories (Camden, NJ);
**
cell lines established at Institute for Brain Research in Developmental Disabilities, Staten Island, NY.

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Table 3
Antibodies
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Antibody Antigen Host Source

7F3 DYRK1A, residues 1160 mouse (33)
8D9 DYRK1A, residues 1160 mouse (31, 32)
R420 DYRK1A, isoform 2 rabbit this study
H143 DRYK1A, residues 621763 rabbit Santa Cruz Biotechnology, Santa Cruz, CA
G19 DRYK1A, N-terminus goat Santa Cruz Biotechnology
Z-5 glutathione S-transferase rabbit Santa Cruz Biotechnology
AC-15 -actin, residues 316 mouse Sigma-Aldrich, St. Louis, MO
B-5-1-2 -tubulin mouse Sigma-Aldrich
N52 neurofilament 200, C-terminus mouse Sigma-Aldrich
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