www.elsevier.com/locate/ybbrc

Biochemical and Biophysical Research Communications 323 (2004) 499–504

BBRC

Regulation of Dyrk1A kinase activity by 14-3-3

Doyeun Kima, Jungyeon Wonb, Dong Wook Shina, Junghee Kanga, Yeon Ju Kima,Su Young Choia, Mi-Kyung Hwanga, Byeong-Woo Jeonga, Gun Soo Kima, Cheol O. Joeb,

Sul-Hee Chunga, Woo-Joo Songa,*

a Division of CNS, Hanwha Chemical R&D Center, Daejon 305-345, Republic of Koreab Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon, 305-701, Republic of Korea

Received 16 July 2004

Available online 3 September 2004

Abstract

Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation

resulting from Down syndrome. In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A

kinase activity. We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction

between the yeast orthologues Yak1p and Bmh1/2p. We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo.

The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status. Functionally, 14-3-3

binding increased Dyrk1A kinase activity in a dose dependent manner in vitro. In vivo, a small peptide inhibiting 14-3-3 binding,

sc138, decreased Dyrk1A kinase activity in COS7. In summary, these results suggest that DYRK1A kinase activity could be regu-

lated by the interaction of 14-3-3.

� 2004 Elsevier Inc. All rights reserved.

Keywords: DYRK1A; 14-3-3; Kinase activity; Down syndrome; Mental retardation

Dual-specificity tyrosine(Y) regulated kinase 1A

(DYRK1A) is a member of a novel DYRKs subfamilywith dual specificities; autophosphorylation for self-acti-

vation on tyrosine residue in the active loop of the cat-

alytic domain, and target protein phosphorylation on

serine/threonine residues [1–3]. To date, seven mamma-

lian DYRKs members have been isolated (DYRK1A,

DYRK1B, DYRK1C; DYRK2; DYRK3; DYRK4A,

and DYRK4B) [2]. Among isoforms the human

DYRK1A gene is localized in the Down syndrome(DS) critical region of chromosome 21, which is associ-

ated with a variety of anomalies of DS, including mental

retardation [4–6]. DYRK1A is expressed in the cerebral

cortex, cerebellum, and hippocampus, which are affected

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.08.102

* Corresponding author. Fax: +82 42 865 6690.

E-mail address: woojoos@yahoo.com (W.-J. Song).

regions of the brain in DS [6]. Transgenic mice overex-

pressing DYRK1A have shown cognitive deficit and lossof learning and memory shown in DS [7,8]. It is also

known that Drosophila minibrain (mnb), the human

DYRK1A orthologue, is involved in postembryonic

neurogenesis, and its mutation causes a reduction of

the brain size due to a decrease in the production of neu-

ronal progeny [9]. Taken together, DYRK1A is consid-

ered to be a gene that is responsible for at least a

portion of mental retardation in relation to DS.Dyrk1A encodes several structural motifs such as

bipartite nuclear localizing signal (NLS), DYRK

homology (DH) box, PEST region, serine/threonine rich

region, and 13 consecutive histidine repeats [1,3].

DYRK1A is localized to the nucleus predominantly by

its canonical NLS sequence [2,10] and is known to phos-

phorylate transcription factors including CREB,

FHKR, STAT3, and GLI1 in the nucleus [11–14]. For

500 D. Kim et al. / Biochemical and Biophysical Research Communications 323 (2004) 499–504

CREB, its phosphorylation mediates the neuronal differ-

entiation of neural progenitor cells [11]. To date, two

different modes of positive regulation for DYRK1A

activity have been reported in response to bFGF and

E1A [11,15]. However, the detailed upstream signaling

that regulates the activity of DYRK1A remainsunknown.

14-3-3 is a large family of acidic 28–33 kDa proteins

conserved from yeast to mammal that is abundant and

widely expressed in all eukaryotic cells [16]. 14-3-3 inter-

acts with over hundreds of various target proteins and

the interaction is predominantly dependent on the phos-

phoserine-containing motif [17,18]. 14-3-3 has attracted

substantial attention because of its pleiotropic biologicaleffects [18]. First, binding of 14-3-3 can modulate the

localization of several target proteins. For example,

14-3-3 translocates phosphorylated cdc25 from nucleus

to cytosol and promotes the retention of phosphorylated

FKHRL1 in cytosol [19,20]. Second, binding of 14-3-3

can regulate the intrinsic catalytic activity of target pro-

teins. 14-3-3 increases the activity of serotonin-N-acetyl-

transferase and Raf-1 kinase, or decreases RSK1 kinaseactivity upon binding [21–23]. Third, 14-3-3 is also in-

volved in other modes of regulation, including scaffold-

ing, protection of proteolysis, dephosphorylation, and

sequestration of target protein [18].

In this study, we identified 14-3-3 as a binding partner

of Dyrk1A through yeast two-hybrid screening. We

characterized the interaction of 14-3-3 with Dyrk1A

and examined the function of the 14-3-3 binding onDyrk1A kinase activity.

Materials and methods

Plasmids. For bacterial expression, 14-3-3 and Dyrk1A were cloned

into pGEX4 and pET25, respectively (Amersham Bioscience, UK).

pEBG (GST fusion vector) and pcDNA3.1a (Invitrogen, USA) were

used for mammalian expression. Deletion mutants were made by PCR

using region specific primers. For single amino acid mutants, site-di-

rected mutagenesis was performed as described in the manual

(Quickchange, Stratagene, USA). Dr. Haian Fu kindly provided sc138

and sc174 constructs, encoding small peptides inhibiting 14-3-3 bind-

ing [24].

Yeast two hybrid. Yeast two-hybrid assay was performed using a

L40 yeast strain, as described previously [25]. Full-length mouse

Dyrk1A cloned into pBHA was used for screening human and rat

brain cDNA libraries (Clontech, USA).

Purification of recombinant proteins. For Dyrk1A, transformed

BL21(DE3) was cultured at 30 �C until OD600 of 0.5, and IPTG (final

concentration, 1 mM) was added for induction. Four hours after

induction, cells were lysed by sonication in lysis buffer [20 mM Tris–Cl

(pH 8.0), 300 mM NaCl, 30 mM imidazole, and 1% protease inhibitor

cocktail]. After centrifugation at 20,000g for 30 min at 4 �C, super-natants were loaded into the Ni–NTA column (Qiagen, Germany),

which has affinity to the intrinsic 13 His repeat of Dyrk1A. The column

was washed with 5 bed volumes of washing buffer (50 mM imidazole in

lysis buffer) and Dyrk1A was eluted with 300 mM imidazole in lysis

buffer (>95% purity). Purification of GST fusion protein was per-

formed as recommended in the manual (Amersham Bioscience).

Preparation of cell lysates. COS7 cells were maintained in high

glucose DMEM supplemented with 10% FBS (Invitrogen). One day

before transfection, cells were seeded at a density of 2 · 105 cells/well in

a 6-well plate. Recommended procedures were followed as described in

the manual (Invitrogen). After transfection, cells were lysed in RIPA

buffer [50 mM Tris (pH 8.0), 150 mMNaCl, 1% NP-40, 0.1% SDS, and

0.5% sodium deoxycholate]. After centrifugation, supernatants were

subjected to immunoblotting, immunoprecipitation, or pull-down as-

say. For dephosphorylation, supernatants were treated with lambda

phosphatase (New England Biolabs, USA) for 2 h at 30 �C.Immunoprecipitation and pull-down. For Dyrk1A immunoprecipi-

tation, 600 lg of protein lysates was incubated with 2.5 lg affinity

purified anti-Dyrk1A antibody (directed against C-terminal fragment

of Dyrk1A) with gentle rocking overnight at 4 �C followed by incu-

bation with protein A resin for 1 h. For GST14-3-3 precipitation, ly-

sate was incubated with glutathione–Sepharose resin (Amersham

Bioscience). Pull down was carried out by adding 5 lg GST14-3-3 (or

its mutant protein) and glutathione–Sepharose resin. Immunoprecipi-

tates or pull-downs were resolved on 10% SDS–PAGE and transferred

to PVDF membrane for immunoblotting.

Dyrk1A kinase assay. The kinase reactions were carried out by

incubating purified Dyrk1A (50 ng) with dephosphorylated MBP

(1 lg, Upstate, USA) in kinase buffer [20 mM Hepes (pH 7.4), 2 mM

Na3VO4, 2 mM DTT, 10 mM MgCl2, and 0.5 lCi [c-32P]ATP (specific

activity of 3000 Ci/mmol, Amersham Bioscience)] for 30 min at 30 �C.MBP was separated on 12% SDS–PAGE and its radioactivity was

quantified using FLA3000 (Fuji, Japan). For in vivo kinase assay,

Dyrk1A immunoprecipitates were used.

Results

To identify Dyrk1A interacting proteins, we carried

out yeast two-hybrid screening in rat and human braincDNA libraries using full-length mouse Dyrk1A as a

bait. We found a total of 38 clones including eukaryotic

transcriptional initiation factor 2, a known substrate of

Dyrk1A. Two independent clones were identified as the

exon 1 of 14-3-3g. Since exon 1 of 14-3-3g was com-

posed of 29 amino acids that are highly conserved in

the 14-3-3 family, we expected that Dyrk1A could bind

other members of the 14-3-3 family. We examined theinteraction of Dyrk1A with 14-3-3e, a brain enriched

isotype of the 14-3-3 family.

Dyrk1A binds 14-3-3 in vitro and in vivo

To examine the interaction of Dyrk1A with 14-3-3 in

vitro, purified Dyrk1A was pulled down by GST14-3-3.

Dyrk1A was co-precipitated with GST14-3-3 in a con-centration dependent manner, but not with GST (Fig.

1A). The interaction between Dyrk1A and 14-3-3 was

also confirmed in vivo. COS7 cells were transfected with

GST or GST14-3-3 and the lysate was incubated with

glutathione–Sepharose resin (Fig. 1B, top panel). Dyr-

k1A was co-precipitated with GST14-3-3 but not with

GST (Fig. 1B, middle panel). However, when Dyrk1A

was immunoprecipitated, only GST14-3-3 was detected(Fig. 1B, bottom panel). It is known that K(lysine)50

residue in the consensus target binding motif is critical

Fig. 1. The interaction between Dyrk1A and GST14-3-3 in vitro and in vivo. (A) Purified Dyrk1A was pulled down by GST or GST14-3-3 (top

panel, amount of Dyrk1A used for pull-down). (B,D) COS7 cells were transfected for GST, GST14-3-3 or GST14-3-3 (K50E) expression and

precipitated with glutathione–Sepharose resin or immunoprecipitated with purified anti-Dyrk1A antibody (B, bottom panel). (C) COS7 lysate was

pulled down by GST, GST14-3-3, or GST14-3-3 (K50E).

D. Kim et al. / Biochemical and Biophysical Research Communications 323 (2004) 499–504 501

for 14-3-3 interaction. We tested whether the K50 resi-

due is essential for the interaction between Dyrk1A

and 14-3-3. Fig. 1C shows that Dyrk1A in the lysate

was pulled down by GST14-3-3 but not by GST14-3-3(K50E) mutant. In vivo, when lysates transfected with

GST14-3-3 or GST14-3-3 (K50E) were precipitated with

glutathione–Sepharose resin, co-precipitation was de-

tected only with GST14-3-3, supporting the in vitro re-

sult (Fig. 1D).

Since the 14-3-3 generally binds its partner in a phos-

phorylation dependent manner [17,18], we tested

whether phosphorylation of Dyrk1A was required forbinding. Cell lysate was treated with lambda phospha-

tase (Fig. 2, middle panel) and pulled down by

GST14-3-3 (Fig. 2, bottom panel). Interaction between

14-3-3 and Dyrk1A was not affected by phosphatase

treatment, suggesting that the interaction was phosphor-

ylation independent.

Fig. 2. Phosphorylation independent binding of Dyrk1A. Lambda

phosphatase treated COS7 lysate was subjected to pull down by

GST14-3-3. Lysate was immunoblotted with anti-Dyrk1A (top panel)

to show the loaded protein or PY99 (middle panel) to detect

phosphorylation.

Binding of 14-3-3 requires N-terminus and K188 residue

of Dyrk1A

To determine the binding domain of Dyrk1A inter-acting with 14-3-3, we generated deletion mutants of

Dyrk1A, as shown in Fig. 3A. COS7 cell lysate express-

ing myc-tagged deletion mutants of Dyrk1A was pulled

down by GST14-3-3 (Fig. 3B). Interaction of C-terminal

deletion mutant with GST14-3-3 was comparable to the

full-length Dyrk1A. However, interaction was dramati-

cally reduced with N-terminal (Dyrk1ADN) and

N- and C-terminal (Dyrk1ADNDC) mutants. Aminoacid 135–763 and 125–606 fragments include DH box

and NLS/ DH box, respectively, suggesting that the N-

terminal domain upstream of NLS is important for

interaction. We also tested whether kinase activity of

Dyrk1A is required for interaction. As shown in Fig.

3C, COS7 lysate expressing kinase mutant of Dyrk1A

(K188R) was not pulled down by GST14-3-3. However,

the interaction with Dyrk1A (Y321F), another kinasemutant that has lost an autophosphorylation activity,

was maintained, indicating that the kinase activity is

not necessarily required.

Binding of 14-3-3 increases Dyrk1A kinase activity in

vitro and in vivo

To examine the regulatory function of 14-3-3 on theDyrk1A activity in vitro, we carried out a Dyrk1A ki-

nase assay using MBP as a substrate. Phosphorylation

of MBP by purified Dyrk1A was increased 2.5-fold in

the presence of GST14-3-3 (Fig. 4A). The kinase activity

Fig. 3. Pull-down for deletion and kinase mutants of Dyrk1A with GST14-3-3. (A) Schematic presentation of Dyrk1A deletion mutants. Boxes

represent the: (1) NLS, (2) DH box, (3) catalytic domain, (4) PEST region, (5) His repeat, and (6) Ser/Thr repeat. (B,C) COS7 cells were transfected

for full-length Dyrk1A, deletion mutants (B) or kinase mutants (C) expression, and lysate was pulled down by GST14-3-3 (top panel). Expression

level of mutants was assessed by immunoblotting (bottom panel).

Fig. 4. Regulation of Dyrk1A kinase activity by 14-3-3. (A) Kinase

activity of Dyrk1A was measured using MBP as substrate in the

presence of GST or GST14-3-3 in vitro. MBP was separated on the

SDS–PAGE and radioactivity of phospho-MBP was quantified. (B)

COS7 cells were double transfected with GST14-3-3 (top panel) and

sc138 or sc174. Lysate was precipitated with glutathione–Sepharose

resin (bottom panel). (C) COS7 cells were transfected with sc138 or

sc174, and Dyrk1A kinase activity was measured as (A). The results

are represented as means ± SE (n = 4, three independent experiment).

*p < 0.05.

502 D. Kim et al. / Biochemical and Biophysical Research Communications 323 (2004) 499–504

was increased in a dose-dependent manner, showing amaximum activity at 5 lg GST14-3-3 (data not shown).

To confirm the activation of Dyrk1A by 14-3-3 in vivo,

the kinase activity was measured for the immunoprecip-

itate of COS7 lysate transfected with GST14-3-3 or

GST14-3-3 (K50E). However, we were not able to detect

a significant difference between GST14-3-3 and GST14-

3-3 (K50E), possibly due to the high level of endogenous

14-3-3 family proteins (data not shown). Conversely, weinvestigated whether the disruption of 14-3-3 binding

affects the kinase activity in vivo. COS7 cells were trans-

fected with sc138, a small peptide inhibitor of 14-3-3

binding, or its mutant sc174. The interaction between

14-3-3 and Dyrk1A was disrupted by sc138, but not

by sc174 (Fig. 4B). The kinase activity of the immuno-

precipitates was decreased approximately 15% in cells

expressing sc138, but not for sc174, suggesting that the

interaction positively regulated the kinase activity ofDyrk1A (Fig. 4C).

Discussion

The binding of yeast Yak1p (DYRK family) and

Bmh1p or Bmh2p (14-3-3 family) was previously re-

ported, suggesting an interaction between mammaliancounterparts [26]. In the present report, we showed the

interaction of 14-3-3 with Dyrk1A, and the positive reg-

ulation of Dyrk1A kinase activity by 14-3-3 binding.

Bmh1/2p binds Yak1p in a phosphorylation-dependent

manner in response to the glucose supply and Dyrk1A

has several consensus candidate sites (RSXpSXP or

RXXXpSXP) for 14-3-3 binding. However, the interac-

tion of Dyrk1A and 14-3-3 was maintained regardlessof Dyrk1A phosphorylation status (Fig. 2), similar to

a few reports on 14-3-3 binding [27,28]. In support of

this result, no serine phosphorylation was reported in

the phosphopeptide mapping study of Dyrk1A [29]

and phosphoserine residue was not detected by immuno-

blotting for the immunoprecipitated Dyrk1A (data not

shown). Furthermore, four candidate serine residue mu-

tants (S20A, S47A, S54A, and S97A) at the N-terminus

D. Kim et al. / Biochemical and Biophysical Research Communications 323 (2004) 499–504 503

did not affect the interaction (data not shown). We also

found that the N-terminus and K188 of Dyrk1A is a re-

quired domain and a critical residue for the interaction,

but Y321 does not play a role in binding (Fig. 3). We

reasoned that the failure of interaction between

K188R mutant and 14-3-3 might be due to the tertiarystructural changes of the N-terminal domain. In fact,

K188 residue constitutes an ATP binding pocket located

near the tertiary structure formed by a N-terminal do-

main different from Y321 [29].

Dyrk1A kinase activity was increased by 14-3-3 up to

2.5-fold in vitro, and was significantly decreased by an

interaction disrupting peptide, sc138 (Fig. 4). It is

known that 14-3-3 increased the activity of serotoninN-acetyltransferase by stabilizing the region involved

in the substrate binding and Raf-1 by stabilizing active

conformation as a cofactor [21,30]. These findings indi-

cate that Dyrk1A activity regulation may be mediated

by increasing or decreasing the stability of the active

structure by 14-3-3. However, an increase of Dyrk1A

activity in vivo was not observed, possibly due to the

abundance of endogenous 14-3-3 in COS7 and HEK293 cell lines used. Similar to our result, overexpression

of 14-3-3 did not increase the specific activity of Raf-1

kinase in NIH3T3 cell line [31], whereas microinjection

of 14-3-3 into Xenopus oocytes could increase the kinase

activity of Raf-1 [22].

Since 14-3-3 is known to translocate the target pro-

tein from the nucleus to cytosol, we tested whether the

presence of 14-3-3 changes the localization of Dyrk1A.However, translocation of Dyrk1A from the nucleus

to cytosol was not observed by overexpression of 14-3-

3 (data not shown). Instead, 14-3-3 was co-localized in

the nucleus with Dyrk1A in some cells highly overex-

pressing Dyrk1A (data not shown). Failure of Dyrk1A

translocation by 14-3-3 from the nucleus to cytosol is

supported by a report that Dyrk1A constitutes a com-

plex subnuclear splicing speckle, composed of sc35 andcyclins [32,33].

Considering implication of DYRK1A in the brain

pathology of DS, it is very important to investigate the

regulation mode of DYRK1A kinase activity. We found

that binding of 14-3-3 could positively modulate the

Dyrk1A kinase activity. Understanding the precise

mechanism of DYRK1A kinase activity regulation in

a structural base would contribute to the developmentof therapeutics to alleviate some forms of DS pathology,

especially mental retardation.

Acknowledgments

We thank J.T. Lee for protein purification. We are

grateful to the financial support of Hanwha ChemicalCorp.

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