Molecular characterization of recombinant mouse adenosine kinase
and evaluation as a target for protein phosphorylation
Bogachan Sahin
1
, Janice W. Kansy
1
, Angus C. Nairn
2,3
, Jozef Spychala
4
, Steven E. Ealick
5
,
Allen A. Fienberg
3,6
, Robert W. Greene
1,7
and James A. Bibb
1
1
The University of Texas Southwestern Medical Center, Dallas, TX;
2
Yale University School of Medicine, New Haven, CT;
3
The Rockefeller University, New York, NY;
4
University of North Carolina, Chapel Hill, NC;
5
Cornell University, Ithaca, NY;
6
Intra-Cellular Therapies Inc., New York, NY;

participant in cellular energy metabolism. In the central
nervous system (CNS), extracellular Ado behaves primarily
as a tonic inhibitory neuromodulator that controls neuronal
excitability through its interaction with four distinct
subtypes of G p rotein-coupled receptors, A
1
,A
2A
,A
2B
,
and A
3
[1]. A
1
receptor signaling in the cholinergic arousal
centers of the basal forebrain and brainstem reduces
cholinergic CNS tone, facilitating the transition from
waking to sleep [2]. A
2A
receptors in the striatum are
involved in the modulation of locomotor a ctivity, p ain
sensitivity, vigilance, and aggression [3]. Caffeine, t he most
widely used psychomotor stimulant substance in the world,
is a well-known Ado antagonist of both A
1
and A
2A
receptor subtypes [4].
Facilitated diffusion of Ado across the cell membrane via

[2,8-
3
H]Adenosine was from Amersham Biosciences.
Protease inhibitors, dithiothreitol, isopropyl thio-b-
D
-gal-
actoside, and ATP were from Roche. [
32
P]ATP[cP] was
from PerkinElmer Life Sciences. The catalytic subunit of
Correspondence to J. A. Bibb, Department of Psychiatry, The
University of Texas Southwestern Medical Center, 5323 Harry Hines
Blvd., NC5.410, Dallas, TX 75390–9070,USA.Fax: + 1 214 6481293;
Tel.: + 1 214 6484168; E-mail:
Abbreviations: AK, adenosine kinase; Ado, adenosine; hAK, human
adenosine kinase; mAK, mouse adenosine kinase.
Note: Nucleotide sequence data for the long and short isoforms of
mouse adenosine kinase are available i n the DDBJ/EMBL/GenBank
databases under the accession numbers, AY540996 and AY540997,
respectively.
(Received 24 M arch 200 4, re vised 29 J une 2 004, a ccepted 1 4 July 2004)
Eur. J. Biochem. 271, 3547–3555 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04291.x
PKA was purified fro m bovine heart as previo usly described
[11]. PKG and cGMP were purchased from Promega;
MAPK, CaMKII, and calmodulin from Upstate; and CK1,
CK2, and Cdk1 from New England Biolabs. Cdk5 and p25
were coexpressed in insect Sf9 cultures using baculovirus
vectors. PKC (a mixture of Ca
2+
-dependent isoforms, a, b

phosphorylation sites were selected for site-directed muta-
genesis using
SCANSITE
software , a we b-bas ed program for
motif prediction (). Site-directed
mutants were generated at these and other sites using a
standard kit (Stratagene) a nd following the m anufacturer’s
recommendations for mutagenic primer design. Mutations
were confirmed by DNA sequencing along both strands,
using primers specific for the T7 promoter and T7 t erminator.
Purification of mAK-L and mAK-S protein
Electrocompetent BL21 (DE3) ce lls were transformed w ith
hybrid pET-28a/16b expression vectors incorporating the
cDNA of mAK-L or mAK-S downstream from a vector-
encoded polyhistidine tag a nd thrombin cleavage site.
Cultures were grown to log phase and induced with
isopropyl thio-b-
D
-galactoside at room temperature for
20 h. Following lysis by French press and centrifugation at
10 000 g, cleared lysates were incubated with Ni-NTA
agarose beads (Qiagen). T he beads w ere w ashed and applied
to an elution column. Bound protein was eluted using a
linear gradient of 0–500 m
M
imidazole. Both AK isoforms
eluted at approximately 150 m
M
imidazole. Samples were
dialyzed overnight i n 1 0 m

activity of 20–50 CiÆmmol
)1
, and recombinant mAK-L or
mAK-S. Reactions were stopped by incubation at 95 °C
and were spotted onto Grade DE81 DEAE cellulose discs.
The discs were washed in 5 m
M
ammonium formate to
remove unphosphorylated adenosine a nd subjected to liquid
scintillation counting.
Immunoblot analysis
Mouse b rain and peripheral tissues were rapidly dissected,
homogenized by sonication, and boiled in 1% SDS.
Appropriate measures were taken to minimize pain or
discomfort in accordance with the Guidelines laid down by
the NIH regarding the care and use of animals for
experimental procedures. Protein concentrations were
determined by BCA assay (Pierce). Twenty-five micro-
grams of total protein f rom each sample was subjected to
SDS/PAGE (15% acrylamide), followed b y electrophoretic
transfer to nitrocellulose membrane and detection by
enhanced chemiluminescence. The blot was screened for
the presence and abundance of AK using a mouse a scites
fluid monoclonal antibody [15]. Known a mounts of
purified recombinant AK were included as standards for
quantification. Results were quantitated using
NIH IMAGE
software.
In vitro
phosphorylation reactions

)1
phospho-
tidylserine, 0.01 mgÆmL
)1
diacylglycerol. PKA reactions
were conducted in 50 m
M
HEPES, pH 7.4, 1 m
M
EGTA,
10 m
M
magnesium acetate, and 0.2 mgÆmL
)1
bovine serum
albumin; PKG reactions in 40 m
M
Tris/HCl, pH 7 .4,
20 m
M
magnesium a cetate, and 3 l
M
cGMP; MAPK
reactions in 50 m
M
Tris/HCl, pH 7.4, 10 m
M
MgCl
2
,and

To calculate reaction stoichiometries, r adiolabeled reaction
products and radioactive standards were quantitated using
IMAGEQUANT
software (Amersham Biosciences). Standards
consisted of 5 lL aliquots of serial dilutions of the reaction
mixtures, with the moles of phosphate defined using the
ATP c oncentration. Division of the signal per mole of
substrate by the signal per mole of phosphate yielded
the reaction stoichiometry (moles phosphate per moles
substrate).
Two-dimensional phosphopeptide map and
phosphoamino acid analysis
Dry gel fragments containing
32
P-labeled phospho-mAK
were excis ed, rehydrated, w ashed, and i ncubated at 3 7 °Cfor
20hin50m
M
ammonium bicarbonate, pH 8.0, containing
75 ngÆmL
)1
trypsin. The supernatant containing the tryptic
digestion products was lyophilized andthe lyophilate washed
up to four times with water and once w ith e lectrophoresis
buffer, pH 3.5 (10% acetic acid, 1% p yridine; v/v/v). T he
final lyophilate was resuspended in electrophoresis buffer,
pH 3.5, a nd 1 0% of the total volume wa s s et aside for amino
acid analysis. The remainder of the sample was spotted on a
TLC plate for one-dimensional electrophoresis. Separation
in the second dimension was achieved by ascending

Results
Two isoforms of AK are expressed in mouse brain
AK was cloned from a mouse brain cDNA library using
primers specific for the 5¢-and3¢-UTRs of human AK
(hAK) [8]. T en randomly selected clones were s ubse-
quently sequenced. Nine of these sequences were identical
and showed extensive homology with the long isoform of
hAK (hAK-L), while one was homologous to hAK-S.
The deduced amino acid sequences (Fig. 1) further
illustrated that, like their human homologues, mAK-L
and mAK-S are identical except at their respective
N-termini, where the first 20 amino acids of mAK-L
(MAAADEPKPKKLKVEAPQA) are replaced by four
residues (MTST) in mAK-S. This results in a length of
361 and 345 amino acids for mAK-L and mAK-S,
respectively.
Mouse and human AK were found to be 89%
homologous. Non-identical residues between the two
species were dispersed throughout the sequence, although
residues known to be i nvolved in catalytic activity, such as
those responsible for substrate and cation binding, were
100% conserved. At the time of this analysis, it was also
noted that only one mouse AK sequence had been
reported to d ate and that this existing sequence corres-
ponded to an N-terminal truncated for m [22]. T hat
sequence has since been replaced in the database with
what is reported here as mAK-L. To the best of our
knowledge, this is the first report of the deduced amino
acid sequence of mAK-S.
In order to study the function and regulation of mouse

Most tissues express more of one AK isoform
than the other
Quantitative immunoblot analysis of AK expression in
mouse b rain and p eripheral tissues using a monoclonal
antibody anti-hAK [15] showed highest levels of AK
expression in the liver, testis, kidney, and spleen (Fig. 3).
AK protein was present at intermediate levels in the brain,
with most forebrain structures and the cerebellum showing
somewhat higher levels of expression than the midbrain and
Ó FEBS 2004 Recombinant mouse AK as a protein phosphorylation target (Eur. J. Biochem. 271) 3549
brainstem. Moreover, in most tissue homogenates, two
protein species of different m olecular mass were detectable
with this antibody. These two closely migrating bands are
Fig. 1. Deduced amino acid sequence a lignment o f the long and short isoforms of human and mouse AK. Sequences are div ided into t wo domains
(yellow and green blocks) based o n crystal structure fo r the shorter splice v ariant of h um an AK [7]. Y ellow blocks constitut e the catalyt ic domain.
The regulatory domain (green blocks) fold s over the c atalytic domain and forms a hydrop hobic pocket for Ado phosphorylation. Residues that
make close contacts w ith Ado are i nd icated by red letters. G reen letters denote residu es that form the A TP/second ary Ad o-b inding site. One Mg
2+
ion is coordina ted betwe en the a ctive s ite a nd this AT P-binding s ite by hydrogen-bonding interactions mediated b y water and the residues
designated by blue letters. Stars indicate nonidentical residues.
Fig. 2. Preparation of active recombinant AK. (A) Purification of
recombinant mAK-L and mAK-S by affinity-column chromatogra-
phy. SDS/PAGE of UIT, uninduced total cellular protein; S10,
supernatant after centrifugation o f cell lysates at 1 0 000 g;P10,
insoluble p ellet after c entrifugation of c ell lysates at 10 000 g;FT,flow-
through, or u nbound protein, af ter incubation of S10 with Ni-NTA
agarose beads; F1, 2 and 3, eluted peak fractions. (B) Lineweaver–
Burke analysis of mAK-L activity. Values represent the average of four
experiments using duplicate samples.
Fig. 3. Quantitative immunoblot analysis of AK expression in mouse

chiometries were 0 .007, 0 .008 and 0.003 mol Æmol
)1
,
respectively, precluding subsequent biochemical analysis.
Similar results were obtained when m AK-S was u sed as the
putative protein kinase substrate (data not shown). In
contrast, all control substrates were efficiently phosphoryl-
ated by their respective p rotein kinases. At 60 min, protein
phosphatase inhibitor-1 was phosphorylated to a s toichio-
metry of 0.99, 0.31, 0.61 and 0 .97 m olÆmol
)1
by PKA,
MAPK, Cdk1 and Cdk5, respectively. Consistent with the
existence of multiple PKC sites in myelin basic p rotein [23],
the PKC-dependent phosphorylation of this control sub-
strate reached a maximal stoichiometry of 2.35 molÆmol
)1
.
Histone H1 was phosphorylated to a stoichiometry of
0.32 molÆmol
)1
by PKG, tyrosine hydroxylase to a stoi-
chiometry of 0.94 molÆmol
)1
by CaMKII, and DARPP-32
to a s toichiometry of 0.49 and 0.92 molÆmol
)1
by CK1
and CK2, respectively.
Phosphorylation of recombinant mouse AK by PKC

mAK-S (data n ot shown).
Mutation of four PKC c onsensus sites to alanine
(Ser48Ala, Ser85Ala, Ser272Ala, and Ser328Ala) had no
Fig. 4. Phosphorylation o f recombinant mAK-L by a panel of protein kinas es. PKC, PKA, PKG, MAPK, CaMKII, CK1, CK2, Cdk1 and C dk5 were
used to phosphorylate mAK-L as well as control substrates in vitro. I 1, protein phosphatase in hibitor-1; MB P, myelin basic protein; H1, histone H1;
TH, tyrosine hydroxylase; D 32, D ARPP-32. The m ultiple H 1 b ands v isible b y C oomassie stain a nd PhosphorIm ager a nalysis o f t he PKG reaction
correspond to degradation p roducts of t he protein. T he two h igher m olecular weight s pecies appearing a s radiolabeled b ands above t he AK signal in
the CaMKII r e action represent autophosphorylation of the different CaMKII isoform s present in this c omme rcial enzyme p reparation. At least one
of these CaMKII bands is also present in the TH lanes. The other is likely too close to the more prominent TH band to be visible.
Ó FEBS 2004 Recombinant mouse AK as a protein phosphorylation target (Eur. J. Biochem. 271) 3551
effect on the phosphorylation of mAK-L by PKC (Fig. 5E).
Mutants generated at the remaining nine conserved serine
residues w ere a lso efficient PKC substrates (data not shown).
In considering these observations, it was realized that in
addition to six histidines and a thrombin cleavage site, the
N-terminal affinity tag encoded by the expression vector
incorporates five serine residues. Indeed, enzymatic removal
of the first N-terminal 17 amino acids by thrombin cleavage
(MGSSHHHHHHSSGLVPR/GSH, t hrombin site indica-
ted by forward slash) substantially diminished the PKC-
dependent phosphorylation of mAK-L (Fig. 5 F). Similarly,
mutation of the five N-terminal serine re sidues in the affinity
tag sequence of m AK-L resulted in a fusion protein that was
no longer phosphorylated by PKC (Fig. 5G).
3552 B. Sahin et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
In this study, we r eport the cDNA and deduced amino acid
sequences for two isoforms of AK expressed in the mouse
brain. To date, the existen ce of AK splice variants has been
described in several mammalian s pecies, namely m ouse

a useful t ool for studying the role of AK in other tissues at
later d evelopmental t ime points. Notably, inhibito rs of AK
have already been used effectively to elevate extracellular
Ado levels [28] and shown so me promise in animal models
of stroke [29], seizure [30], and pain and inflammation [31].
Therefore, AK continues to be the subject of intensive study
for the development of neuroprotective, cardioprotective,
and analgesic agents, as well as drugs to treat sleep disorders
and enhance vigilance.
Although pharmacological and biochemical studies point
irrefutably to t he importance of AK in Ado homeostasis, t he
question of whether AK activity is regulated remains largely
unanswered. Insulin has been shown to induce AK expres-
sion in rat l ymphocytes [32]. Studies in the b rain have
suggested that A K activity e xhibits diurnal variations[33,34].
Most recently, akainic acid-induced mouse m odel of e pilepsy
was used to demonstrate that AK expression is up-regulated
in the epileptic hippocampus, c oincident with p ronounced
astrogliosis, which m ay partly explain the postlesion increase
in AK immunoreactivity in this region [24]. Thus, several
lines of evidence indicate that AK levels and enzyme activity
are m odulated in a number of systems, most likely through
the transcriptional and/or translational control o f AK
expression. However, it remains unclear whether post-
translational mechanisms also exist for the direct r egulation
of AK activity. A better understanding of AK regulation,
with regard to g ene expression as well as protein s tructure
and function, may reveal specific signaling pathways that
control this e nzyme and provide new targets for drug d esign.
A number of factors could be responsible for the possible

(A) Time-course analysis of the phosphorylation of mAK-L by PKC.
The radiographic image shown in the middle panel was used to derive
the p lotted v alues f or phosphate i ncorporation. (B) Phosphorylation of
mAK-L and mAK-S by PKC in vitro. The two panels represent SDS/
PAGE analysis of Coomassie-stained (top) and
32
P-labeled ( bottom)
mAK-L a nd mAK-S. Reaction t imes are in dic ated at t he top.
(C) Lineweaver–Burke analysis of PKC phosphorylation o f mAK-L.
The plot represents the results of four reactions conducted under
identical linea r c onditions using duplicate samples. (D) Phosp hopep -
tide mapping (PPM) and phosphoamino acid analysis (PAAA) of
mAK-L p reparative ly phosphorylated by PKC. (E) Site-directed
mutagenesis analysis of PKC phosphorylatio n of mAK-L. The Coo-
massie stain and autoradiogram depict various forms of mAK-L
phosphorylated by PKC and subjected to SDS/PAGE. The results of
four in vitro phosphorylation reactions are shown in which PKC was
used to phosphorylate Ser fi Ala mutants a t four PKC consensus sites
for 60 min. The stoichiom etry of each reaction is quantified in the
histogram as a percentage of the stoichiometry of PKC-dependent
phosphorylation of wild-type mAK-L. (F) The effect of thrombin
cleavage on the phosphorylation of mAK-L by PKC. SDS/PAGE
analysis of Coomassie-stained (top) and
32
P-labeled (bottom) mAK-L
is shown. Reaction times are indicated at the top. (G) The effe ct of fi ve
Ser fi Ala mutations in the N-terminal affinity tag on the phos-
phorylation of mAK-L by PKC. The two panels rep resent SDS/PAGE
analysis of Coomassie-stained (top) and
32

Defense (JAB and AAF), the Department of Veterans Affairs (RWG),
and the Ella McFadden Charitable Trust Fund at the Southwestern
Medical Foundation (JAB).
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