Báo cáo khoa học: Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation potx

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;

7
Veterans Administration Medical Center, Dallas, TX, USA
The regulation of adenosine kinase (AK) activity has the
potential to control intracellular and interstitial adenosine
(Ado) concentrations. In an effort to study the role of AK in
Ado homeostasis in the central nervous system, two iso-
forms of the en zyme wer e cloned f rom a mouse b rain cDNA
library. F ollowing overexpression in bacterial cells, the cor-
responding proteins were purified to homogeneity. Both
isoforms were enzymatically active and found to possess K
m
and V
max
values in agreement with kinetic parameters des-
cribed for other forms of AK. The distribution of AK in
discrete brain regions and various peripheral tissues was
defined. To investigate the possibility that AK activity is
regulated by protein phosphorylation, a panel of protein
kinases was screened for ability to phosphorylate recom-
binant mouse AK. Data from these in vitro phosphorylation
studies suggest t hat AK is most likely not an efficient s ub-
strate for PKA, PKG, CaMKII, CK1, CK2, MAPK, C d k1,
or Cdk5. PKC was found to phosphorylate recombinant
AK efficiently in vitro. Further analysis revealed, however,
that this PKC-dependent phosphorylation occurred at one
or more serine residues associated with the N -terminal
affinity tag used for protein purification.
Keywords: adenosine kinase; adenosine r egulation; protein
serine/threonine kinases; CNS.
Adenosine (Ado) is a potent biological mediator and a key

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

equilibrative nucleoside t ransporters closely c ouples baseline
Ado concentrations in the intracellular and extracellular
compartments [5]. Adeno sine kinase ( AK), which catalyzes
the transfer of the c-phosphate from ATP to the 5¢-hydroxyl
of Ado, generating AMP and ADP, is one of several
enzymes responsible for maintaining steady-state Ado levels
[6]. The structure of AK has been determined at 1.5 A
˚
resolution and c onsists of one large and one small a/b
domain and two Ado binding sites [7]. AK has a low K
m
value [8] that falls within the range of extracellular Ado
levels (25–250 n
M
) [9], suggesting that the reaction it
catalyzes may be the primary route of Ado metabolism
under physiological conditions. Moreover, AK inhibitors
are e ffective pharmacological reagents for increasing inter-
stitial Ado levels [10]. Thus, it is likely that mechanisms that
might regulate A K activity w ould be i mportant in the
modulation of extracellular Ado concentrations.
Materials and methods
Chemicals and enzymes
All chemicals were from Sigma, except where indicated.
Deoxyoligonucleotides were obtained from Integrated
DNA Technologies, I nc. R estriction and DNA modifying
enzymes were from New England Biolabs. Electrocompe-
tent bacteria were from Life Technologies, Inc. Cloning and
expression vectors were from Invitrogen and Novagen.
Site-directed mutagenesis reagents were from Stratagene.

[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

and c) w as purified from rat b rain [12]. Recombinant protein
phosphatase inhibitor-1 and DARPP-32 were generated
as previously described [ 13,14]. Recombinant tyrosine
hydroxylase w as kindly p rovided by P. Fitzpatrick and
C. Daubner, Texas A&M University. Purified histone H1
and myelin basic protein were from Upstate Biotechnology.
TLC plates were from Analtech (microcrystalline cellu-
lose, for phosphoamino acid analysis) and Bodman
(polyethyleneimine-impregnated cellulose, for phospho-
peptide mapping). Biotinylated thrombin and streptavidin
agarose were from Novagen.
Molecular cloning and site-directed mutagenesis
Long and short forms of mouse AK (mAK-L and mAK-S)
were amplified by PCR f rom a mouse b rain cDNA library
(courtesy of L. Monteggia, UT Southwestern, Dallas, TX).
Primers: 5¢-GGTGCATATGGCAGCTGCGG for the
5¢ end; 5¢-TCCACTCCACAGCCTGAGTT for the 3 ¢ end.
PCR p roducts were TA-cloned into the bacterial vector pCR
II-TOPO (Invitrogen) and subjected to automated fluores-
cent DNA sequencing using primers specific for the T7 and
Sp6 prom oters. For protein expression, a 5¢-primer i ncluding
an NdeI restriction site and a 3¢-primer containing a BamHI
restriction site were u sed to subclone mAK-L a nd mAK-S
cDNA sequences into a hybrid bacterial expression vector
based on p ET-16b and inc orporating the multiple cloning
region of pET-28a (Novagen). Primers: 5¢-CGTGGGGT
GCATATGGCAGCTGCG for the 5 ¢ end of mAK-L;
5¢-GTAGGTGCACATATGACGTCCACC for t he 5¢ end
of mAK-S; 5¢-ATATAGGATCCTCAGTGGAAGTC
TGG for the 3¢ end of both clones. Consensus PKC

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

M
Tris/HCl, pH 7.5, and 1 m
M
dithiothreitol, with two changes of buffer. Eluted and
dialyzed protein (10 lg) was analyzed for purity by SDS/
PAGE (15% acrylamide). In the final set of experiments
(Fig. 5F), the N-terminal affinity tag was removed using
biotinylated thrombin (Novagen) according to the manu-
facturer’s recommendations.
AK activity assays
Kinetic analysis o f AK activity was performed under
empirically defined linear steady-state conditions. Reactions
were carried out at 37 °C in a final v olume of 20 lL.
Reaction mixtures contained 50 m
M
Tris/HCl, pH 7 .5,
100 m
M
KCl, 5 m
M
MgCl
2
,5m
M
b-glycerol phosphate,
3m
M
ATP, dilutions of [2,8-
3
H]adenosine with a specific

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

All reactions were carried out at 30 °C in a final volume of
at least 30 lL containing 10 l
M
substrate, 100 l
M
ATP,
and 0.2 m CiÆmL
)1
[
32
P]ATP[cP]. The PKC reaction solu-
tion included 20 m
M
MOPS,pH7.2,25m
M
b-glycerol
phosphate, 1 m
M
sodium orthovanadate, 1 m
M
dithiothre-
itol, 1 m
M
CaCl
2
,10m
M
MgCl
2
,0.1mgÆmL

)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

20 m
M
EGTA; and Cdk5 reactions in 30 m
M
MOPS,
pH 7.2, and 5 m
M
MgCl
2
. F or C aMKII, CK1, CK2, and
Cdk1, reaction buffers provided b y the suppliers were used.
As positive controls, reactions were conducted using
proteins previously defined as physiological substrates for
each protein kinase. Specifically, p rotein phosphatase
inhibitor-1 was used in the PKA, MAPK, Cdk1 and
3548 B. Sahin et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Cdk5 reactions [13,16]; myelin basic protein in the PKC
reaction [17]; histone H1 in the P KG react ion [18]; tyrosine
hydroxylase i n the CaMKII reaction [19]; and DARPP-32
in the CK1 and CK2 reactions [20,21]. Time-course
reactions were performed by removing 10 lL aliquots
from the reaction solution at various time points and
adding an equal volume of 5· SDS protein sample buffer
to stop the reaction. Kinetic parameters were determined
using the results of four experiments performed under
empirically defined linear steady-state c onditions. In all
cases, [
32
P]phosphate incorporation was assessed by SDS/
PAGE (15% acrylamide) and PhosphorImager analysis.

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

chromatography. R esulting phosphopeptide maps were
visualized by autoradiography. Smearing was consistently
observed in the first dimension when microcrystalline
cellulose TLC plates (Analtech) were used. After t esting a
number of d ifferent TLC plates, buffer compositions, and
electrophoresis conditions, this issue was resolved by the use
of polyethyleneimine-impregnated cellulose TLC plates
(Bodman). To our knowledge, this electrophoretic separ-
ation p roblem may be unique to AK, as a number of other
phosphoproteins similarly analyzed by phosphopeptide
mapping have shown little or no smearing on microcrystal-
line cellulose TLC plates.
For phosphoamino acid analysis, the a liquot set aside in
the previous step was hydrolyzed at 100 °Cfor1hin6
M
HCl under an N
2
atmosphere. The reaction was stopped by
a sixfold dilution in water and the mixture was lyophilized.
The lyophilate was resuspended in electrophoresis buffer,
pH 1.9 (8% acetic acid, 2 % formic acid; v/v/v) and spotted
on a microcrystalline cellulose TLC plate along with
phosphoserine, -threonine, and -tyrosine standards. Elec-
trophoresis was performed over half the length of the TLC
plate using electrophoresis buffer, pH 1.9, at which point
the plate was transferred into the pH 3.5 buffer and
electrophoresis was carried out to completion. A 1% (v/v)
ninhydrin solution in acetone was sprayed onto the plates to
visualize the phosphoamino acid standards. Samples were
visualized by autoradiography.

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

AK in vitro, both isoforms were s ubcloned into a pET
expression vector encoding an N-terminal polyhistidine tag
for affinity purification. Recombinant protein was purified
to homogeneity by affinity-column chromatography. SDS/
PAGE analysis of the pure fractions indicated an a pparent
molecular weight o f 45 and 43.5 kDa for polyhistidine-
tagged recombinant m AK-L and m AK-S, respectively
(Fig. 2 A). Moreover, in vitro AK activity assays demon-
strated that the two recombinant proteins were enzymati-
cally active, efficiently catalyzing the phosphorylation of
AdotoAMP(formAK-L,K
m
¼ 20 ± 4 n
M
; V
max
¼
16 ± 1.6 nmolÆmin
)1
Ælg
)1
, n ¼ 8) (Fig. 2B). No significant
difference was noted between mAK-L and mAK-S with
respect to K
m
and V
max
(data not shown). These kinetic
parameters were also in agreement with previously reported
values for other forms of AK [8].

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

brain and peripheral tissues. The three lanes on the far right were used
to blot 10, 50 and 100 ng of pure r ecombinant mAK-S for quantifi-
cation pu rp oses. Recombinant mAK -S s tandards have a higher
apparent molecular weight than m AK-Sinthesamplelanesdueto
N-terminal polyhistidine tags. Quantification of relative AK abun-
dance in each tissue examined is a lso shown.
3550 B. Sahin et al. (Eur. J. Biochem. 271) Ó FEBS 2004
likely to represent the long and short isoforms of the
enzyme. Many of the tiss ues included in this a nalysis also
showed a prevalence of one isoform over the other. For
instance in the spleen, the short isoform is the predominant
AK species , whereas in the t estis and kidney, the long
isoform is more abundant. Most brain regions, with the
exception of the cerebellum, express detectable levels of only
the short isoform. In t he cerebellum, both isoforms are
present at nearly equal levels.
Phosphorylation of recombinant mouse AK
by a protein kinase panel
Motif prediction analysis of the mouse AK sequence
indicated t he pres ence of putative phosphorylation sites
for several protein kinases, including PKA, PKC, CaMKII,
CK1 and CK2 (). T o investigate the
possibility that AK activity may be regulated by protein
phosphorylation, a panel of these protein kinases and others
was tested f or ability t o phosphorylate r ecombinant mouse
AK in vitro (Fig. 4). PKC was able to phosphorylate mAK-
L efficiently. PKA, PKG, MAPK, CK2, and Cdk1 did not
detectably phosphorylate mAK-L. Faint radiolabeling of
mAK-L could be detected in reaction mixtures for CaM-
KII,CK1,andCdk5.However,maximalreactionstoi-

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

A time-course phosphorylation reaction conducted using
an excess of PKC and 10 l
M
AK displaye d linear
conversion of substrate to phosphoprotein over the first
5 m in and n ear s aturation b y 2 0 min, with a maximal
stoichiometry greater than 0.30 molÆmol
)1
(Fig. 5A).
mAK-L and mAK-S served as equally efficient substrates
for PKC in vitro (Fig. 5B). Kinetic analysis of the PKC-
dependent phosphorylation of mAK-L revealed a K
m
of
6.9 ± 1.1 l
M
and V
max
of 68 ± 3 lmolÆmin
)1
Ælg
)1
for this
reaction (Fig. 5C, n ¼ 8). S imilar values were obtained
using the short isoform as a substrate (data not shown).
A phosphopeptide m ap of mAK-L p reparatively phos-
phorylated by PKC showed t wo major sp ots (Fig. 5D, first
panel). Phosphoamino acid analysis of the same material
indicated that this phosphorylation occurs at serine
(Fig. 5 D, second panel). S imilar results were obtained with

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

[24,25], rat [26] and human [8,27]. A search for multiple
forms of AK in other species is likely to generate similar
results.
Recent immunohistochemical studies have shed light on
the pattern of brain AK expression, with a roughly
homogenous distribution reported in astrocytes t hroughout
the brain, in addition to pockets of high neuronal e xpression
in the olfactory bulb, striatum, and brainstem [24]. In
agreement, the immunoblot analysis shown here indicates
that AK levels are roughly equivalent in most brain regions,
with midbrain and brainstem structures showing somewhat
lower levels than the cerebellum and various components of
the forebrain. Furthermore, one or the o ther AK isoform
predominates in most tissues, including the brain, where the
short isoform is prevalent. The functional significance of
this isoform preference at the level of tissues and whole
organs remains unknown. Although no difference was
observed in enzymatic activity between recombinant
mAK-L and mAK-S, it is possible that in vivo the two
molecules are functionally distinct in some other important
respect, such as transcription al and/or translational r egula-
tion, rate of turnover, s ubcellular l ocalization, or association
with as yet undefined regulatory factors.
The most abundant nucleoside kinase in mammals, AK
has emerged as a key e nzyme in the regulation of interstitial
Ado a nd intracellular adenylate levels in the C NS and
periphery. A K-knockout mice undergo normal e mbryo-
genesis, but develop microvesicular hepatic steatosis within
4 d ays of b irth, dying by the end of two weeks with fatty
liver [25]. Conditional gene knockout may therefore provide

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

regulation of AK at the post-translational level, including
protein stability, subcellular localization, regulatory binding
partners, and post-tr anslational modifications such as
protein phosphorylation. In the present study, we report
that in vitro AK does not serve as an efficient substrate for
representatives of several major classes of protein serine/
threonine kinases. A lthough C aMKII, CK1, and Cdk5
were found to phosphorylate AK w eakly, the maximal
stoichiometry achieved in these reactions remained below
0.01 molÆmol
)1
. T hese low levels of phosphorylation effect-
ively preclude further biochemical characterization, such as
the identification of phosphorylation sites or the assessment
of a possible e ffect of AK phosphorylation on AK activity.
Furthermore, they strongly suggest t hat these reactions are
unlikely to occur in vivo or otherwise be physiologically
relevant. Taken together, our findings indicate that AK is
unlikely to b e r egulated by any of the protein k inases
investigated here.
On the o ther hand, it is important to note that our screen
was by n o means exhaustive, and although t he protein
kinases tested in this study represent most of the p rincipal
classes of protein serine/threonine kinases, the possibility
remains that an untested, perhaps unidentified, protein
kinase phosphorylates AK. Future studies utilizing more
broad-based strategies, such as immunoprecipitation of AK
from radiolabeled cells or tissue preparations, may reveal
AK-specific regulatory pathways of this nature.
Fig. 5. Phosphorylation of recombinant mouse AK by PKC in vitro.

(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

P-labeled (bottom) mAK-L
and a quintuple m utant o f mAK-L (5XS>A ) incorporatin g serine-to-
alanine mutations at the five serine residues of the N-terminal affinity
tag. Reaction times a re indicated a t the to p.
Ó FEBS 2004 Recombinant mouse AK as a protein phosphorylation target (Eur. J. Biochem. 271) 3553
In addition to these central observations, our studies have
produced several findings of technical significance. The
results reveal a potentially important hazard in the use o f
the pET vector system for the recombinant expression of
putative PKC substrates, and perhaps substrates of other
protein kinases. With regard to the analysis of phospho-
proteins by thin-layer chromatography, it should b e noted
that the novel use of polyethyleneimine-impregnated cellu-
lose TLC plates was essential to the generation of good
phosphopeptide maps using AK. These observations
may be of interest to other investigators studying AK and
PKC.
Acknowledgements
The authors would like to thank Lisa Monteggia at UT Southwestern
Medical Center for providing the mouse brain cDNA library used in
these experiments, Paul Fitzpatrick and Colette Daubner at Texas
A&M University for providing recombinant t yrosine hydroxylase for
use in CaMKII phosphorylation reactions, and Donna Hanson of
Bodman Industries for TLC materials and technical assistance
regarding TLC of AK phosphopeptide. This work was supported by
funding from the Medical Sc ientist T raining Program at UT
Southwestern Medical Center (BS), the National Cancer Institute
(JS), t he National Institute of Drug Abuse (JAB), t he National Alliance
forResearchonSchizophreniaandDepression(JAB),theNational
Institute of Mental Health (ACN and RWG), the Department of

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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