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 Sahin1, Janice W.. To investigate the possibility that AK

Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation

Bogachan Sahin1, Janice W Kansy1, Angus C Nairn2,3, Jozef Spychala4, Steven E Ealick5,

Allen A Fienberg3,6, Robert W Greene1,7and James A Bibb1

1

The University of Texas Southwestern Medical Center, Dallas, TX;2Yale University School of Medicine, New Haven, CT;

3

The Rockefeller University, New York, NY;4University of North Carolina, Chapel Hill, NC;5Cornell University, Ithaca, NY;

6

Intra-Cellular Therapies Inc., New York, NY;7Veterans 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 enzyme were cloned from a mouse brain cDNA

library Following overexpression in bacterial cells, the

cor-responding proteins were purified to homogeneity Both

isoforms were enzymatically active and found to possess Km

and Vmaxvalues 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 that AK is most likely not an efficient sub-strate for PKA, PKG, CaMKII, CK1, CK2, MAPK, Cdk1,

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 regulation; 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 protein-coupled receptors, A1, A2A, A2B,

and A3[1] A1receptor signaling in the cholinergic arousal

centers of the basal forebrain and brainstem reduces

cholinergic CNS tone, facilitating the transition from

waking to sleep [2] A2A receptors in the striatum are

involved in the modulation of locomotor activity, pain

sensitivity, vigilance, and aggression [3] Caffeine, the most

widely used psychomotor stimulant substance in the world,

is a well-known Ado antagonist of both A1 and A2A

receptor subtypes [4]

Facilitated diffusion of Ado across the cell membrane via

equilibrative nucleoside transporters closely couples baseline

Ado concentrations in the intracellular and extracellular

compartments [5] Adenosine 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 consists of one large and one small a/b domain and two Ado binding sites [7] AK has a low Km value [8] that falls within the range of extracellular Ado levels (25–250 nM) [9], suggesting that the reaction it catalyzes may be the primary route of Ado metabolism under physiological conditions Moreover, AK inhibitors are effective pharmacological reagents for increasing inter-stitial Ado levels [10] Thus, it is likely that mechanisms that might regulate AK activity would be important 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, Inc Restriction 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 [2,8-3H]Adenosine was from Amersham Biosciences Protease inhibitors, dithiothreitol, isopropyl thio-b-D -gal-actoside, and ATP were from Roche [32P]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: james.bibb@utsouthwestern.edu

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 in the DDBJ/EMBL/GenBank

databases under the accession numbers, AY540996 and AY540997,

respectively.

(Received 24 March 2004, revised 29 June 2004, accepted 14 July 2004)

PKA was purified from bovine heart as previously 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 Ca2+-dependent isoforms, a, b

and c) was purified from rat brain [12] Recombinant protein

phosphatase inhibitor-1 and DARPP-32 were generated

as previously described [13,14] Recombinant tyrosine

hydroxylase was kindly provided 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 from a mouse brain cDNA library

(courtesy of L Monteggia, UT Southwestern, Dallas, TX)

Primers: 5¢-GGTGCATATGGCAGCTGCGG for the

5¢ end; 5¢-TCCACTCCACAGCCTGAGTT for the 3¢ end

PCR products 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 promoters For protein expression, a 5¢-primer including

an NdeI restriction site and a 3¢-primer containing a BamHI

restriction site were used to subclone mAK-L and mAK-S

cDNA sequences into a hybrid bacterial expression vector

based on pET-16b and incorporating the multiple cloning

region of pET-28a (Novagen) Primers: 5¢-CGTGGGGT

GCATATGGCAGCTGCG for the 5¢ end of mAK-L;

5¢-GTAGGTGCACATATGACGTCCACC for the 5¢ end

TGG for the 3¢ end of both clones Consensus PKC

phosphorylation sites were selected for site-directed

muta-genesis usingSCANSITEsoftware, a web-based program for

motif prediction (http://scansite.mit.edu) Site-directed

mutants were generated at these and other sites using a

standard kit (Stratagene) and following the manufacturer’s

recommendations for mutagenic primer design Mutations

were confirmed by DNA sequencing along both strands,

using primers specific for the T7 promoter and T7 terminator

Purification of mAK-L and mAK-S protein

Electrocompetent BL21 (DE3) cells were transformed with

hybrid pET-28a/16b expression vectors incorporating the

cDNA of mAK-L or mAK-S downstream from a

vector-encoded polyhistidine tag and 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) The beads were washed and applied

to an elution column Bound protein was eluted using a

linear gradient of 0–500 mMimidazole Both AK isoforms

eluted at approximately 150 mM imidazole Samples were

dialyzed overnight in 10 m Tris/HCl, pH 7.5, and 1 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 of AK activity was performed under empirically defined linear steady-state conditions Reactions were carried out at 37C in a final volume of 20 lL Reaction mixtures contained 50 mM Tris/HCl, pH 7.5,

100 mM KCl, 5 mM MgCl2, 5 mM b-glycerol phosphate,

3 mM ATP, dilutions of [2,8-3H]adenosine with a specific activity of 20–50 CiÆmmol)1, and recombinant mAK-L or mAK-S Reactions were stopped by incubation at 95C and were spotted onto Grade DE81 DEAE cellulose discs The discs were washed in 5 mM ammonium formate to remove unphosphorylated adenosine and subjected to liquid scintillation counting

Immunoblot analysis Mouse brain 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 from each sample was subjected to SDS/PAGE (15% acrylamide), followed by electrophoretic transfer to nitrocellulose membrane and detection by enhanced chemiluminescence The blot was screened for the presence and abundance of AK using a mouse ascites fluid monoclonal antibody [15] Known amounts of purified recombinant AK were included as standards for quantification Results were quantitated usingNIH IMAGE software

In vitro phosphorylation reactions All reactions were carried out at 30C in a final volume of

at least 30 lL containing 10 lM substrate, 100 lM ATP, and 0.2 mCiÆmL)1[32P]ATP[cP] The PKC reaction solu-tion included 20 mM MOPS, pH 7.2, 25 mM b-glycerol phosphate, 1 mMsodium orthovanadate, 1 mM dithiothre-itol, 1 mM CaCl2, 10 mM MgCl2, 0.1 mgÆmL)1 phospho-tidylserine, 0.01 mgÆmL)1 diacylglycerol PKA reactions were conducted in 50 mMHEPES, pH 7.4, 1 mMEGTA,

10 mMmagnesium acetate, and 0.2 mgÆmL)1bovine serum albumin; PKG reactions in 40 mM Tris/HCl, pH 7.4,

20 mM magnesium acetate, and 3 lM cGMP; MAPK reactions in 50 mMTris/HCl, pH 7.4, 10 mMMgCl2, and

20 mM EGTA; and Cdk5 reactions in 30 mM MOPS,

pH 7.2, and 5 mMMgCl2 For CaMKII, CK1, CK2, and Cdk1, reaction buffers provided by the suppliers were used

As positive controls, reactions were conducted using proteins previously defined as physiological substrates for each protein kinase Specifically, protein phosphatase inhibitor-1 was used in the PKA, MAPK, Cdk1 and

Cdk5 reactions [13,16]; myelin basic protein in the PKC

reaction [17]; histone H1 in the PKG reaction [18]; tyrosine

hydroxylase in 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 conditions In all

cases, [32P]phosphate incorporation was assessed by SDS/

PAGE (15% acrylamide) and PhosphorImager analysis

To calculate reaction stoichiometries, radiolabeled reaction

products and radioactive standards were quantitated using

IMAGEQUANTsoftware (Amersham Biosciences) Standards

consisted of 5 lL aliquots of serial dilutions of the reaction

mixtures, with the moles of phosphate defined using the

ATP concentration 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 32P-labeled phospho-mAK

were excised, rehydrated, washed, and incubated at 37C for

20 h in 50 mMammonium bicarbonate, pH 8.0, containing

75 ngÆmL)1trypsin The supernatant containing the tryptic

digestion products was lyophilized and the lyophilate washed

up to four times with water and once with electrophoresis

buffer, pH 3.5 (10% acetic acid, 1% pyridine; v/v/v) The

final lyophilate was resuspended in electrophoresis buffer,

pH 3.5, and 10% of the total volume was set 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 Resulting phosphopeptide maps were

visualized by autoradiography Smearing was consistently

observed in the first dimension when microcrystalline

cellulose TLC plates (Analtech) were used After testing a

number of different 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 problem 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 aliquot set aside in

the previous step was hydrolyzed at 100C for 1 h in 6M

HCl under an N2atmosphere 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¢- and 3¢-UTRs of human AK (hAK) [8] Ten randomly selected clones were subse-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 involved 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 date and that this existing sequence corres-ponded to an N-terminal truncated form [22] That 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 subcloned 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 apparent molecular weight of 45 and 43.5 kDa for polyhistidine-tagged recombinant mAK-L and mAK-S, respectively (Fig 2A) Moreover, in vitro AK activity assays demon-strated that the two recombinant proteins were enzymati-cally active, efficiently catalyzing the phosphorylation of Ado to AMP (for mAK-L, Km¼ 20 ± 4 nM; Vmax¼

16 ± 1.6 nmolÆmin)1Ælg)1, n¼ 8) (Fig 2B) No significant difference was noted between mAK-L and mAK-S with respect to Kmand Vmax(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 brain and peripheral 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

brainstem Moreover, in most tissue homogenates, two protein species of different molecular mass were detectable with this antibody These two closely migrating bands are

Fig 1 Deduced amino acid sequence alignment of the long and short isoforms of human and mouse AK Sequences are divided into two domains (yellow and green blocks) based on crystal structure for the shorter splice variant of human AK [7] Yellow blocks constitute the catalytic domain The regulatory domain (green blocks) folds over the catalytic domain and forms a hydrophobic pocket for Ado phosphorylation Residues that make close contacts with Ado are indicated by red letters Green letters denote residues that form the ATP/secondary Ado-binding site One Mg2+ ion is coordinated between the active site and this ATP-binding site by hydrogen-bonding interactions mediated by 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 of cell lysates at 10 000 g; P10,

insoluble pellet after centrifugation of cell lysates at 10 000 g; FT,

flow-through, or unbound protein, after 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 recombinant mAK-S for quantifi-cation purposes Recombinant mAK-S standards have a higher apparent molecular weight than mAK-S in the sample lanes due to N-terminal polyhistidine tags Quantification of relative AK abun-dance in each tissue examined is also shown.

likely to represent the long and short isoforms of the

enzyme Many of the tissues included in this analysis 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 testis 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 the 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 the presence of putative phosphorylation sites

for several protein kinases, including PKA, PKC, CaMKII,

CK1 and CK2 (http://scansite.mit.edu) To investigate the

possibility that AK activity may be regulated by protein

phosphorylation, a panel of these protein kinases and others

was tested for ability to phosphorylate recombinant 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, and Cdk5 However, maximal reaction

stoi-chiometries were 0.007, 0.008 and 0.003 molÆmol)1,

respectively, precluding subsequent biochemical analysis

Similar results were obtained when mAK-S was used as the

putative protein kinase substrate (data not shown) In

contrast, all control substrates were efficiently

phosphoryl-ated by their respective protein kinases At 60 min, protein

phosphatase inhibitor-1 was phosphorylated to a

stoichio-metry of 0.99, 0.31, 0.61 and 0.97 molÆmol)1 by PKA, MAPK, Cdk1 and Cdk5, respectively Consistent with the existence of multiple PKC sites in myelin basic protein [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)1by CaMKII, and DARPP-32

to a stoichiometry 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 lM AK displayed linear conversion of substrate to phosphoprotein over the first

5 min and near saturation by 20 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 Km of 6.9 ± 1.1 lMand Vmaxof 68 ± 3 lmolÆmin)1Ælg)1for this reaction (Fig 5C, n¼ 8) Similar values were obtained using the short isoform as a substrate (data not shown)

A phosphopeptide map of mAK-L preparatively phos-phorylated by PKC showed two major spots (Fig 5D, first panel) Phosphoamino acid analysis of the same material indicated that this phosphorylation occurs at serine (Fig 5D, second panel) Similar results were obtained with mAK-S (data not shown)

Mutation of four PKC consensus sites to alanine (Ser48Ala, Ser85Ala, Ser272Ala, and Ser328Ala) had no

Fig 4 Phosphorylation of recombinant mAK-L by a panel of protein kinases PKC, PKA, PKG, MAPK, CaMKII, CK1, CK2, Cdk1 and Cdk5 were used to phosphorylate mAK-L as well as control substrates in vitro I1, protein phosphatase inhibitor-1; MBP, myelin basic protein; H1, histone H1;

TH, tyrosine hydroxylase; D32, DARPP-32 The multiple H1 bands visible by Coomassie stain and PhosphorImager analysis of the PKG reaction correspond to degradation products of the protein The two higher molecular weight species appearing as radiolabeled bands above the AK signal in the CaMKII reaction represent autophosphorylation of the different CaMKII isoforms present in this commercial enzyme preparation 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.

effect on the phosphorylation of mAK-L by PKC (Fig 5E).

Mutants generated at the remaining nine conserved serine

residues were also 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, thrombin site indica-ted by forward slash) substantially diminished the PKC-dependent phosphorylation of mAK-L (Fig 5F) Similarly, mutation of the five N-terminal serine residues in the affinity tag sequence of mAK-L resulted in a fusion protein that was

no longer phosphorylated by PKC (Fig 5G)

In this study, we report the cDNA and deduced amino acid

sequences for two isoforms of AK expressed in the mouse

brain To date, the existence of AK splice variants has been

described in several mammalian species, namely mouse

[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 throughout

the brain, in addition to pockets of high neuronal expression

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 other 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 transcriptional and/or translational

regula-tion, rate of turnover, subcellular localizaregula-tion, or association

with as yet undefined regulatory factors

The most abundant nucleoside kinase in mammals, AK

has emerged as a key enzyme in the regulation of interstitial

Ado and intracellular adenylate levels in the CNS and periphery AK-knockout mice undergo normal embryo-genesis, but develop microvesicular hepatic steatosis within

4 days of birth, dying by the end of two weeks with fatty liver [25] Conditional gene knockout may therefore provide

a useful tool for studying the role of AK in other tissues at later developmental time points Notably, inhibitors of AK have already been used effectively to elevate extracellular Ado levels [28] and shown some 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 the importance of AK in Ado homeostasis, the question of whether AK activity is regulated remains largely unanswered Insulin has been shown to induce AK expres-sion in rat lymphocytes [32] Studies in the brain have suggested that AK activity exhibits diurnal variations [33,34] Most recently, a kainic acid-induced mouse model of epilepsy was used to demonstrate that AK expression is up-regulated

in the epileptic hippocampus, coincident with pronounced astrogliosis, which may 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 modulated in a number of systems, most likely through the transcriptional and/or translational control of AK expression However, it remains unclear whether post-translational mechanisms also exist for the direct regulation

of AK activity A better understanding of AK regulation, with regard to gene expression as well as protein structure and function, may reveal specific signaling pathways that control this enzyme and provide new targets for drug design

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-translational 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 Although CaMKII, CK1, and Cdk5 were found to phosphorylate AK weakly, the maximal stoichiometry achieved in these reactions remained below 0.01 molÆmol)1 These low levels of phosphorylation effect-ively preclude further biochemical characterization, such as the identification of phosphorylation sites or the assessment

of a possible effect of AK phosphorylation on AK activity Furthermore, they strongly suggest that these reactions are unlikely to occur in vivo or otherwise be physiologically relevant Taken together, our findings indicate that AK is unlikely to be regulated by any of the protein kinases investigated here

On the other hand, it is important to note that our screen was by no means exhaustive, and although the protein kinases tested in this study represent most of the principal 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 plotted values for phosphate incorporation (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 and mAK-S Reaction times are indicated at the top.

(C) Lineweaver–Burke analysis of PKC phosphorylation of mAK-L.

The plot represents the results of four reactions conducted under

identical linear conditions using duplicate samples (D)

Phosphopep-tide mapping (PPM) and phosphoamino acid analysis (PAAA) of

mAK-L preparatively phosphorylated by PKC (E) Site-directed

mutagenesis analysis of PKC phosphorylation 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 at four PKC consensus sites

for 60 min The stoichiometry 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 effect of five

Ser fi Ala mutations in the N-terminal affinity tag on the

phos-phorylation of mAK-L by PKC The two panels represent SDS/PAGE

analysis of Coomassie-stained (top) and32P-labeled (bottom) mAK-L

and a quintuple mutant of mAK-L (5XS>A) incorporating

serine-to-alanine mutations at the five serine residues of the N-terminal affinity

tag Reaction times are indicated at the top.

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 of

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 be 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 tyrosine 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 Scientist Training Program at UT

Southwestern Medical Center (BS), the National Cancer Institute

(JS), the National Institute of Drug Abuse (JAB), the National Alliance

for Research on Schizophrenia and Depression (JAB), the National

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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