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: 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 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 (http://scansite.mit.edu). 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 (http://scansite.mit.edu). 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. 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Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation Bogachan Sahin 1 , Janice W. Kansy 1 ,. hAK, human adenosine kinase; mAK, mouse adenosine kinase. Note: Nucleotide sequence data for the long and short isoforms of mouse adenosine kinase are available