Identification of Two Active Functional Domains of Human Adenylate Kinase 5

FEBS Letters 583 (2009) 2872–2876

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Identiﬁcation of two active functional domains of human adenylate kinase 5

Nicola Solaroli *, Christakis Panayiotou, Magnus Johansson, Anna Karlsson

Department of Laboratory Medicine, F68, Karolinska Institute, S-14186 Huddinge, Sweden article info abstract

Article history: A full length cDNA that partially corresponded to human adenylate kinase 5 (AK5) was identified Received 6 July 2009 and shown to encode for two separate domains. The full length protein could be divided in two dis- Revised 23 July 2009 tinct functional domains, a previously unidentified domain of 338 amino acids and a second domain Accepted 27 July 2009 of 198 amino acids that corresponded to the protein characterized as AK5, now called AK5p2. The Available online 3 August 2009 first domain, AK5p1, phosphorylated AMP, CMP, dAMP and dCMP with ATP or GTP as phosphate Edited by Miguel De la Rosa donors similarly to AK5p2. Our data demonstrate that human AK5 has two separate functional domains and that both have enzymatic activity. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Adenylate kinase Nucleotide metabolism

1. Introduction chondrial matrix [6]. AK5 is a cytosolic isoform initially detected exclusively in brain, when only a limited number of tissues were Phosphoryl transfer reactions are critical in cellular metabolism investigated, but low levels of AK5 expression is also detected in and to generate and modulate metabolic signals [1]. Enzymes that other tissues [7]. The recently characterized isoform AK6 is a nucle- catalyze the reversible phosphoryl transfer between nucleoside tri- ar enzyme but without any experimentally confirmed expression phosphates and nucleoside monophosphates, resulting in the con- profile [8]. AK7 is reported to be associated with ciliar function version of the monophosphate to its corresponding diphosphate since a mutation in the AK7 gene in a rat model recently was re- form are named nucleoside monophosphate kinases (NMPKs). ported to cause ciliar dysfunction and a phenotype similar to the Based on the differences in substrate specificity, NMPKs are human syndrome called primary ciliary dyskinesia [9]. divided into subgroups consisting of adenylate kinases (AKs), Thus, the AK isoforms have been found in different intracellular uridylate–cytidylate (UMP–CMP), guanylate kinases and thymidyl- compartments and with tissue specific levels of expression to serve ate (dTMP). While there are several AKs and guanylate kinases the specific needs of different cellular functions. The AKs also differ present in mammalian tissues there was until recently only one in molecular weight, kinetic properties and nucleotide specificity identified UMP-CMP kinase and one dTMP kinase. A second [10]. The common substrate recognized by all AKs is AMP but UMP–CMP kinase has been identified and shown to have a mito- AK5 and AK6 also phosphorylate CMP and dCMP [11,8]. In addition, chondrial location and there still may be a mitochondrial dTMP all isoforms are coded by separate genes, localized to different kinase to be found [2,3]. chromosomes. In the present study we have identified yet another Adenylate kinase catalyzes the nucleotide phosphoryl exchange specific characteristic of one of the AK isoforms. We demonstrate reaction AMP + ATP 2 ADP, and thus regulates adenine nucleotide that the human AK5 has two separate functional domains and that ratios in different intracellular compartments. There are seven dif- both domains show similar adenylate kinase activity. ferent adenylate kinases identified and six of these enzymes have been thoroughly characterized. AK1, AK2 and AK3 are expressed 2. Materials and methods at higher levels than the other AKs and have a more general expression in most tissues although AK1 is dominant in skeletal 2.1. Cloning and sequencing muscle [4]. AK2 is the main mitochondrial adenylate kinase and is located in the intermembrane space [5], while the other mito- The IMAGE clone 4816351 was from Geneservice. The encoded chondrial adenylate kinases, AK3 and AK4 are located in the mito- proteins (AK5, AK5p1 and AK5p2) were expressed in Escherichia coli. Nine primers flanking the three possible open reading frames were designed. Three forward primers for pET16b vector were: 50- * Corresponding author. Address: Karolinska Institute, Department of Laboratory TCGTCATATGCTCGAAATGAACACCAACGATGCCAA-30,50-TCGTCAT- Medicine, F68, Karolinska University Hospital, S-14186 Huddinge, Sweden. Fax: +46 ATGCTCGAAATGTGTTCTAAGCCCGAAGA-30,50-TCGTCATATGCTCG- 8 7795383. 0 E-mail address: [email protected] (N. Solaroli). AAATGGGAGGTTTCATGGAAGA-3 . Two reverse primers for pET16b

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.07.047 N. Solaroli et al. / FEBS Letters 583 (2009) 2872–2876 2873 vector were: 50-CAGCCGGATCCTCGATTCAGAAAATAGAGTCAATAG- sheets were autoradiographed (Personal Molecular Imager FX) 30,50-CAGCCGGATCCTCGATTTAAGTGTCCTCTCCAAATA-30. and analyzed/edited with Quantity One v4.6 software. For Two forward primers for pEGFP-N1 vector were: 50-CTCAA- Michaelis–Menten kinetics, non-radiolabeled products were sepa- GCTTCGAATTTATGAACACCAACGATGCCAA-30,50-CTCAAGCTTCG- rated and quantified by reversed-phase HPLC using a ChromolithTM AATTTATGTGTTCTAAGCCCGAAGA-30. Three reverse primers for column as described [13]. pEGFP-N1 vector were: 50-GTCGACTGCAGAATTCGGAAAATAGAGT- CAATAGCTG-30,50-GTCGACTGCAGAATTCGTTAAGTGTCCTCTCCAA- 2.4. In vitro translation ATA-30,50- GTCGACTGCAGAATTCGCAGTCCATTCAAAAGGCTCT-30. The PCR products were cloned into the XhoI site of pET-16b vec- Fresh precursor proteins (AK5, AK5p1 and AK5p2) were synthe- tor or the EcoRI site of pEGFP-N1 vector using the In-Fusion Dry- sized (PURExpress In Vitro Protein Synthesis Kit) in the presence of Down Cloning. The plasmids were sequenced by MWG-Biotech to [35S]methionine following the manufacturer’s instructions. For verify the DNA sequences. protein quantification 2 ll of each reaction was loaded in a 4– 12% SDS–PAGE gel. The gel was washed in Gel-Dry Drying solution, 2.2. Expression and purification dried with Dry Easy Membrane and used for autoradiographic detection. The plasmids were transformed into the E. coli strain Rosetta (DE3) and single colonies inoculated into LB-medium with 2.5. Cell culture and transfection 100 lg/ml ampicillin. The bacteria were grown at 37 °C and protein expression was induced at OD600 0.8 with 1 mM isopropyl- HeLa cells were seeded in Ibidi l-Slides and cultured in Dul- 1-thio-b-D-galacto-pyranoside for 12 h at 27 °C. The expressed pro- becco’s Modified Eagle’s Medium with 10% (v/v) fetal bovine serum, teins were purified using TALON Metal Affinity Resin as described 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were grown by the manufacturer. Purity of the protein was verified by SDS– at 37 °C in a humidified incubator with 5% CO2. Plasmids were PAGE gel and concentration was determined with Bradford Protein transfected into cells using FuGENE HD transfection reagent as de- Assay using BSA as standard. The protein was aliquoted and stored scribed by the manufacturer. GFP fluorescence was observed in a at 80 °C. Leica DMI-6000B with Leica Application Suite AF software v1.8.

2.3. Enzyme assay 3. Results

The substrate specificity of the purified enzymes was assayed 3.1. Identification of two functional domains of AK5 by thin layer chromatography (TLC) as described [12]. The assay 32 was performed with 1 lCi [c- P]ATP (Perkin–Elmer), recombinant A cDNA with the Walker A motif that partially corresponded to enzyme and concentrations of xMP or dxMP indicated. The TLC the previously characterized human adenylate kinase 5 (AK5) was

Fig. 1. (A) Map of the full length AK5 protein (two arrows show where the two variants start). (B) Alignment of AK5p1 and AK5p2 with human adenylate kinases (black boxes indicate completely conserved amino acid residues and different shades of grey boxes indicate different levels of conserved residues. * Indicate amino acids involved in substrate binding or in the active site pocket definition). (C) Phylogenetic tree of the human adenylate kinases. 2874 N. Solaroli et al. / FEBS Letters 583 (2009) 2872–2876 identified in a human genome library. The gene was localized on chromosome 1 and consisted of 14 exons with a total length of 277.92 kb. Two main variants were reported (BC036666 and BC033896) which differed in the N-terminus where one variant had an additional short sequence of 26 amino acids (TAG) (Fig. 1A). The full length protein of 536 amino acids contained two distinct functional domains; a previously unidentified domain of 338 amino acids and a second domain of 198 amino acids that corresponded to the protein already characterized as AK5 [7]. The open reading frame contained three main ATG start codons, which with the common single stop codon, could result in transcripts of different sizes (Fig. 1A). The sequence of the two distinct proteins suggested to be closely related to known AKs. The first domain Fig. 2. Affinity assay of AK5p1 with variable concentrations of AMP or dAMP in was named AK5p1 and the second domain, previously considered [c-32P]ATP phosphotransferase assay. (The control reaction, lane C, is without to be the full length AK5, was renamed AK5p2. AK5p1, differently addition of substrate.) from AK5p2, contained 107 amino acids after the second ATG but without any recognizable targeting signal or other known functional domain. After the AK5p1 domain there was a second stretch bands showed an intensity that was proportional to the 35 of 60 amino acids before the ATG of AK5p2. The sequence compar- [ S]methionine present in each sequence (Fig. 3A). The three en- ison between AK5p1 and AK5p2 showed an identity of 36% and a zymes, at similar concentrations, were tested for their capacity to general homology of 64% (Fig. 1B). AK5p1 also showed an identity phosphorylate AMP, dAMP, CMP and dCMP. Full length AK5 of 35%, 24%, 26% and 27%, respectively, with AK1, AK2, AK3 and showed an increased capacity to phosphorylate AMP as compared AK4, as represented by the phylogenetic tree (Fig. 1C). The highest to the single subunits (Fig. 3B), while with dAMP and CMP as phos- level of identity of the novel AK5p1 domain with known adenylate kinases was within the glycin rich p-loop structure, the NMP binding domain and the LID domain. The two domain gene structure of AK5 was also found to be conserved in other mammals (in mice the predicted amino acid sequence has 94% identity, data not shown).

3.2. Expression and characterization of the different forms of AK5

The full length protein and the AK5p1 protein domain alone were expressed in E. coli, with and without the initial fragment of 26 amino acids, as described in Methods. For an initial screening of substrate specificity AMP, CMP, GMP, UMP, dAMP, dCMP, dGMP, TMP and dUMP were tested in a TLC assay using ATP or GTP as phosphate donors (Table 1). AK5p1 phosphorylated AMP, CMP, dAMP and dCMP with ATP or GTP as phosphate donors. At the assay conditions used, and at lower concentrations of substrate, AK5p1 showed generally a higher affinity for AMP compared to dAMP (Fig. 2). The relative efficiency of CMP and dCMP was 15% compared to AMP. No significant differences were found between the enzymes with or without the initial 26 amino acid fragment. To investigate if there were synergic effects of the two subunits the full length AK5, AK5p1 and AK5p2 were synthesized by in vitro translation. We used [35S]methionine to visualize the synthesized proteins and to compare the relative amount. As expected the

Table 1 Substrate speciﬁcity of the recombinant enzymes using ATP or GTP as phosphate donor.

Substrate (1 mM) Phosphate donor (5 mM) AK5 AK5p1 AK5p2a ATP GTP ATP GTP ATP GTP AMP +++ ++ +++ ++ +++ +++ CMP +++++++ GMP UMP Fig. 3. (A) SDS–PAGE gel of the in vitro synthesized proteins. Lane 1, 2, 3 contain, dAMP +++ ++ +++ ++ +++ respectively, AK5 (63.2 kDa), AK5p1 (41.3 kDa) and AK5p2 (21.9 kDa). (B) TLC assay dCMP + + + + + of AK5 (lane 1, 4, 7), AK5p1 (lane 2, 5, 8) and AK5p2 (lane 3, 6, 9) without substrate dGMP or with AMP/dAMP. (Relative levels of phosphorylation expressed in relation to% of TMP AK5 with AMP/dAMP phosphorylation.) (C) TLC assay of AK5 (lane 1, 4, 7), AK5p1 dUMP (lane 2, 5, 8) and AK5p2 (lane 3, 6, 9) without substrate or with CMP/dCMP. (Relative levels of phosphorylation expressed in relation to percentage of AK5 with a Van Rompay et al. [7]. CMP/dCMP phosphorylation.) N. Solaroli et al. / FEBS Letters 583 (2009) 2872–2876 2875 phate acceptor the difference of full length AK5 and the AK5p1 and TAG produced proteins exclusively localized into the cytosol AK5p2 enzymes was minimal (Fig. 3B and C). (Fig. 4B).

The Km and Vmax kinetic parameters for the substrates AMP and dAMP for AK5p1 showed the highest affinity for AMP with a Km of 4. Discussion 172 lM while the Km for dAMP was >2000 lM. The Vmax for AMP as substrate was 226 pmol/lg/min while the Vmax for dAMP was only The many members of the AK family are likely to reflect not 39 pmol/lg/min, resulting in AMP to be more than 60-fold more only the necessity for cells in general to maintain cellular adenine efficient as substrate for AK5p1 as compared to dAMP (Table 2). nucleotide pools but also that a fine tuning of this demand is We were not able to measure the AK5p1 kinetic parameter for important for specialized cell functions. The differences in enzy- the substrates CMP or dCMP using ATP as phosphate donor, and matic activity, subcellular location, tissue distribution and expres- not for AMP, dAMP, CMP or dCMP using GTP as phosphate donor. sion levels suggest both general and very specialized needs of AK activity in specific cellular processes. Recent studies have shown 3.3. AK5. subcellular localization the involvement of AKs in severe medical conditions [10]. Heart function in AK1 knockout mice displayed accelerated loss of car- The subcellular prediction software used did not indicate any diac contractions on ischemic challenge [14]. Mutations of the gene putative targeting signal in the cDNA sequences (data not shown). encoding for AK2 have been identified in individuals with reticular This was confirmed by in vivo expression of the enzyme as fusion dysgenesis, an autosomal recessive form of severe combined protein with the Green fluorescent protein (GFP). The previously immunodeficiency [15]. Another recent study has shown that sen- studied AK5p2 was shown to have a cytosolic/nuclear localization sorineural deafness is associated with reticular dysgenesis caused and therefore we decided to investigate the full length protein and by AK2 deficiency [16]. the AK5p1 with and without the 26 amino acid initial TAG In contrast to the multi-tissue expression profiles of most other (Fig. 4A). The cells transfected with wild type GFP, full length AKs, AK5 has the unique property of being highly expressed in AK5 and AK5p1 with the initial TAG showed a uniform distribution brain [7]. Autoantibodies targeted against AK5 have been impli- in the cytosol/nucleus; on the other side the lacking of the 26 aa cated in two cases of limbic encephalitis [17]. In the present study we analyzed two transcript variants of the AK5 gene; the first one represents a shorter transcript (3526 bp) that encodes the longest Table 2 Kinetic properties of recombinant AK5p1 with ATP as phosphate donor (2.5 mM) and isoform (562 aa) and the second transcript has instead a different, 0 AMP or dAMP as acceptor. (Data from 4 to 5 measurements ± S.E.) and longer 5 sequence, and use a downstream start codon, as compared to variant 1, which leads to a shorter isoform (536 aa). Iso- Substrate K (lM) V (pmol/lg/min) V /K m max max m form 1 has 26 amino acids extra in the N-terminal compared to AMP 172 ± 29 226 ± 6 1.31 isoform 2, while the remaining part of the sequence is identical dAMP >2000 39 ± 3 <0.02 in both isoforms. These 26 amino acids and the different isoforms

Fig. 4. (A) Plasmid vector constructs (pEGFP-N1) used to express AK5, AK5 w/o TAG, AK5p1 and AK5p1 w/o TAG. (B) HeLa cells expressing pEGFP-N1 (control), AK5, AK5 w/o TAG, AK5p1 and AK5p1 w/o TAG (cells were contra-stained with Mitotracker). 2876 N. Solaroli et al. / FEBS Letters 583 (2009) 2872–2876 have been analyzed with different software for the prediction of References protein subcellular localization, but none of them could identify possible targeting signals, such as the nuclear localization [1] Okajima, T., Fukamizo, T., Goto, S., Fukui, T. and Tanizawa, K. (1998) Exchange of nucleoside monophosphate-binding domains in adenylate kinase and UMP/ sequence (NLS). CMP kinase. J. Biochem. 124, 359–367. As far as enzymatic activity is concerned, both the full length [2] Van Rompay, A.R., Johansson, M. and Karlsson, A. (1999) Phosphorylation of AK5 and its first functional domain AK5p1 phosphorylated AMP, deoxycytidine analog monophosphates by UMP–CMP kinase: molecular dAMP, CMP and dCMP with ATP and GTP as phosphate donors. characterization of the human enzyme. Mol. Pharmacol. 56, 562–569. [3] Xu, Y., Johansson, M. and Karlsson, A. (2008) Human UMP–CMP kinase 2, a The previously characterized AK5p2 phosphorylated AMP, CMP novel nucleoside monophosphate kinase localized in mitochondria. J. Biol. and dAMP when ATP was used as phosphate donor and AMP, Chem. 283, 1563–1571. CMP and dCMP with GTP as phosphate donor. Interestingly, [4] Janssen, E., de Groof, A., Wijers, M., Fransen, J., Dzeja, P.P., Terzic, A. and Wieringa, B. (2003) Adenylate kinase 1 deficiency induces molecular and AK5p2 cannot phosphorylate dAMP in the presence of GTP which structural adaptations to support muscle energy metabolism. J. Biol. Chem. is different from AK5p1. Our results show that the substrate spec- 278, 12937–12945. ificity of AK5, in contrast to all the human AKs characterized so far, [5] Fukami-Kobayashi, K., Nosaka, M., Nakazawa, A. and Go, M. (1996) Ancient divergence of long and short isoforms of adenylate kinase: molecular is dependent on the phosphate donor. All the other AKs have a sub- evolution of the nucleoside monophosphate kinase family. FEBS Lett. 385, strate specificity that is independent of the phosphate donor as 214–220. they phosphorylate predominantly AMP irrespective of phosphate [6] Noma, T., Fujisawa, K., Yamashiro, Y., Shinohara, M., Nakazawa, A., Gondo, T., Ishihara, T. and Yoshinobu, K. (2001) Structure and expression of human donor present. mitochondrial adenylate kinase targeted to the mitochondrial matrix. The AKs are characterized by their high level of conservation in Biochem. 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The high degree of identity and close evolutionary Mutation of murine adenylate kinase 7 underlies a primary ciliary dyskinesia relationship of AK5p1 and AK5p2 with AK1 is clearly shown by a phenotype. Am. J. Respir. Cell Mol. Biol. 40, 305–313. [10] Dzeja, P. and Terzic, A. (2009) Adenylate kinase and AMP signaling networks: phylogenetic tree analysis of all the known human adenylate metabolic monitoring, signal communication and body energy sensing. Int. J. kinases (Fig. 1C). Mol. Sci. 10, 1729–1772. Although a high level of similarity between the different iso- [11] Van Rompay, A.R., Johansson, M. and Karlsson, A. (2000) Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside forms of adenylate kinases is a dominant feature of this enzyme monophosphate kinases. Pharmacol. Ther. 87, 189–198. family, important differences have also been shown in previous [12] Lust, J. and Sahud, M.A. (1972) Contributions of anion-exchange thin-layer studies. In the present study we have identified yet another spe- chromatography to the study of free nucleotides in tissues. J. Chromatogr. 71, 127–133. cific characteristic of one of the AK isoforms. Our data demonstrate [13] Curbo, S., Amiri, M., Foroogh, F., Johansson, M. and Karlsson, A. (2003) The that human adenylate kinase 5 has two separate functional do- Drosophila melanogaster UMP–CMP kinase cDNA encodes an N-terminal mains and that both domains have similar adenylate kinase activ- mitochondrial import signal. Biochem. Biophys. Res. Commun. 311, 440– 445. ity. The advantage of different activities in one single enzyme is not [14] Pucar, D. et al. (2002) Adenylate kinase AK1 knockout heart: energetics and obvious, but the property of having two transcripts from the AK5 functional performance under ischemia-reperfusion. Am. J. Physiol. Heart Circ. gene, resulting in one or two functional and independently active Physiol. 283, H776–H782. proteins, may be physiologically significant. To our knowledge this [15] Pannicke, U. et al. (2009) Reticular dysgenesis (aleukocytosis) is caused by mutations in the gene encoding mitochondrial adenylate kinase 2. Nat. Genet. is the only AK with this particular feature. In the nucleotide metab- 41, 101–105. olism of Trypanosoma brucei there is gene duplication with tandem [16] Lagresle-Peyrou, C. et al. (2009) Human adenylate kinase 2 deficiency causes a repeats of the adenosine kinase gene. However, a comparison with profound hematopoietic defect associated with sensorineural deafness. Nat. Genet. 41, 106–111. this parasite may be difficult since it lacks the de novo synthesis [17] Tuzun, E., Rossi, J.E., Karner, S.F., Centurion, A.F. and Dalmau, J. (2007) and has a different pathway of nucleotide metabolism [18]. Adenylate kinase 5 autoimmunity in treatment refractory limbic encephalitis. If this novel mechanism will prove to regulate the level of AK J. Neuroimmunol. 186, 177–180. [18] Luscher, A., Onal, P., Schweingruber, A.M. and Maser, P. (2007) Adenosine activity in specific cells will be a subject for future investigations. kinase of Trypanosoma brucei and its role in susceptibility to adenosine antimetabolites. Antimicrob. Agents Chemother. 51, 3895–3901. Acknowledgements

This work was supported by grants from the Swedish Cancer Society and the Swedish Research Council.