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A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

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A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties
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  Biochem. J. (2014)  457 , 323–334 (Printed in Great Britain) doi:10.1042/BJ20131174  323 A robust methodology to subclassify pseudokinases based on theirnucleotide-binding properties James M. MURPHY* † 1,2 , Qingwei ZHANG ‡ , Samuel N. YOUNG*, Michael L. REESE § 3 , Fiona P. BAILEY¶, Patrick A. EYERS¶,Daniela UNGUREANU  , Henrik HAMMAREN  , Olli SILVENNOINEN  , Leila N. VARGHESE* † , Kelan CHEN* † , Anne TRIPAYDONIS*,Natalia JURA**, Koichi FUKUDA †† , Jun QIN †† , Zachary NIMCHUK ‡‡ 4 , Mary Beth MUDGETT §§ , Sabine ELOWE¶¶,Christine L. GEE  , Ling LIU*** 5 , Roger J. DALY*** 5 , Gerard MANNING ††† , Jeffrey J. BABON* †  and Isabelle S. LUCET ‡ 1,2 *The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia † Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia ‡ Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia § Department of Microbiology and Immunology, Stanford University, Stanford, CA 24305-5124, U.S.A.¶Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, U.K.  School of Medicine and Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere 33014, Finland**Cardiovascular Research Institute and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158-9001, U.S.A. †† Department of Molecular Cardiology, Lerner Research Institute, NB20, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A. ‡‡ Department of Biology, California Institute of Technology, Pasadena, CA 91125, U.S.A. §§ Department of Biology, Stanford University, Stanford, CA 24305-5020, U.S.A.¶¶Centre de Recherche du Centre Hospitalier Universitaire de Qu´ebec and and Facult´e de M´edicine, D´epartement de P´ediatrie, Universit´e Laval, Qu´ebec G1V 4G2, Canada  Australian Synchrotron, Clayton, Victoria 3168, Australia***Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia ††† Genentech, 1 DNA Way, MS 93, South San Francisco, CA 94010, U.S.A. Protein kinase-like domains that lack conserved residues knownto catalyse phosphoryl transfer, termed pseudokinases, haveemerged as important signalling domains across all kingdomsof life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinasedomains have been attributed unexpected catalytic functions,often amid controversy. We established a thermal-shift assay as abenchmark technique to define the nucleotide-binding propertiesof kinase-like domains. Unlike  in vitro  kinase assays, this assay isinsensitive to the presence of minor quantities of contaminatingkinasesthatmayotherwiseleadtoincorrectattributionofcatalyticfunctions to pseudokinases. We demonstrated the utility of thismethod by classifying 31 diverse pseudokinase domains into fourgroups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotidebinding enhanced by cations. Whereas nine pseudokinases boundATP in a divalent cation-dependent manner, over half of thoseexamined did not detectably bind nucleotides, illustrating thatpseudokinase domains predominantly function as non-catalyticprotein-interaction modules within signalling networks and thatonly a small subset is potentially catalytically active. We proposethat henceforth the thermal-shift assay be adopted as the standardtechnique for establishing the nucleotide-binding and catalyticpotential of kinase-like domains.Key words: nucleotide binding, non-catalytic protein-interactiondomain, protein kinase, pseudoenzyme, pseudokinase. INTRODUCTION Although first described in sea urchins [1], examples of pseudokinasedomainsarenowknownacrossallkingdomsoflife.In previous years, essential functions in signal transduction havebeenattributedtotheseproteinkinase-likedomains,principallyasmodulators of the catalytic activities of   bona fide  protein kinasesor as scaffolding proteins that nucleate the assembly of signallingcomplexes [2–14]. Often amid controversy, several pseudokinasedomains have been reported to exhibit a low catalytic activity,yet the importance of this enzymatic activity to the protein’sbiological function and the ubiquity of this phenomenon acrossall pseudokinases remain unclear.Pseudokinase domains were srcinally predicted to be devoidof any catalytic activity owing to the absence of one or more of the three crucial residues known to catalyse phosphoryl transfer Abbreviations:AMP-PNP,adenosine5 ′ -[ β , γ  -imido]triphosphate;BPK1,bradyzoitepseudokinase1;CASK,calcium/calmodulin-dependentserineproteinkinase; CCK4, colon carcinoma kinase 4; CH2, calponin homology 2; CRN, CORYNE; DAP, N  ′ 2 ′ -(4-aminomethyl-phenyl)-5-fluoro- N  ′ 4 ′ -phenyl-pyrimidine-2,4-diamine; EphB6, Ephrin type-B receptor 6; ErbB3, v-erb-b2 avian erythroblastic leukaemia viral oncogene homologue 3; HER3, human epidermalgrowth factor receptor 3; ILK, integrin-linked kinase; IRAK, interleukin-1-receptor-associated kinase; ITC, isothermal titration calorimetry; JAK, Januskinase; JH, JAK homology; KSR2, kinase suppressor of Ras 2; MLKL, mixed lineage kinase domain-like; NRBP1, nuclear receptor-binding protein 1;PEAK1, pseudopodium-enriched atypical kinase 1; PTK7, protein tyrosine kinase 7; ROP, rhoptry protein kinase; Ror1, receptor tyrosine kinase-like orphanreceptor; RT-PCR, real-time PCR; RYK, receptor-like tyrosine kinase; SgK, sugen kinase; STRAD α , STE20-related kinase adaptor  α ; TARK1, tomato atypicalreceptor-like kinase 1; TRB2, tribbles pseudokinase 2; TYK2, tyrosine kinase 2; ULK4, unc-51 like kinase 4; VRK3, vaccinia-related kinase 3. 1 These authors contributed equally to this work. 2 Correspondence may be addressed to either of these authors (email jamesm@wehi.edu.au or isabelle.lucet@monash.edu). 3 Present address: Department of Pharmacology, University of Texas, Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041,U.S.A. 4 Present address: Department of Biological Sciences, Virginia Tech, 220 Ag Quad Lane, Blacksburg, VA 24061, U.S.A. 5 Present address: Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800,Australia. c  The Authors Journal compilation  c  2014 Biochemical Society    B   i  o  c   h  e  m   i  c  a   l   J  o  u  r  n  a   l  w  w  w .   b   i  o  c   h  e  m   j .  o  r  g  324  J. M. Murphy and others Figure 1 Pseudokinase domains selected for characterization of nucleotide binding ( A ) Schematic cartoon of the eukaryotic protein kinase domain illustrating the amino acid motifs thought to be required for phosphoryl transfer that are usually absent from pseudokinase domains(depictedinthesamestyleas[71]).( B )Purifiedpseudokinasedomains( ∼ 1 µ g)wereresolvedbyreducingSDS/PAGEandthen wereCoomassieBlue-stained.Molecularmassmarkersareindicatedon the left-hand side. In each case, the predominant species present in the preparation is the pseudokinase domain. ( C ) Multiple sequence alignment of canonical catalytic motifs in PKA (proteinkinase A), a model serine/threonine protein kinase, and JAK2(JH1), a model tyrosine kinase, with the 31 pseudokinase domains examined in the present study. Consensus sequences of the catalyticmotifs and the protein kinase secondary structure are depicted as described previously ([72] and [73]).  ϕ , hydrophobic;  δ , hydrophilic;   , large hydrophobic; X, any amino acid. Subdomains arelabelled according to the nomenclature proposed in [15]. The G-loop and the VAIK, HRD and DFG motifs of catalytically active protein kinases and their pseudokinase counterparts are boxed inred, and residues thought to be essential for robust catalytic activity are shaded in green. Details of the species of srcin, domain boundaries and expression strategy are given for each proteinstudied, with further details presented in the Experimental section and Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570323add.htm). It should be noted that equivalent resultswere obtained for BubR1 with the domain boundaries of residues 720–1043 (results not shown). in active protein kinases (Figure 1A). These residues are locatedwithin highly conserved motifs: (i) the VAIK motif, where thelysine residue in the  β 3 strand of the N-lobe positions the ATP α - and  β -phosphates for catalysis; (ii) the HRD motif in thecatalytic loop, which crucially contributes the catalytic asparticacid residue; and (iii) the DFG motif within the activation loopcontributesanasparticacidresidueinvolvedinbindingMg 2 + thatstabilizes the bound ATP [15,16]. More recently, further consid-eration has been given to the integrity of the [GSA]xGxx[GSA]motif (where x is any amino acid) in the glycine-rich loop (‘G-loop’ or ‘P-loop’) that connects the  β 1 and  β 2 strands of thekinase domain N-lobe, since the flexibility of this loop is a keyfactor in promoting ATP binding [17]. Despite the absence of oneor more of the three canonical catalytic residues, previous studieshave reported nucleotide binding and enzymatic activities for anumber of pseudokinases [14,18–21], raising the prospect that itmight be a more general phenomenon of pseudokinases.A prerequisite underlying whether a pseudokinase mightexhibitcatalyticactivityistodefinewhetheritbindstonucleotidesin the presence of divalent cations. Having extensively reviewedthe methods available to characterize the nucleotide-bindingproperties of pseudokinases [22], we used the most robust c  The Authors Journal compilation  c  2014 Biochemical Society  Nucleotide binding by pseudokinases  325 technique, the thermal-shift assay, to examine nucleotide anddivalent cation binding by pseudokinase domains  in vitro .This assay obviates the need for specific fluorescently taggednucleotides, such as TNP-ATP [2(3)- O -(2,4,6-trinitrophenyl)adenosine 5-triphosphate], which have been reported to bindtargets with affinities several hundred-fold greater than ATP insome cases [23] or non-specifically fluoresce in the presenceof BSA (P.A. Eyers and D. Ungureanu, unpublished work).Additionally, the assay monitors the thermal denaturation of themajor protein species, so any minor contaminants, including co-purified protein kinases, that would lead to misleading resultsin radiometric  in vitro  kinase assays do not contribute to thesignal.Usingthisassay,weexperimentallydefinedthenucleotide-and divalent cation-binding properties of a collection of 31diverse pseudokinase domains, including almost half of thehuman pseudokinome, and found that the majority of examinedpseudokinases did not detectably bind nucleotides. The findingsof the present study are consistent with the idea that thesedomainspredominantlyservenon-enzymaticfunctionsincellularsignalling networks. EXPERIMENTALRecombinant protein expression and purification The sources of cDNA templates used in the preparation of theexpression constructs are shown in Supplementary Table S1 (athttp://www.biochemj.org/bj/457/bj4570323add.htm). Insect cellexpressionwasperformedusingSf21cells[exceptforJAK1(JH2)(where JAK is Janus kinase and JH2 is JAK homology 1),TYK2(JH2) (where TYK2 is tyrosine kinase 2) and HER3(human epidermal growth factor receptor 3)/ErbB3 (v-erb-b2avian erythroblastic leukaemia viral oncogene homologue 3),which used Sf9 cells] as described previously [20] starting fromthe pFastBac HTb or pFastBac1 vectors (LifeTechnologies) or apAceBac1-derived vector (ATG Biosynthetics) to generate His 6 -tagged proteins. Bacterial expression constructs were preparedanalogouslytoincorporateN-terminalHis 6  tags.VRK3(vaccinia-relatedkinase3)andCRN(alsoknownasCORYNE)wereclonedinto pProEX HTb (LifeTechnologies); SgK495 [sugen kinase495; also known as STK40 (serine/threonine kinase 40)] andTRB2 [also known as TRIB2 (tribbles pseudokinase 2)] werecloned into pET30 Ek/LIC (Novagen); SgK223 and SgK269[also known as PEAK1 (pseudopodium-enriched atypical kinase1)] were cloned into pCOLD IV (Clontech); and ROP2 (rhoptryprotein kinase 2) and BPK1 (bradyzoite pseudokinase 1) werecloned into pET28a (Clontech), and expression was performedaccording to established protocols [24]. Typically, proteins werepurified using a standardized procedure [20], before Superdex-200 gel-filtration chromatography (GE Healthcare) in 200 mMNaCl, 20 mM Tris/HCl, 10 % (v/v) glycerol and 0.5 mM TCEP[tris-(2-carboxyethyl)phosphine] (pH 8.0).The expression and purification of JAK2(JH1) [25,26], ILK(integrin-linked kinase)– α -parvin CH2 (calponin homology 2)domaincomplex[27],HER3/ErbB3[4],MviN[28],ROP5B I  [29],JAK2(JH2) [20] and STRAD α  (STE20-related kinase adaptor  α )[30] were performed as described in their respective references.Typically, proteins were concentrated to 2 mg/ml or higher, snap-frozen in liquid N 2  and stored at  − 80 ◦ C until required. Proteinconcentrations were estimated on the basis of absorbance at280 nm and the calculated molar absorption coefficients. Thermal-shift assay for nucleotide binding Thermal-shift assays were performed using a Corbett Real TimePCR machine with proteins diluted in 150 mM NaCl, 20 mMTris/HCl (pH 8.0) and 1 mM DTT to 2–5 µ M and assayed withthe appropriate concentration of ligand in a total reaction volumeof 25 µ l. SYPRO Orange (Molecular Probes) was used as a probewithfluorescencedetectedat530 nm.Thetemperaturewasraisedin 1 ◦ C per min steps from 25 ◦ C to 95 ◦ C and fluorescencereadings were taken at each interval. Nucleotide- or cation-binding experiments were assessed relative to a buffer control.Two generic inhibitors, DAP  {  N  ′ 2 ′ -(4-aminomethyl-phenyl)-5-fluoro-  N  ′ 4 ′ -phenyl-pyrimidine-2,4-diamine [31]; supplied bySYNthesis Med Chem }  and VI16832 [32], were assessed relativeto a 2 % (v/v) DMSO control. With the exception of the titrationexperiments,nucleotideconcentrationsof0.2 mM,divalentcationconcentrations of 1 mM, and DAP or VI16832 concentrations of 40 µ M were used in each experiment. For each well, samplefluorescence was plotted as a function of increasing temperature.The melting temperature ( T  m ) corresponding to the midpointfor the protein unfolding transition was calculated by fitting thesigmoidal melt curve to the Boltzmann equation using GraphPadPrism, with  R 2 values of  > 0.99. Data points after the fluorescenceintensity maximum were excluded from fitting. Changes in theunfolding transition temperature compared with the control curve(  T  m ) were calculated for each ligand (nucleotides +− cations). Apositive   T  m  value indicates that the ligand stabilizes the proteinfrom denaturation, and therefore binds the protein. A minimumof two independent assays was performed for each protein andrepresentative data are shown for each. ITC ITC (isothermal titration calorimetry) was performed using aMicroCal instrument (GE Healthcare). ATP (0.5 mM with noadded divalent cations) was titrated into a solution of 50 µ Mhuman MLKL (mixed lineage kinase domain-like) pseudokinasedomain at 25 ◦ C. Both samples were prepared in 200 mM NaCl,20 mM Hepes and 5 % (v/v) glycerol (pH 7.5). RESULTSSelection and preparation of target pseudokinase domains A diverse group of pseudokinase domains were selected for ex-pression, purification and nucleotide-binding studies to representa cross-section of domains lacking different combinationsof catalytic residues from the VAIK, HRD and DFG motifs(Figure 1).Wealsoexamined theproteins Ror1(receptor tyrosinekinase-like orphan receptor 1), BubR1 [also known as BUB1B(BUB1 mitotic checkpoint serine/threonine kinase B)] and RYK(receptor-like tyrosine kinase), which despite containing the keyresidues of the VAIK, HRD and DFG motifs did not exhibitcatalytic activity in earlier studies [33–36]. In addition to the 23human and two mouse pseudokinases chosen for examination,we also analysed the bacterial pseudokinase MviN [28], twoplant pseudokinases, CRN (CORYNE) [37] and TARK1 (tomatoatypical receptor-like kinase 1) [38], and three  Toxoplasmagondii  pseudokinases, ROP5B I  [29], ROP2 [39] and BPK1 [40].In most cases, pseudokinases were overexpressed and purifiedfrom insect cells (Figures 1B and 1C). All were prepared eitherwith a His 6  tag or proteolytically cleaved to remove globularaffinity tags, such as GST and MBP (maltose-binding protein),since these domains will themselves undergo denaturation inthe thermal-shift assay and may obscure detection of ligandbinding to pseudokinase domains. Because some pseudokinasedomains are unstable in their apo forms, we sought to testwhether the assay was amenable to examining ligand binding c  The Authors Journal compilation  c  2014 Biochemical Society  326  J. M. Murphy and others within protein complexes by examining ILK co-expressed andpurified in complex with the CH2 domain of   α -parvin [27,41].Coomassie Blue-stained SDS/PAGE gels of the recombinantproteins examined in the present study are shown in Figure 1(B). A thermal-shift assay for examining nucleotide and cation binding Initially, we established an assay to evaluate nucleotide andcation binding to pseudokinase domains. The assay, based onan earlier method [42], monitors the thermal denaturation of atest protein by measuring the fluorescence arising from bindingof the dye SYPRO Orange to hydrophobic patches that becomeexposed during denaturation. Ligand binding to a protein isknown to enhance a protein’s thermal stability, which manifestsin an elevated melting temperature ( T  m ) that can be used tomeasure ligand binding. Relative to many other methods thathave previously been explored to characterize nucleotide bindingto kinase-like domains [22], the thermal-shift assay offers thefollowing advantages: (i) modest quantities of recombinantprotein (2–5 µ g per condition) can be used; (ii) an RT-PCR (real-time PCR) instrument, a common piece of laboratory equipment,can be used; (iii) depending upon the RT-PCR instrument, 96or 384 conditions can be tested simultaneously; (iv) the bindingof any ligand, even a simple divalent cation such as Mg 2 + , canbe assessed without fluorescent tagging of the ligand; (v) theassay is extremely sensitive, being able to detect interactions withaffinities in the 10 − 4 M range; and (vi) the presence of minorquantities ofcontaminatingkinaseswill notaffecttheassay,sincethe denaturation measurement is dominated by the major species.We validated our assay using human MLKL, whose mouseorthologue had already shown ATP binding in this assay [43].We measured ATP binding to human MLKL by both thermal-shift assay and ITC, which yielded comparable  K  d  values( ∼ 15 µ M; Supplementary Figures S1A and S1B at http://www.biochemj.org/bj/457/bj4570323add.htm). These studies, coupledwith those reported previously for other kinases [44], provideimportant validation that the magnitudes of the thermal shiftscorrelate with ligand-binding affinities and thus that the thermal-shift assay provides an accurate representation of ligand binding.To gauge the sensitivity of the assay, we examined whether theassay could detect ADP–Mg 2 + binding to the active tyrosinekinase domain of JAK2 (the ‘JH1’ domain), a domain wehave previously shown to bind ADP–Mg 2 + with a  K  d  value of  ∼ 100 µ M [45]. As shown in Supplementary Figures S1(C) andS1(D), we could indeed detect binding of JAK2(JH1) to a varietyof nucleotides in the presence of Mg 2 + , including ADP. On thebasisofthesepreliminarystudies,weestimatedthatthisassaycandetect ligand binding down to sub-millimolar  K  d  values. Indeed,a recent study using an alternative technique demonstrated thatEphB6 (Ephrin type-B receptor 6) binds GTP–Mg 2 + with a  K  d valueof  > 500 µ M[46],andthisinteractionwasalsosuccessfullydetected in our assay (Figure 3C). Classification of pseudokinases We proceeded to qualitatively assess the propensity of eachof 31 recombinant pseudokinase domains (Figure 1) to binddivalent cations (Mg 2 + or Mn 2 + ), nucleotides  { AMP, ADP, ATP,AMP-PNP (adenosine 5 ′ -[ β , γ  -imido]triphosphate) and GTP }  orboth cation and nucleotide together. We also examined bindingto two promiscuous ATP-competitive inhibitors, DAP [31] andVI16832 [32] (structures shown in Supplementary Figures S1EandS1F),asameansofevaluatingwhethertheproteinscontainedan accessible, potentially druggable, pocket equivalent to thenucleotide-binding site of a catalytically active kinase. On thebasis of our experience using this technique to screen for smallmolecule kinase inhibitors, we deemed a   T  m  value of   3 ◦ C tobe a robust indicator of ligand binding.On the basis of their ligand-binding properties in the thermal-shift assay, we categorized the 31 pseudokinases into fourclasses: (i) devoid of detectable nucleotide or cation binding;(ii) nucleotide binding; (iii) cation binding; and (iv) nucleotideand cation binding. As summarized in Table 1, more than half (16) of the pseudokinases did not detectably bind nucleotidesor cations in our assay (Figure 2 and Supplementary Figure S2at http://www.biochemj.org/bj/457/bj4570323add.htm). Interest-ingly, although we did not detect nucleotide or cation bindingfor TYK2(JH2) and IRAK3 (interleukin-1-receptor-associatedkinase 3) (Figures 2E and 2F), both proteins were capable of binding the inhibitors DAP and VI16832, indicating the presenceofanintactATP-bindingcleftandraisingthepossibilitythatthesedomains may bind nucleotides at affinities below the sensitivityoftheassay.AsshowninFigure3,fourproteinsboundnucleotidealone [MLKL, EphB6, STRAD α  and ULK4 (unc-51 like kinase4); Class 2] and two bound cations alone (SgK269 and ROP2;Class 3). It should be noted that the thermal shifts observed forClass 3 pseudokinases in the presence of nucleotides and cationscan be accounted for by the presence of Mn 2 + alone (Figures 3Fand3G).Fewerthanone-thirdofthepseudokinasedomainstested(9 of 31) bound nucleotide and cation: JAK1(JH2), JAK2(JH2),ILK, HER3/ErbB3, SgK071, CRN, ROP5B I , IRAK2 and TARK1(Figure 4 and Supplementary Figure S3 at http://www.biochemj.org/bj/457/bj4570323add.htm). Of note, our assay was amenableto examining the nucleotide and cation binding of pseudokinaseswithin complexes with other protein-interaction domains. Wewere able to assay ILK (Figure 4C and Supplementary FigureS3C), even though its stability relies on being in complex withthe  α -Parvin CH2 domain.RYK contains each of the three canonical residues present inactive protein kinases, but is known to be catalytically inactive[36] and did not detectably bind nucleotides or cations in ourassays. To test whether the highly conserved phenylalanineresidue within the TFVK sequence, the counterpart of aconventional kinase’s VAIK motif, might occupy the adenine-binding pocket and prevent nucleotide binding, we examineda TFVK > VAVK mutant RYK by thermal shift. Although thismutation did not result in detectable nucleotide binding, themutant was able to engage the promiscuous ATP-competitiveinhibitor DAP, whereas wild-type RYK did not (Figures 2B and2C). ATP binding by a second protein, EphB6, was not enhancedbythepresenceofdivalentcations(Figure3C),leadingustoquerywhethertheR 813 LGsequenceinplaceofthecanonicalDFGmotif might serve a role in binding the  γ  -phosphate of ATP, effectivelysupplanting the conventional Mg 2 + . Introduction of the R813Dmutation abrogated the direct binding of ATP by EphB6 in theabsence of cations, but conferred an unexpected ability to bindATP in the presence of Mn 2 + (Figure 3D).ItissurprisingthatBubR1,whichcontainsthecatalyticresiduestypical of an active protein kinase, did not bind nucleotidesin the presence or absence of divalent cations (Figure 2A). Toensure that this was not a consequence of the domain boundaries(residues 724–1043) we used initially, we also characterized alonger construct (residues 720–1043), similar to that used in [35],which contains the kinase extension domain that is critical forcatalytic activity of the related  bona fide  protein kinase BUB1[47]. We expressed BubR1 in both Sf21 and  Escherichia coli cells and found that all preparations behaved equivalently in thethermal-shift assay (results not shown), confirming the inabilityof BubR1 to detectably bind to nucleotides or divalent cations. c  The Authors Journal compilation  c  2014 Biochemical Society  Nucleotide binding by pseudokinases  327 Figure 2 Thermal denaturation curves for selected Class 1 (non-binding) pseudokinase domains Thermal denaturation curves of the Class 1 pseudokinases, BubR1 ( A ), wild-type RYK ( B ), CASK ( D ) and MviN ( G ) (upper panels) with the corresponding histograms depicting the   T  m  values ineach condition (lower panels), as derived from analysis of upper panel curves. Although thermal denaturation of TFVK > VAVK RYK ( C ) and IRAK3 ( F ) was not affected by nucleotides or cations,TYK2(JH2) ( E ) exhibited modest, but consistent, thermal shifts between experiments in the presence of ATP–Mg 2 + , ATP–Mn 2 + or GTP–Mg 2 + . Thermal shifts for all three of these proteins weredetected in the presence of the inhibitor VI16832 and/or DAP. A colour key for the curve labelling is to the right-hand side of the curves. In the histograms, bars are coloured according to the colour keywhen  T  m > 3 ◦ C and pale blue otherwise. Thermal denaturation curves for the other Class 1 pseudokinases, Ror1, PTK7/CCK4, SgK223, SgK495, TRB2, GCN2 [also known as EIF2AK4 (eukaryotictranslation initiation factor 2- α  kinase 4)], VRK3, BPK1, NRBP1 and SCYL1 (SCY1-like 1), are shown in Supplementary Figure S2 (at http://www.biochemj.org/bj/457/bj4570323add.htm). c  The Authors Journal compilation  c  2014 Biochemical Society
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