Genetic analysis of protein kinase B (AKT) in Drosophila

Genetic analysis of protein kinase B (AKT) in Drosophila
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  Brief Communication 599 Genetic analysis of protein kinase B (AKT) in Drosophila  Brian E. Staveley*, Laurent Ruel † , Jing Jin † , Vuk Stambolic † , Fabrizio G.Mastronardi*, Pascal Heitzler ‡ , James R. Woodgett † and Armen S. Manoukian* The decision between survival and death is animportant aspect of cellular regulation duringdevelopment and malignancy. Central to this regulationis the process of apoptosis, which is conserved inmulticellular organisms [1]. A variety of signallingcascades have been implicated in modulation ofapoptosis, including the phosphatidylinositol (PI) 3-kinase pathway. Activation of PI 3-kinase is protective,and inhibition of this lipid kinase enhances cell deathunder several conditions including deregulatedexpression of c-Myc, neurotrophin withdrawal andanoikis [2–7]. Recently, the protective effects of PI 3-kinase have been linked to its activation of thepleckstrin homology (PH)-domain-containing proteinkinase B (PKB or AKT) [8]. PKB/AKT was identifiedfrom an oncogene, v- akt  , found in a rodent T-celllymphoma [9]. To initiate a genetic analysis of PKB, wehave isolated and characterized a Drosophila  PKB/AKTmutant (termed Dakt1 ) that exhibits ectopic apoptosisduring embryogenesis as judged by induction ofmembrane blebbing, DNA fragmentation andmacrophage infiltration. Apoptosis caused by loss ofDakt function is rescued by caspase suppression but isdistinct from the previously described reaper  / grim  / hid  functions. These data implicate Dakt1 as a cell survivalgene in Drosophila  , consistent with cell protectionstudies in mammals. Addresses: *Divisions of Cell and Molecular Biology, and † Experimental Therapeutics, Ontario Cancer Institute, Department ofMedical Biophysics, University of Toronto, 610 University Avenue,Toronto, Ontario M5G 2M9, Canada. ‡ Institut de Genetique et deBiologie Moleculaire et Cellulaire, 1 rue Laurent Fries, BP163, 67404Illkirch cedex, France.Correspondence: Armen S. ManoukianE-mail: armenm@oci.utoronto.caReceived: 31 December 1997 Revised: 2 April 1998 Accepted: 3 April 1998 Published: 27 April 1998Current Biology 1998, 8:599–602 © Current Biology Ltd ISSN 0960-9822 Results and discussion Molecularanalyseslocalized   Dakt1 towithin ≈ 30kbofthe   stubbloid          (   sbd          )gene[10,11];thisplaces   Dakt1 within   Df(3R)sbd          45   [12].WethereforefocusedourhuntforaDakt1mutanttolethalsthatfailedtocomplementthisdeficiency.AsDakt1isubiquitouslyexpressed[10,11],weutilizeda GAL4     transgenedrivenbythe armadillo promoter( arm–GAL4     )[13],whichallowedexpressionofDakt1inafairlyubiquitouspatternusinganupstreamactivatingsequence   Dakt1 ( UAS–Dakt1 )transgene.Screeningacollec-tionofmutantsmappingto   Df(3R)sbd          45   ,onelarvallethalmutation, l(3) 89B q  1 [14],wasidentifiedthatcouldberescuedbythecombinationofthe arm–GAL4     and UAS–Dakt1 transgenes( arm–Dakt1 ;seeMaterialsandmethods).AsPKB/AKTandDakt1haveasimilarityof      76.5%attheamino-acidlevel[10,11,15],wealsotestedwhetherbovinePKB/AKTcouldrescue l(3) 89B q  1 mutantsusinga UAS–PKB/AKT          transgene.The arm–PKB/AKT          com-binationwaseffectiveatrescuing l(3) 89B q  1 (seeMaterialsandmethods),albeitwithlowerefficiency.Thisdemon-stratesthat l(3) 89B q  1 (simplifiedto‘ q  ’)encodesDakt1,whichisthefunctionalhomologofPKB/AKT.Immunoblot analysisof Dakt1 from wild-type andhomozygous q  larvae showed no significant differencesinexpression (Figure1a), so q  isnot aprotein-null allele of      Dakt1. ADakt1 kinase activity assay, however, revealed asignificant lossof Dakt1 kinase activity in homozygous q  larvae (Figure1b). Since q  encodesDakt1 protein, wefocused our search for DNAlesionsassociated with q  tothe coding region of   Dakt1 and sequenced the   Dakt1 coding region from genomicDNAisolated from q  larvae.Asacontrol, we also sequenced   Dakt1 DNAfrom anotherlethal mutation, l(3) 89B o 1 (simplified to ‘ o ’), isolated inthe same mutagenesisscreen as q  [14]. Comparison of thesequencesof several independent isolateseach of wild-type, q  and o Dakt1 revealed asingle consistent pointmutation unique to the q  DNAsequence. Thissequencechange resulted in asingle amino-acid change, F327I(Figure1c). Thisphenylalanine residue isacore residuein subdomain VIIof the kinase catalyticdomain, formingthe ‘DFG’ motif which ishighly conserved among proteinkinasesincluding mammalian PKBproteins. Given oursequence analysisof q  along with our kinase assay results,we conclude that q  encodesanon-functional Dakt1kinase. To confirm thishypothesis, we generated the cor-responding amino-acid change in bovine PKB(F293I) andassessed itseffectson PKBkinase activity. The F293Ichange producesakinase that isinactive even in the pres-enceof the agonist pervanadate (Figure1d).As   Dakt1 isamaternallyexpressedgene[11],wetestedthematernalcontributionofDakt1usinggermlineclone(GLC)analysis[15].GLC q  femalesproducedembryoslackingvariousportionsoflarvalcuticleattheendof        embryogenesis.TherangeofphenotypesdependedonthelevelofzygoticDakt1activityintheembryo.WithoutanyzygoticexpressionofDakt1, q  GLCembryosproducedonlyafewscrapsofcuticle(Figure2b).When q  GLCembryosexpressedsomezygoticDakt1,someventralcuticlewasproduced(Figure2c).ThisphenotypewasextensivelysuppressedbyexpressionofDakt1usingourheat-shockinducible    hs-Dakt1 transgene(Figure2d).AsmammalianPKB/AKThasbeenimplicatedinanti-apop-toticactivity[16],wedecidedtotest   Dakt1 GLCembryosforevidenceofapoptosis.Acridineorange(AO)staininghasbeenshowntobeagoodindicatorofapoptosis(andnotnecrosis)in   Drosophila ,detectingcellulareventssuchasmembraneblebbing[17].AOstainingof         Dakt1 GLCembryosshowedextensiveapoptosiscomparedtowild-typeembryos(Figure2e,f).ConfirmationofaneffectonapoptosiswasperformedusingaTUNELassayin   Dakt1 embryostodetecttheincidenceofDNAfragmentation[18].   Dakt1 embryosshowedextensiveDNAbreakageasassayedbyTUNEL  insitu .IncidenceofTUNELsignalin   Dakt1 GLCembryosprecedestheinitiationofthesignalinwild-typeembryos(Figure3a,b).TUNELsignalaccumu-latedduringdevelopmentof         Dakt1 embryostoengulfthemajorityoftheembryo(datanotshown).Duringapoptosis, 600 Current Biology , Vol 8 No 10 Figure 1 Analysis of Dakt1 protein expression andactivity in wild-type and homozygous q  (Dakt1q) larvae. (a) Immunoblot analysis ofDakt1 protein from wild-type and q  larvaeusing anti-Dakt1 antibody (see Materials andmethods). (b) Results of the Dakt1 kinaseactivity assay from wild-type and q  larvae [14]. (c) Schematic diagram of the Dakt1 amino-acid sequence showing the F327I change inthe q  allele of Dakt1 and the sequence of therelevant region in the bovine (b) and human(h) PKB homologs. (d) The correspondingF293I sequence change in bovine PKBcreates a kinase-dead protein. Testing of thekinase activity and expression of the PKBF293I mutant and wild-type PKB in HeLa S3cells (see Supplementary material publishedwith this paper on the internet): + denotesaddition of the kinase agonist pervanadate. 1251007550    D  a   k   t   1   k   i  n  a  s  e  a  c   t   i  v   i   t  y   (   %   ) Dakt1PKB kinase assay (d)(b)(a) Anti-HA immunoblotting[ 32 P]GSK3 peptideHAPKBDakt1qDakt1bPKBhPKB α hPKB β KVADIGLCKKVADFGLCKKITDFGLCKKITDFGLCKKVADIGLCK Kinase consensus (c) Subdomain VII XXXDFGXXX    W   i   l  d    t  y  p  e   D  a   k   t  1  q    W   i   l  d    t  y  p  e   D  a   k   t  1  q    U  n   t  r  a  n  s  f  e  c   t  e  d   + +    H  A  –   P   K   B   H  A  –   P   K   B    F  2  9  3   I 250 Current Biology Figure 2 The phenotype of Dakt1 GLC mutants. (a) First instar cuticle of a wild-type larva. (b) Phenotype of a Dakt1 GLC embryo in theabsence of zygotic Dakt1 activity, showingalmost complete loss of cuticle. (c) Phenotypeof a Dakt1 GLC embryo with zygotic Dakt1activity, showing complete loss of head anddorsal structures. Only a portion of the ventralcuticle remains and does not show extensivedefects in segmentation. (d) Phenotype of a Dakt1 GLC embryo rescued with hs-Dakt1 induced maternally and zygotically. Note thereappearance of most cuticle structures,demonstrating a functional rescue of thephenotype. (e) Acridine orange (AO) stainingof a wild-type late stage 12 embryo. Note thesignal present in the amnioserosa and inregions of the head. (f) AO staining of a stage12 Dakt1 GLC embryo showing a ubiquitousAO signal.  macrophagesconvergetoengulfcellularfragmentsbyphagocytosis.WeusedanantibodytoCroquemort(Crq),the   Drosophila homologofCD36,todetectmacrophagesinembryos[19].Consistentwithourpreviousobservations,   Dakt1 embryosexhibitedasignificantincreaseinCrqexpressioncomparedtowild-typeembryos(Figure3c–f).Thisexpressionisatthecellsurfaceandfocusedontheapoptoticcells(Figure3f).Itappears,therefore,thatlossof      Dakt1activityresultsinprematureandectopicapoptosiswiththecharacteristicsofmembraneblebbing,DNAfrag-mentationandmacrophage-mediatedendocytosis.Todate,thedeficiencyofthe reaper       ( rpr       ),       grim and    hid          genes(   Df(3L)H99 )istheonlymutationthatblocksapopto-sisin   Drosophila [18].Overexpressionofanyofthesethreegenesresultsinectopicapoptosisinembryos[18,20,21].Wewerethereforecuriousastowhether   Dakt1 mutantsresultedinapoptosisthroughthemis-expressionof       rpr       ,       grim or    hid          .Thiswasfoundnottobethecase,as   Dakt1 mutantembryosdidnotshowoverexpressionofthesegenes( rpr       ,Figure3g,h;    hid          and       grim ,datanotshown).Tofurtherexaminethepossibilityofaninteractionbetween   Dakt1 andthesegenes,wegenerateda   Df(3L)H99FRT          82ß   l(3) 89B q  1 chromosomeandmadeGLCs.Consistentwithourexpressionstudies,lossof       rpr       ,       grim and    hid          in  H99 didnotsuppressthephenotypeof         Dakt1 GLCembryos(Figure3i).TheseresultssuggestthatDakt1andH99modulateapoptosisviadistinctmechanisms.TotesttheinvolvementofcaspasesinDakt1-mediatedapoptosis,weexpressedthebaculoviralcaspase-inhibitoryproteinp35in   Dakt1 embryos.Ectopicp35hasbeenshowntoblockcaspaseactivityandtosuppressapoptosisin   Drosophila [22],and    hs-p35   effectivelyblockedapoptosisin   Dakt1 embryos(Figure3j),demonstratingtherequirementforcaspaseactivityinthisprocess.OurepistasistestssuggestthatDakt1doesnotfunctionupstreamofthe rpr       ,       grim and    hid          genefunctionsintheembryo.Itispossible,though,thatDakt1mightberegu-latedbythe rpr       ,       grim and    hid          genes(atthe  H99 locus)andinfactactdownstreamofthesegenes.Thispresentstwopossibilities.First,Dakt1andthe  H99 locusrepresentinde-pendentpathways.Second,the  H99 locusmightrepressDakt1function.ThisstudythusprovidesthefirstgeneticevidenceimplicatingPKBasananti-apoptoticfactor. Materials and methods Drosophila strains and genetic experiments  Dakt1 and bovine PKB cDNAs were cloned into pUAST P-elementtransformation vector and injected into w  1118  embryos to generate trans-formants. For adult rescue experiments, we used a combination of arm–GAL4  with UAS–Dakt1 ( arm–Dakt1 ) or with UAS–PKB  ( arm–PKB  ). The arm–Dakt1 transgene resulted in a rescue of 34.1%and arm–KB resulted in a rescue of 17.8%. Rescue was scored as thepercentage of flies with a homozygous q  chromosome (the expectednumber is zero). The heat-shock inducible hs–Dakt1 line was generatedusing the pHS Casper P-element vector. GLC analysis for 3R using FRT  82  β  was performed as in [15]. For determining the extent ofzygotic/paternal rescue, l(3)89Bq  1 GLC females were crossed to wild-type, l(3)89Bq  1 , TM3  , Sb  , l(3)89Bq  2  and Df(3R)sbd  45  males and theembryonic phenotypes were compared. As l(3)89Bq  1 and Df(3R)sbd  45  chromosomes behaved identically, we believe that l(3)89Bq  1 is a genetically null allele of Dakt1 . A second Dakt1 allele(termed q  2  ) was also used for these experiments. As q  2  was induced onthe TM3 chromosome, however, we were unable to perform GLC experi-ments using this allele. To rescue q  GLC embryos, q  GLC females werecrossed to hs-Dakt1 ; q   /  TM3  males and the progeny were heat-shockedthree times during embryogenesis for 5min each. A Df(3L)H99 FRT  82  β  l(3)89Bq  1 chromosome was constructed using standard recombination Brief Communication 601 Figure 3 Monitoring apoptosis markers in Dakt1 GLC embryos. (a) TUNELsignal in a stage 11 wild-type embryo. Only a few cells are positive forthe TUNEL signal at this stage. (b) TUNEL signal in a stage 8 Dakt1 GLC embryo. Even by stage 8, extensive DNA fragmentation asassayed by TUNEL has begun in the absence of Dakt1. (c) A stage 11wild-type embryo showing the presence of Crq-expressingmacrophages near sites of apoptosis (e.g. presumptive amnioserosa). (d,e) A stage 11 Dakt1 GLC embryo showing the extensive andwidespread presence of macrophages as assayed by the Crq signal. (f) Close-up of (e). The expression of Rpr is not appreciably altered in (g) stage 10 q GLC embryos compared to (h) wild-type stage 10embryos. (i) The cuticle phenotype of Dakt1 GLC embryoshomozygous for H99  is similar to that of Dakt1 mutants. (j) Cuticlephenotype of Dakt1 GLC embryos expressing ectopic baculoviral p35from the hs-p35 transgene. This indicates suppression of the Dakt1 GLC phenotype by p35  but not the H99  deficiency.  and then crossed to FRT  82  β  ovoD  males for the induction of GLCs. GLCfemales were then crossed to the Df(3L)H99FRT  82  β  l(3)89Bq  1 chro-mosome and the progeny analyzed. A 1kb Bam  HI fragment of pBSPScontaining the entire coding region of Autographa californica  p35 (fromLois Miller) was cloned into the Bgl  II site of pHS Casper and trans-formed. For the epistasis experiment, q  1 GLC females were crossed to hs-p35  ; q  1  /  TM3  males and the progeny were collected onto nylonsieves. Embryos were then staged (stages 10–12), heat-shocked at37°C in a waterbath for 8min and allowed to recover at 25°C. Embryoswere then allowed to develop cuticle and analyzed. Dakt1 antibody production, immunoblots and kinase assays  Rabbit antisera against Dakt1 were raised against an olihistidine–Dakt1(His–Dakt1) fusion protein. His–Dakt1 was constructed by cloning a1590bp fragment of the Dakt1 complete coding region (530 aminoacids) into the Bam  HI site of the pET15b vector (Novagen). Fusion pro-teins were produced in E. coli strain BL21(DE3) and purified from bac-terial lysates through binding to nickel-chelating resin according tomanufacturer’s instructions (Novagen). Rabbits were immunized subcu-taneously with purified recombinant proteins in complete Freund’s adju-vant followed by booster injections at 4 week intervals. Larvaehomozygous for q  were identified by following the TM6B balancer. Drosophila embryos were lysed in Gentle Soft buffer (20mM PIPESpH7.4, 10mM NaCl, 0.5% NP-40, 5mM EDTA, 0.05% 2-mercap-toethanol, 5mg/ml leupeptin, 1mM benamidine, 0.5mM NaF and100mM Na vanadate). The lysates were normalized for total proteinbefore immunoprecipitation or separation by SDS–PAGE forimmunoblotting. For western blots, Dakt1 proteins were visualized usingthe ECL system (Amersham). For Dakt1 immunoprecipitations, 5mlrabbit polyclonal antiserum were added to the cell lysates for 2h at 4°C.Immunocomplexes prebound to protein A–Sepharose (Sigma) werewashed five times with lysis buffer. In vitro Dakt1/HA–PKB kinaseassays were measured by the capacity of the kinases to phosphorylate aGSK3 peptide substrate corresponding to the sequence in GSK3 β sur-rounding the Ser9 that is phosphorylated by PKB. Dakt1 immunocom-plexes were incubated at 30°C with 30mM GSK3 peptide in thepresence of 50mM [ 32 P]ATP in 50mM PIPES, pH7.4, 10mM MgCl 2 and 1mMEGTA. The phosphorylated peptides were separated fromunincorporated [ 32 P]ATP by Tricine-SDS–PAGE and quantified byanalysis on a Molecular Dynamics Phosphorimager. Analysis of embryos and cuticles  Cuticles were cleared in Hoyer’s medium before photography andanalysis. For acridine orange (AO) staining, embryos were dechorion-ated with bleach and layered with 5mg/ml AO in PBS and heptane for5min. Embryos were then removed from the mix and dropped ontoHalocarbon oil for analysis and photography. Terminal deoxynu-cleotide transfer mediated dUTP–biotin nick-end labelling (TUNEL)assays in embryos were performed as in [20,21] and the embryoswere layered through mountant (70% glycerol/30% 0.1M Tris, pH8)and mounted for photography. Anti-Crq staining and the antibody aredescribed in [19]. The rpr  , grim  and hid  probes were generated andlabeled with digoxigenin using PCR with specific primers to thesegenes and Drosophila  genomic DNA. Hybridization and detection in situ  was as in [23]. Supplementary material  Additional methodological detail is published with this paper onthe internet. Acknowledgements B.E.S. and L.R. contributed equally to this work. We thank P. Tsichlis for Dakt1 cDNA and Lois Miller for p35  DNA. We also thank Nathalie Franc foranti-Crq antibody and helpful advice and Hermann Steller and John Abramsfor rpr  , grim  and hid  cDNAs. We are indebted to Kristin White, Rich Binariand Sam Scanga for discussion and advice. This work was supported bygrants from MRC of Canada (A.S.M. and J.R.W) and by the HowardHughes Medical Institute (J.R.W.). References 1.Jacobson MD, Weil M, Raff MC: Programmed cell death in animaldevelopment. Cell 1997, 88: 347-354.2.Harrington WA, Bennett MR, Fanidi A, Evan GI: c-Myc-inducedapoptosis in fibroblasts is inhibited by specific cytokines. EMBO J  1994, 13: 3286-3295.3.Yao R, Cooper GM: Requirement for phosphatidylinositol 3   ′ ¢ kinase in the prevention of apoptosis by nerve growth factor. Science 1995, 267: 2003-2006.4.Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C, Coffer P,Downward J, Evan G: Suppression of c-Myc-induced apoptosis byRas signalling through PI(3)K and PKB. Nature 1997, 385: 544-548.5.Kennedy SG, Wagner AJ, Conzen SD, Jordan J, Bellacosa A, TsichlisPN, Hay N: The PI 3-kinase/Akt signaling pathway delivers ananti-apoptotic signal. 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Genes Dev 1996, 10: 1773-1782.22.Hay BA, Wassarman DA, Rubin GM: Drosophila homologs ofbaculovirus inhibitor of apoptosis proteins function to block celldeath. Cell 1995, 83: 1253-1262.23.Manoukian AS: Detection of mRNA in situ  : techniques for studyinggene expression in Drosophila melanogaster  tissues. In mRNAFormation and Function. Edited by Richter JD. New York: AcademicPress; 1997:361-370. 602 Current Biology , Vol 8 No 10  Genetic analysis of protein kinase B (AKT) in Drosophila  Brian E. Staveley, Laurent Ruel, Jing Jin, Vuk Stambolic, Fabrizio G.Mastronardi, Pascal Heitzler, James R. Woodgett and Armen S. Manoukian Current Biology 27 April 1998, 8 :599–602 S1 Materials and methods Sequence analysis of q Wild-type, homozyous q  1 and o  1 larvae were isolated and subjected toDNA extraction. The coding region of Dakt1 was then amplified byPCR from each of the DNA sources using Dakt1 -specific oligos span-ning the coding region and subjected to sequencing by the AMGENEST program. Experiments on bovine PKB  The bovine PKB F293I mutant was generated in the pALTER-1 vector(Promega) using the oligonucleotide 5 ′ -CATCAAGATCACCGA-CATAGGCCTGTGCAAGGAGGGC-3 ′ . This PKB F293I mutant wastagged at the 5 ′ end with the haemagglutinin (HA) epitope and thensubcloned into the pcDNA3 expression vector (Stratagene). Wild-typePKB, PKB F293I and vector plasmid as control were each introducedinto HeLa S3 cells by cationic liposome-mediated transfection. Cellswere then grown in 10% serum for 20h followed by either stimulationwith pervanadate (100mM, 15min) or mock treatment with buffer.HA–PKB and HA–PKB F → I proteins were immunoprecipitated fromcell lysates by using 12CA5 anti-HA antibody/protein G–Sepharose(Pharmacia Biotech) and assayed as described above for PKB activity.The amount of PKB and PKB F293I expressed in each sample wasdetermined by immunoblotting with anti-HA antibody (Figure1d). Supplementary material
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