Drayer, Anneke Lyndsay

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   University of GroningenPhosphoinositide-specific Phospholipase C in Dictyostelium discoideum,A molecular geneticapproach to study its function and biochemical propertiesDrayer, Anneke LyndsayIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1995Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Drayer, A. L. (1995). Phosphoinositide-specific Phospholipase C in Dictyostelium discoideum,A moleculargenetic approach to study its function and biochemical properties. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 08-06-2022120 8. SummarySUMMARYPhosphoinositide-specific phospholipaseC (PLC) is generally considered to be one ofthe central efÍector enzymes intransmembrane signall ing, generating thesecond messengers Ins(1,4,5)P3 anddiacylglycerol. PLC activity in the cellularslime mould Dictyostelium discoideum wasshown to be regulated via cAMP receptorsand G-proteins (1). Previous studies havesuggested Ins(1,4,5)P3 and diacylglycerolplay a role in chemotaxis and development.In this thesis the role oÍ the enzyme PLC inDictyostelium disoideum groMh, chemotaxisand development has been investigated bygenerating a cell l ine which no longercontains PLC activity.Comparison oÍ the processes involved insignal transduction between difÍerentorganisms shows that many pathways andproteins have been evolutionary conserved(chapter 7), The Íact that many componentsare genetically conserved in higher and lowereukaryotes was used to search Íor a PLCgene in Dictyostelium (chapter 2). Primersencoding highly conserved amino acidregions in mammalian and Drosophila PLCisoÍorms were used in the polymerase chainreaction to identiÍy PLC in Dictyostelium. OnePLC-like sequence was Íound; the deducedamino acid sequence of the full proteinresembles mammalian PLC6. A mutant wilh adisrupted PLC gene (plc- ) was generatedand analyzed Íor development and cAMP-mediated responses (chapter 3). lt wasdemonstrated that PLC activity was absent inplc cells, and unexpectedly, no deÍect ingrowth, chemotaxis or development wasobserved. Although no PLC activity could bedetected in the mutant, the level oÍIns(1,4,5)Ps was found to be near ly  normal .This suggests there are alternative pathwaysto generate this second messenger besidesPLC-mediated hydrolysis of Ptdlns(4,5)Pz.The deduced amino acid seouence oÍDictyoslelium PLC does not predict anymembrane-spanning regions, yet thesubstrate and proteins interacting with PLCsuch as cAMP receptors and activated G-proteins are membrane-associated. Inchapter 4 localisation oÍ PLC wasinvestigated, as the cellular distribution oÍ theenzyme may provide a way to regulate itsactivity. Eighty percent of the PLC proteinwas localized to the soluble fraction oÍ bothresting and cAMP-stimulated cells, whereasthe highest amount oÍ PLC activity wasmeasured in the pafi iculate Íraction.Particulate PLC was Íound to be regulated bya heat-stable component from the solublefraction, which was required to express Íullenzyme activity. These studies suggest onlya small oart oÍ the available PLC in a cell is ina suitable location to hydrolyse Ptdlns(4,5)Pzin the particulate f raction, whereby anadditional, unidentiÍ ied, Íactor in the solubleÍraction is required to activate particulateD l  aIn chapter 5 an account is given oÍ thevarious procedures employed to producerecombinant PLC and subsequently tomeasure activity in the recombinant enzyme.Expression oÍ Díctyostelium PLC inEscherichia coll was successÍul in producingsuÍÍicient amount of the protein to raiseantibodies. Init ially most oÍ the recombinantPLC was retrieved as an insolublecomponent in inclusion bodies in E.col|which did not show enzyme activity.Changing culture conditions and expressionvectors lead to the production oÍ soluble,recombinant PLC. UnÍodunately however, itproved not possible to obtain activerecombinant Dictyostelium PLC, althoughisrb imoÍwiin lthtpr(Íoracïn n iH v lEF.thenotof ttPLC(^ - ^ l4 t  t d lI n s ( 1in pltcannunidehowesugg(perÍorh ighlyoomal ikeRedu<on Srothercel lsactivit 'WASdevelcdata ir8. Summary 121r second messenger besidesydrolysis of Ptdlns(4,5)P2.,d amino acid sequence oÍ)LC does not predict anyrning regions, yet thelroteins interacting with PLCreceotors and activated G-membrane-associated. Inrcalisation oÍ PLC wasthe cellular distribution oÍ therovide a way to regulate itspercent oÍ the PLC proteinr the soluble fraction oÍ bothvlP-stimulated cells, whereasnount oÍ PLC activity wasthe oadiculate Íraction.was Íound to be regulated by:omoonent from the solublewas required to express Íull. These studies suggest onlyre available PLC in a cell is inon to hydrolyse Ptdlns(4,5)Pzrlate Íraction, whereby anentiÍ ied, Íactor in the solubleuired to activate particulatei an account is given oÍ thelures employed to produce)LC and subsequently toy in the recombinant enzyme.I Dictyostelium PLC inI was successÍul in producinglnt oÍ the protein to raiseally most oÍ the recombinantrtrieved as an insolubleinclusion bodies in E.coli,rl show enzyme activity.re conditions and expressionr the production of soluble,"C. UnÍortunately however, itlossible to obtain activetictyostelium PLC, althoughvarious substrates and reaction conditionswere tested.Ca2* is an important regulator of all PLCisoÍorms. The amino acid seouence oÍDictyostelium PLC predicts an EF-hand Ca2*-binding motiÍ, a domain Íound in many Ca2*-modulated proteins.^ln chapter 6 the functionoÍ this domain in Ca"-dependent PLC activitywas analyzed by introducing points mutationsinto the EF-hand. P/c- cells were used inthese studies to express the mutated PLCproteins in their native environment. lt wasdemonstrated that this domain is importantfor enzyme activity, as the maximal enzymeactivity decreased with increasing number ofpoint mutations. However, mutation oÍ theEF-hand domain did not aÍÍect the aÍfinitv ofthe enzyme Íor Ca2*, demonstrating thiá isnot the site that mediates Ca'*-dependenceof the PLC enzyme reaction.PLC-independent Ins(1,4,5)P3 ÍormationOne oÍ the surprising results in theanalysis of the plc mutant is thatIns(1,4,5)P3 levels are only slightly decreasedin plc- cells compared to unmutated cells. ltcannot be completely excluded that another,unidentif ied, PLC isoÍorm is present,however, all genetic and biochemical datasuggest this is not the case. PCR reactionspedormed with various primers encodinghighly conserved regions in the A and Bdomains of PLC did not yield any other PLC-like sequence than the one reponed.Reduced stringency-hybridization experimentson Southern blots gave no indications forother  PLC sequences.  In  addi t ion,  in  p lc-cells no basal or receptor-stimulated PLCactivity could be measured, and PLC activitywas absent during the completedevelopmental programme. Together thesedata indicate the PLC gene which was clonedaccounts for all detectable PLC activity inDictyostelium.How is Ins(1,4,5)Ps generated in plccells? Mass determination oÍ inositolphosphate isomers indicated many possibleprecursors for Ins(1,4,5)Ps (chapter 3). lt ispossible that the phosphorylation of lowerinositol phosphates or the dephosphorylationof higher inositol polyphosphates couldprovide lns(1,4,5)P3. Recently, a putativeroute to supply Ins(1,4,5)Ps has beenidentiÍ ied using higher inositol poly-phosphates as substrate instead ofPtdlns(4,5)Pz. ln Dictyostelium lysates,lns(1,3,4,5,6)P5 was shown to be degradedto Ins(1,4,5)Pa wi th both Ins(1,3,4,5)Pa andlns(1,4,5,6)P+ ês in termediates (2) .Ins(1,3,4,5,6)Ps can be Íormed by stepwisephosphorylation oÍ inositol, independently oÍIns(1,4,5)Ps Íormat ion (3) .  In  p/c  cel ls  thelevel  oÍ  Ins(1,3,4,5,6)Ps i  lower than incontrol cells (6 compared to 20 pmol/1 07cells, see chapter 3), consistent with thehypothesis  that  Ins(1,3,4,5,6)P5 could serveas a source for  Ins(1,4,5)P3.The occurence of an alterative pathway togenerate Ins(1,4,5)Ps could expla in thenormal phenotype oÍ the p/c- mutant. Thenovel phosphatase activit ies were found inplc- as well as wild-type cells, indicating it isnot a result of disrupting the PLC gene, but isa normally occuring pathway in Dictyostelium.The role oÍ this pathway in signaltransduction has sti l l  to be established.Interestingly, mammallan cells are alsocapable oÍ  synthesis ing Ins(1,4,5)P3 bydephosphorylation of higher inositol poly-phosphates with the enzyme multiple inositolpolyphosphate phosphatase (MIPP) (2,4).This could implicate that PlC-independentformat ion of  Ins(1,4,5)P3 is  a widely occur ingevent in lower as well as higher organisms.122 8. SummaryA rofe Íor PLC in Dictyostelium?The results obtained in the describedstudies clearly demonstrate PLC is notessential for normal growth, chemotaxis anddevelopment in Dictyostelium discoideum.Then why has PLC been evolutionaryconserved in Dictyostelium? lt is possible thatlhe plc- cells are unique in that they havebeen careÍully cultured to survive, andemploy salvage pathways to adapt to the newsituation in the absence oÍ PLC. On the otherhand, there could be an advantage for cellsto have PLC in their original surroundings,which is not necessary in laboratoryconditions. Culturing of cells in less optimaland more stressÍul surroundings andcomparing survival rates of plc andunmutated strains might give an answer, butcould require many generations.In chapter 1 a summary oÍ data from theliterature was presented which suggested arole Íor PLC in Dictyostelium chemotaxis anddevelopment. Many oÍ the discrepancies canbe explained by the fact lhat plc- cells stillcontain the second messengersdiacylglycerol and Ins(1,4,5)P3. Thesecomoounds can sti l l  inÍluence chemotaxisand development, but PLC is not essential Íorproducing these messengers.Analysis oÍ mutants Frigid A, with a deÍectin the u-subunit oí the Ga2 protein, andFrigid C, with an unidentiÍied defect, havedemonstrated the Frigid C gene product andGaZ are essential for chemotaxis anddevelopment. lt has also been demonstratedthat PLC is regulated by these components(1). The Íact that chemotaxis anddevelopment are normal in plc- cellsindicates other effector enzymes besidesPLC are regulated by Frigid C and Gs2.Besides its defect in PLC activation, mutantFrigid A is also deÍective in cAMP-stimulatedactivation oÍ adenylyl cyclase and guanylylcyclase. lt has been established that adenylylcyclase and intracellular cAMP play anessential role in development, while guanylylcyclase and cGMP are involved inchemotactic movement. Therefore, impairedchemotaxis and developmenlin Frigid Acan,at least partly, be attributed to defectiveregulation oÍ guanylyl and adenylyl cyclase.ln mutant Frigid C chemotaxis is absentunder physiological conditions, but cells arenot completely deÍective in chemotacticmovement. Approximately 100-fold highercAMP concentrations are required to inducea chemotactic response. This could be aresult of the reduced number of cAMPreceptors. The only deÍect at the secondmessenger level in Frigid C was Íound to beactivation oÍ PLC. The plc- mutantdemonstrates activation of PLC is notessential for chemotactic movement anddevelopment, and it is Íeasible that the Frigid Cgene product regulates another, unidentiÍ iedeÍfector enzyme besides PLC.It has been argued that a l ink existsbetween inhibit ion oÍ PLC and antagonism oÍchemotaxis (5). Partial antagonists oÍ cAMPsuch as 3'NH-cAMP and 8-CPT-cAMP bind tosurface cAMP receptors and induce a cGMPresponse at a concentration proportional totheir binding activity. Chemotaxis however, isinduced only at very high concentrations. Atlow concentration these compounds inhibitcAMP-induced chemotaxis. In wild-type cells3'NH-cAMP and 8-CPT-cAMP decreaseIns(1,4,5)Pg levels due to inhib i t ion oÍ  PLCactivity (6). lt has been suggested thatinhibit ion oÍ PLC afÍects chemotaxis. Asudden decrease in Ins(1,4,5)P3 could afÍectCa2* homeostasis, leading to inhibit ion oÍchemotaxis. In Go1 -null cells, in whichinhib i t ion oÍ  PLC does not  occur ,  Ins(1,4,5)P3levels remain unchanged aÍter stimulationwith 3 NH-cAMP (6).8. Summary 123n established that adenylylacellular cAMP play an;velopment, while guanylyliMP are involved inment. Therefore, impairedvelopment in Frigid A can,e attributed to deÍectiveylyl and adenylyl cyclase.C chemotaxis is absentI conditions, but cells areleÍective in chemotacticximately 100-Íold higherns are required to induceponse. This could be aluced number of cAMply deÍect at the secondFrigid C was Íound to be.C. The plc- mutantvation oÍ PLC is notTotactic movement andis Íeasible that the Frigid CIates another, unidentiÍ iedsides PLC.rgued that a l ink existsÍ PLC and antagonism oÍfiial antagonists oÍ cAMP'and 8-CPT-cAMP bind tortors and induce a cGMpcentration proportional to'. Chemotaxis however, isy high concentrations. Atthese compounds inhibitnotaxis. In wild-type cells8-CPT-cAMP decreasedue to inhibit ion oÍ PLCi been suggested thatafÍects chemotaxis. AIns( 1,4,5)Ps could aÍÍectleading to inhibit ion oÍ11-nul l  ce l ls ,  in  whichgs not  occur ,  Ins(1,4,5)Psranged after stimulationcc+(,,,/.*y"" '"'\l"nue{\'k":>,6:*" :". cHEMorAX,s)"lt r-> :^'á tt @3NHcAMi ll \*"ïu a rl^\\r\EFigure 1: Model Íor regulat ion oÍ chemotaxis by second messengers.PLC activity in Dictyostelium discoideum is regulated by a stimulatory pathway and an inhibitory pathway.The guanylyl cyclase (GC) activation pathway (light grey arrows) operates via cARl and G-cr2. Thechemotactic antagonist 3'NH-cAMP selectively inhibits PLC activity, but activates guanyl cyclase. In cellswith a disrupted DdPLC gene, no PLC activity is present, yet cells do contain Ins(1,4,5)Ps anddiacylglycerol which are proposed to be synthesized by alteÍnative routes (double hatched arrows).Cytosol ic Ca" can be supplied by release Írom Ins(1,4,S)P3-sensit ive and Ins(1,4,5)p3-insensit ive stores(open box), or by receptor-mediated Ca'" influx accross the membrane (open arrow). Mutants showingabbeÍant chemotaxis and isolated genes are indicated by italics. Mutant K/-í0 is defective in receptoi-stfmulated guanylyl cyclase activity, while KI-9 has strongly reduced guanylyl cyclase levels. Mutant SÍrnFhas a defect in cGMP-phosphodiesterase activity resulting in enhanced and prolonged Íeceptor-mediatedCGMP elevation, and a prolonged chemotactic response. Mutant FgdC is deÍective in receptor stimulationof PLC activity. The question mark indicates a putative, unidentiÍied effector enzyme regulated by theFgdC gene product.Abbreviations: Rs, stimulatory receptor; Ri, inhibitory receptoÍ; Gs, stimulatory G-protein; Gi, inhibitory G-protein; InsP3, inositol 1,4,s-tr isphosphate; InsP", other phosphorylated rnyeinositol compound; DG,diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine.124 L Summary9.1 0 .1 11 2In this cell l ine 3'NH-cAMP is not anantagonist of chemotaxis, supporting thehypothesis that inhibition of PLC activitycauses antagonism of chemotaxis. Accordingto this hypothesis the chemotactic defect inmutant Frigid C could also be caused by thedeÍect in PLC regulation, as cAMP does notstimulate, but inhibits PLC activity.Chemotaxis in plc- cells on the other handwould not be aÍfected as Ins(1,4,5)P3 levelsremain normal.In Figure 1 the regulatory componentsand second mesengers involved inchemotaxis as discussed above, aresummarized in a model. The secondmessengers cGMP and Ca2*/diacylglycerolplay a role in chemotaxis by regulating thereorganization oÍ myosin with thecytoskeleton and actin polymerization,respectively. As can be seen Írom Figure 1,Ins(1,4,5)Pg is not the only Íactor involved inregulating cytosolic Ca2' levels. Ca2* can bereleased Írom intracellular Ins(1,4,5)Ps-insensitive stores, and cAMP-stimulated Ca'-uptake can occur independently oÍ G-proteinactivation. Diacylglycerol can be produced byother signal activated phospholipases otherthan PLC-induced Ptdlns(4,5)P2 hydrolysis.ln plc- cells normal chemotaxis can beattributed to the presence oÍ PLC-independent routes to generate these secondmessengers. However, many questionsconcerning PLC activity in wild type cells andregulation of Ins(1,4,5)Ps levels remainunanswered.  The near ly  normal  Ins(1,4,5)P3levels in p/c- cells suggest Ins(1,4,5)P3 isessential Íor viabil ity. On the other hand, it ispossible that the contribution oÍ PLC in theIns( l ,4 ,5)P3 product ion oÍ  wi ld{ype cel ls  ismuch lower than generally expected.Concerning PLC- independent  Ins(1,4,5)P3Íormation, do plc- cells show a more activedegradat ion oÍ  Ins(1,3,4,5,6)Ps to produceIns(1,4,5)Ps as suggested by the reducedlns(1,3,4,5,6)P5 levels? Have plc- cellsadapted to the absence of PLC activity bygenerating new paths Íor Ins(1,4,5)Psproduction? Furthermore, the role oÍ receptorstimulated lns(1 ,4,5)Ps ^ production inregulating intracellular Ca" levels has to beestablished. lt wil l be interesting to see iÍintracellular Ca2* levels are regulateddiÍÍerently in plc- cells than in wild type cells,and iÍ this explains the inhibit ion oÍchemotaxis by partial antagonists in wild typecel ls .Concluding, disruption oÍ the PLC genehas demonstrated PLC activity is notessential Íor chemotaxis and development inDictyostelium discoideum. Unexpectedlyhowever, many questions have been raisedwhich remain to be answered.REFERENCES1 . Bominaar AA and Van Haastert PJM (1 994),Biochem. J.  297:  189-1932. Van Dijken P, De Haas JR, Craxton A, ErneuxC, Shears SB and Van Haastert PJM,acceoted J. Biol. Chem.3. Stephens LR and lrvine RF (1990), Nature346: 580-5834.5.6.Craxton A, Al i  N and Shears SB (1995),Biochem. J. 305: 491-498Bominaar AA (1995), ThesisBominaar AA and Van Haastert (1994), J. CellS c i .  1 0 4 : 1 8 1 - 1 8 5

Phosphoinositide-specific Phospholipase C in Dictyostelium discoideum,A molecular genetic approach to study its function and biochemical properties

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Phosphoinositide-specific Phospholipase C in Dictyostelium discoideum,A molecular genetic approach to study its function and biochemical properties

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