ONCOLOGY REPORTS 1: 1269-1275, 1994
Hornall and Xenopus MOlS mRNA are highly conserved but show different patterns of expression in adult tissues
DIETER KüBELT, THOMAS KARN, BJÖRN HOCK, UWE HOLTRlCH, ANDREAS 8RÄUNINGER, GEORG WOLF, KLAÜS STREBHARDT and HELGA RÜBSAMEN-WAlGMANN
Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Hau~. PaulcEhrlich-Str. 42-44, 60596 Frankfurt, Germany Received Au güst 30, 1994; Accepted September 23, 1994
Abstract. Phosphorylution of human .p34'.'"'1 at Thr 161 seems to be necessary for its cutulytic activity. CAK (cdk activating kinase) contuining p4o~w15 t'rom Xenopu.,· egg extracts phosphorylates and activuü!s p34"''"1 in u cyclin dependent manner at Thr 161. We describe the cDNA sequence coding for human MO 15, which predicts a serine/threonine kinase of 346 aa. Despite ·the high homology of91% between the human und Xenopus proteins we observed a rather different mRNA distribütion in adult tissues: In cantrast to ubiquitously expressed human MO 15- transcripts M015-mRNAexpression in Xenopus is restri<.:ted to oocytes indicating a differenrcellular rote in these two phylogerietically distant species, By virtue of the homology to members of the family of cell cycle kinase genes we examilled MOI.S mRNA expression for its correlation to the proliferative activity .of cells .. Stia!'uh1tion of lymphocyies showedMOIS mRNA expressTontobe independent of mitotic activity.
lntroduction
Growth factors bind to speci tic cell surface. molecules such as receptor-tyrosine kinases to initiate a signal transductimt cascade that ultimately triggers the transcription of illlmediate and delayed early reSp(mse genes. Late in G1-
phase growih factor induced signals activate the cell-cycle ma.chinery, which contalns cyclins und cyclin-dependend kinases (cdk) as key components (for a review see ref. I).
The protein cdkl or p34«k1 was found to hc the product of a
celt
divisimi·gcne, L"dc2 or Scl!iz.o.va!'c/uu'omvces pombe und CDC28of Sac:churomyc:es cerevüiae re~pectively. The kinase activity of these proteins is required for two key tnuisitions. in the cell cycle, namely the passag~ through Stl}rt and the G2 toM transition.The regulation of p34<Jd has heen shown to be complex, 'invölving subunit rearrangements as weil as molecular
Correspoiulence to: Dr Klaus· Strebhtm.h. Chemotherapeutisches Forschungsinstitut,. Georg-Speyer- Haus, Paui-Ehrl ich-Str. 42-44, 60596 Frankfurt, Germany
Key words: p4UM01\ cell cycle, cdc::?.-related genes, phosphorylation, r!!gulation
mildil"icatiun. p34«k! physically associates with cydins, which Jtre required forits activity, phosphörylation (2) anu
subst~atespecillcity (3~6). The mitolic cyclins A and B were originally identificd as protcins whose leve.ls oscillate throughout the cell cycle (7). Connections between the cyclins as cell cycle n!gulators and cellular Iransformation provide evidence that disturbances in oorma.l cell cycle control play a · critical rote in oncogenesis (for a review see . ref. 8).
The reversible phosphorylation of profeins is recognized to be a maj6r rllechanism for the conlrol of intracellular events in eukaryotic cells: Three rnajor sites of phosphorylatiori (Thr .14,Tyrl5 andThr 161)have been identitied in the human p34"Jd protein: The dephösphorylation of Thr 14 and Tyr 15 as weil asphosphorylation of Thr 161 are required Tor its activation. Negative regulation of mitosis occurs by phosphorylation of p34"'1d on Tyr 15 by the wee , ..
protein kinl}se with serine-, threonine" and tyrosine- phosphorylaürig activities (9,1 0). This Ieads to the inactivation of the p34<dc2~cyclin B complex (10,11). Various obs.ervations suggest that the product of the cdc25 gene dephosphorylates Thr 14 and Tyr 15 thereby activating p34"''"2(for a review see ref. 12). The other phosphorylation of human p34«'"2 whiCh is necessary for its catalytic activity occurs at Thr 161 (corresponding to Thr 167 in Scliiz.o~
sacdwromyc:es pimtbe), a residue which is highly conserved in the cdk-family. Site~directed.mutagenesis of this site provides a form of p34<Jcl thät lost its catalytic activity and can act in a dominant-negative fashion. (13,14 ). In somatic ccils of.vertebmtcs and L"ellcxtracts i"roan Xenopu~~ eggs.
formatiön of cyclin B-p34<Jd complexes requires phösphorylation of '(hr 161 (15; 16}. Complexesof p34"'1"2 . with cyclin B are inactivated at thetransition from metaphase . to anaphase. Subsequently, cyclins are degraded and Thr 161
is dephosphorylated ( 1.7). Thr 161 is located within the autophosphorylation domain öfp34<dd but neither p34"d"1 alone nor complexes with cyclin B show auto- phosphorylation activity. ln conträst, a kinase activity distim:l from p34<Jd has been showri 'to phosphorylate Thr 161 in p34"''"2 ( 16). CAK (cdk-l.tctivating kinase) from Xenopus egg extracts and starfish oocytes specitlcally phosphorylates and activates p34•"•2 and p33c''~2 in a 9yclin-dependent nianner at Thr 16l·and Thr 160 respectively (18~21).lnthe pu('ification procedure of CAK the starfish' homologue ofXenopus p40M015 was found to remain assQciated to a'sl!cond
1270 KOBEI.T et al: HUMAN AND xenotms MO is mRNA polypeptide. This peptide possibly is a regulatory subunit
required to confer catalytic activity to p40M01 5 ( 19).
Protein-kinase genes are oftenexpresscd at low Jrequcncy and thacforc difficult to ünalyzc. This ohstaclc can bc ovcn.:ome hy üsing llcgcncratc oligonucleotidc primcrs hascd on conserved motifs of the kinase dumains for thc PCR amplifiCation of reverse~transcribed mRNA (22c26). In the present study we identifieda gene coding for a proti~in kinase from embryonie tissues in a PCRcmediatcd approach. The cDNA representing the complcte open reading frame. which
·. is likcly thc human countcrpart of MO 1.5, was isolatcd from a cDNA lihrary hased tin huinan RNA. ln conlrastto thc Xemlpus counterparl. the e)(prcssion ofhuman MO 15 mRNA . is ubiqüitous in adult tissucs suggcsting a. different roJe .o(
MOlS in adult tissues of thcsc phylogcnctically distaut
· spccics. ·
Materials and melhods
IJNA seqtrellcill~. 0.6 pmol of DNA, 4 plittil primcr and a Ta4 DycDcoxy Tcrminätor Cycle Scqucncing Kil from Applied Biosystems wcre used according to thc maimfacturer's protocoL The products of the sequencing rcactions were purified by phcnolkhloroform cxtraction and analyzcd on an Applicd Biosystem 373 A DNA s~qucnccr.
The primers uscd for amplification of thc cDNA were ECO- VHRDL (5'~TTTGGAAITCGTNCAYMGNGAYYT~3')and P6l2DEA
I
(5'-TTGGAATTCATCCCNNN NNNCCACACATC~3') (23).
Aminoacid sequences were aiigned with the GAP programof HUSAR (Heidelbcrg UNIX Sequencc Analysis Rcsource, Deutsches Krebsfm:schungsz.cntrum, Hcidelbcrg).
PCR; RT-PCR with RNA isolütcd l'rtimXc•llopus~lissucswas
pcrformed according .to the fQIJowiilg schcdulc.: 95"C for I min, 60°C for 2 min and 72"C for J min and a final
· incubation for I 0 min at T}:c.
RNA i.mlation ancl Nortltem h/ots. Xcitilfllt.\·-lissucs wcrc.
homogcnized in a guc:u1itlinium isothiocyanatc solutibn ünd RNAwas isolated by centrifugation through ä 5.7 M (:sCI cushjon./ 20 J.lg total RNA was elcctri>phorcsed in a denaluring agarose/fi>rmaldehydc gel and transferred to a nitrocellulose membi·ane.
Pur~{ic(tlion (~f recatitl}iflant MO 15"tn·otein. GST-MO 15 was constnict!!d. by cloning tl)e PCR-amplified opcn reading·
frame ofMOI5 into the plasmid pGEX-2T (EcoRI/Hindlll).
lts
orientatimt was checked hy PCR and sequencing;Synthe~·is of recot1tbinant proteins from transfonned .bactcria was induced with I 00 J.!M isopropyl-ß~D~galactoside (IPTG) al 30"C for 16 h; After.harvcstand Iysis ofthe cells (he lysate was sonicated and centrifuged at 18.000g for 30 min. ·seads were collected hy ccntrifugation an<J.washcd twicc in Iysis liuffer. GST-MO 15 was dutcd with 5 mM rcduccd gluthathione (Sigma) in 50 mM Tris-HCJ pH 8.0, I mM DTT. Sampies were analyz.cd hy 12(1/n SDS-pagc.
For
thromt>in digestihn of.GST-MOJ 5. I J.lg of the fl1sion protein Mund tn gllitathiilll·ScptHII'o~c wns washt:ll with huffl~t·, ,Jigcstcd with 10 ~llof IOtJ/mlthromhin at 2W'C l'nr 2 h anll lhc supci'n:1tant was an:ilyzcd hy SDS-p:igc.
Screetiing (~f'/11111UIII cDNA libraries: An oligo(dT) primed cDNA lihrary from a primary.human squamous cell lung c(!rcinlllila in Iambda gi I 0 was screened under high- stringcncy conditions (42°C, 50% formmnidc) with a probe corrcsponding to nl 514-666 of human MO 15. A random . primcd cDNA lihrary in Iambda Max Urom human thymus was screencd undcr the same conditions with tw() probes corresporiding to 1it 514-666 and nt 4~262 of human MO 15 respectivcly.
Rcstllts
Cloning und clwraderh.ation f~f/umian MO 15 cDNA. cDNA from human embryonie tissucs was ai11plified u·sing the prinicrs<ECO-VJ.IRDL and P612DEAI) corrcsponding to tlw highly conscrvcd aniino aCid motifs from subdomüins VI and IX (VHRDL and DVWXXG. rcspcctivcly)ofprotcin kinases (27). The PCR products wcre Iigated into the Bluescript KSIH+I vcctor (Stra(agcnc). Transformation of competcnt JMI 09 cclls yicldcd 2XO rccrlmhinant clones encompassing 7 unknown protcin kinascs. One of thcse cloncs was closcly related' to human cell cycle kinases such as cdk 1-3, PSSALRE and in particular to MÖ 15 from Xenopus. This clone, dcsignatcd K2. was uscd as a probe to. scrcen 1.8x I 0'' .recomhinünt doncs from an oligo(dT) primcd cDNA library in Iambda gtl 0 from a primary human squamousccll lung carciitoma and a randqm primed cDNAiibrary inlambda Max I f1:om hum<lnthymus. 5 dones ranging from 200 bp to I 040 bp were. obtained. Determination of the nuclcotide sequencc of all twerlapping clones and verification of the
sequenc~ hy PCR and. sequencing from normal tissue revcaled a combined scqi.1encc of 1274 bp with a single open
re~tding Jramc of l!ßR nt nmging from a putative start cödon
· <lt. position I 04 .to an in-fnunc stop codon at. position I 142 and prctlicting a protcin of 39 kDil which contains 346 aa (Fig.J). A stop codon in thc sarne rcading frame but upstrcmil of thc putative in.itiation codon was idcntilied. We . vcrilicd the prcdktcd m~,Jecular wcight bf .the K2~protcin by using thc Escltericltia mli J M I ()l) straitt anti pGEX as vcctor lo oyerexpress recomhinant protein (Fig. 2).
A computer search indicated that the complete open reading frame ofthe putative K2cprotein is homologaus to a VariC!y of protcin kinascs prcscnt in thc Swissprot- and PIR- protein data hasc (EMBL) indicating that ii hclongs to this dass of enzynies. Using .thc sequence of thc complete open reading frame wc. determined the identity of the K2-protein to be 43% to the human p34<dc~. 44.9% to cdk3, 46;4% to PSSALREand 49.9% to p33<dk~. The aminocterrninal portiein of the putative K2-polypeptide reveals conserved sequence motit's of serine/threonine kinases. The consensus ~equence GXGXXG found in subdomain I. (27) öf nucleotide bindirig proteins and kinases is modified ·as GXGXXA~motif. In uddition hl thcovcraiJ hömology.don1ains tobe h.ighly
· conscrvcd within thc l~tmily ofcdc-rcluted kinuses, such as the PSTAIRE motif or the rcsiducs whosc phosphorylation
<lJlpears -to hc signilic<illt for thc activity of verlebrate cdc2- relatcd gene products. arc. partially'detectable in K2: 4 out of 7 uu t'!'cui•thcflSTAH~E·mütif urc i~lcntlcuJJn K2. Incmmust tn Thr 161 ol' pJ4•··""~. which hus acorrcspnmling rcsidue in K2. thc phnsphorylataJ}Ic rcsiducsThr 14 and Tyr I~ werc
ONCOLOGYREPORTS 1: 1269-1275, 1994
_, .
.
CCCGGTGGACGGAAGTGGGTGT'rGGAGGCTT:r.AAGGTAGCTTTAAATTcGTGTTGTCCTG GGAGCTCGCCCTTTTCGGCTGGAGTCGGGCTTTACGGCGCCGGATGGCTCTGGACGTGAA M A L D V K GTCTCGGGCAAAGCGTTATGAGAAGCTGGACTTCCTTGGGGAGGGACAGTTTGCCACCGT
s RA KR v E K L o E" L
BE Bo
FII
T vTTACAAGGCCAGAGATAAGAATACCAACCAAATTGTCGCCATTAAGAAAATCAAACTTGG Y K A R D K N T N Q I V A I K K I K L G ACATAGATCAGAAGCTAAAGATGGTATAAATAGAACCGCCTTAAGAGAGATAAAATTATT
H R . S E A K D G I jN .it T 'A
:tt Jl
EJ I K L LACAGGAGCTAAGTCATCCAAATATAATTGGTCTCCTTGATGCTTTTGGACATAAATCTAA Q E · L S H P N I I G L L D .A F G H K S N TATTAGCCTTGTCTTTGATTTTATGGAAACTGATCTAGAGGTTATAATAAAGGATAATAG
I S L V F D F M E T ·.D L t V I I K D N S TCTTGTGCTGACACCATCACl\CATCAAAGCCTACATGTTGATGACTCTTCA.AGGATTAGA L V L T P S H I K A Y M L M
T
L Q G L E ATATTTACATCAACATTGGATCCTACATAGGGATCTGAAACCAAACAACTTGTTGCTAGAY L H Q H W I L H R D L K P N N L L L D TGAAAATGGAGTTCTAAAACTGGGAGATTTTGGCCTGGCCAAATCTTTTGGGAGCCCCAA E N G V L K L A
itMMiJ
L A K . S F G S P N TAGAGCTTATACACATCAGGTTGTAACCAGGTGGTATCGGGCCCCCGAGTTACTATTTGG R A Y T H Q V V T R W Y R A P E L L ~ G AGCTAGGA TGTATGGTGTÄGGTGTGGACATGTGGGCTGTTGGCTGTATATTAGCAGAGT'l' A RM
Y G V G V D M W A V G C I L A E L ACTTCTAAGG(;TTCCTTTTTTGCCAGGAGATTCAGACCTTGATCAGCTAACAAGAATATT L L R V P F L P G D S D .L D Q L T R I F TGAAACTTTGGGCAC:bCCAACTGAr:;,GAACAGTGGCCGGAC•ATGTGTAGTCTTCC.AGAT'l'A E T L G T P T E E Q W P D M C S L P ~ Y TGTGACATGTAAGAGTTTCCCTGGAATACCTTTGCATCACATCTTCAGTGCAGCAGGAGA V T C K S F P G . I . p' L ~ H I F S A A G D CGACTTACTAGATCTCATACAAGGCTTATTCTTATT'l'AA.TCCATGTGCTCGAATTACGGCD L L D
L
I Q G L F L F N P C A R I T A CAC.I\CAGGC ACTGAAAATGAAÖTATTTCAGTAATCGGCCAGGGCCAACACCTGGATGTC AT Q A L · I< M. K Y F S N R P G P T P .G C Q GCTGCCAAGACCAÄACTG'l'CCAGTGGAAACC'I'TAAAGGAGCAAGCAAATCCAGC'l"l"T'C;Gc L . P R P N C . P V E T L K E Q A N P A L A AATAAAAAGGAAAAGAACAG.Z:..GGCCTTAGAACAAGGAGGA TTGCCCAAGAAAGTAATTTT I K R K R T E A L E Q G G L P K K L I F T,XAAAGAGÄACACTGGÄCAACAT'I"TTACTACTGAGGGAAATA"GCCAAAAAGGCAAATAAT GGAAAAATAGTAAACATTAAGTAAATGCTGTAGAAGTGAGTTTGTAAATATTCTACACAT GTAAAATATGTAAA
Figurc I. Nudco.tidc ,•mll prclli.:tcll :unino ;o.:ill SCl(UCii<:cs of hum;on MO I S indullin~; parlialuntranslatell SCl(U<:nccs. Thc suirt codon is in hold type, slopcollons ;orc unll.:rlinc.J. Thc llighly conscrvcd motifs of thc kinasc domain (27) ar.: shown in invcrtcd l.:llcrs: thc inv;triant r.:sillues !DFGl implicatcd in A TP-binlling. and the conscitsus sel(ucncc GXGXXA of scrinclthrcuninc kinascs rcl;otcd lo cdc2 (33 ). Thc amino adds sluiwing hmnology with thc cdc2 PSTAIRE motif(34) .an~ shaded.
notfound in K2. This comparisoli dcmonstratcs that K2 docs not bclong to the cdc2-family of polypcptidcs.
In contrast, homology of the K2-protcin wasJound to be 86o/o identical and 91% similar (including conservative amino. acid exchanges). to p4()Mli15 from Xenop11s( 18). Dueto
· th~ high degree ofhomology and the same domain iopology.
(Fig. 3); it seems very likely that. K2 represents the human
homologuc t>f the Xeiwp11s gene MO 15. Therel'ore, wc propose tocall il M015 as weiL The genewas isolated in parallel by two groups (36,37) calling it (HS)CAK I and STK I respectively. Only vcry short stretches of starfish MO 15 are known ( 19)': These are highly conserved compared to the human countcrpart. Mos1 amino acid Substitutions between human and Xenhp11s p40M015 are located in the
1272
KüBELT ctal: HUMAN AND xellof>ri.f MO 15•mRNA---MALDVKSRAKRYEKLDFLGEGQFATVYKARDKNT MEGIAARGV •• R ... Q •.••••• · ... . .NQIVAIKKIKLGHRSEAKDGINRTAI.REIKLLQELSHPNI.
DR .-. •.••••••••• •. A ~ • N • ...••...• ~ .; .. -· .. , ., ....
.IGLLDAFGHKSNISLVFDFMETDLEvUKDNSLVLTPSHI
-• •· • • • • • • • • •· • • •••••••• _ •••••.••• _ • T •.•• ·-. f; • A. ~
~YMLHTLQGLEYLHQHWILHRDLKPNNLLLDENGVLKLA .S •• •-• ·---~-"!' •••• • HL ••••• •.• •••••• _. • .~ ••• -•••.•
DFGLAKSFGSPNRAYTHQVVTRWYRAPELLFGARMYGVGV·.··
••• : • •••••••.• I . . . • • • • . -•.• ~-s ••• ~ •••••• -._ •••
DMWAVGCILAELLI..JtVPFLPGDSDLDQLTRIFETLGTPTE
... ·- .. -· ... ·- .. -.
-··-... .
EQWPD~CSLPDYVTCKSFPGIPLHHIFSAAGDDLLDLIQG
•••• G-. S •• _ ••.• --.cAF· •• · ••• T: • .. L •• I. e •.•••.• E. L ••
LFLFNPCARITATQALKMKYFSNRPGPTPGCQLPRPNCPV
•• T~. • ••• C •. ;·s ••• RKR.:•• •.• A •••• N:L .•.•••• SI ETLKEQANPALAIKRKRTEALEQGGLPKKLIF
.A •••• Q.LN. G ••••• ~. GMD. KDIA ••• S.
Figure 2. Deduccd ;unirHi acid scqucncc ofhuman· MO 15 ;uul ils comparisi111 wilh the· Xc'IIOf>ll.> hnmi>lnguc. Only thc rcsiducs dillering hetwccn htiman and Xc•lloflll-' scqucm:cs arc shown for' lhc I aller. ( iaps arc indicalcd hy hyphcns.
1 2
67.5kD -··
40 kb
···.-&..._,·
····.··· ....Figure J. Expression and puriricatiiur nf rcconibinanl MOf5,prt>tcin frnm haclcria .. SDS-j1<tgi: of(iST-MO 15 <lanc .I l arul (iST-MOI5 alkr lhromhin.
digc~lii>n Cl<tnc 2!.
curhoxytcrminal porl'ion of lhc protcln. Morcovcr. thc aminotcrminus of p40"0'~ fr11111 XcuoJ1U.I' .j~ hy 6 aa Ionger lhanthc human polypcptidc .. Tiic ATP:himling domhin o( thc MOI5 prritcin is idctllical comparcd lo ils starfish and Xt'llopus countcrparls (I l{ 19 ). Thc following motifs in suhdomains VI dii<DLKI'N.aa U.';·f•lll anti VIII t<iSPNI<ÄYTII. aa Hd-171>. whidt ari· characltristic l'or ccrtain scrincitht·collilil~ li11asl·s. <trc <tlmost 'idcntical hctwccn lhc countcrparls,ofMOI.".
AKT :1 I RAC-alpha I PKB AKT-21 RAG-beta
'---.,..._~~ cAPK ' - - - . . . - ' - - ' - - - - DMH
·,.--.--'--"----'~ CaMi< · r - - - i ,..--'...,L,.,---,...---,--PSK·C3
' - - - ' - PSK-Hl
'---~---~-PLK
'--'-'----'---...,...--....;... p78K CDK2 .
CDK3
CDK1/CDC2Hs r--t--,_;. CDK5 I PSSALRE
PCTAIREcl PCTAIRE-2
PCTAIRE•3 ri'---'----'--....;..·~mm
'--"---~--GTAP58 CDK4 I PSK-J3 CDK6 I PLSTIRE '---~~- CHED
' - - - ' - - - CKII·alpha ERK'l ERK·2
L
---'----[======:-;
PSK'H2 PSK·GlC·RAF I RAF-1
'---.-~'---'-"-~-t-B·RAF A·RAF
r----..,--.--~-- PIM-1
'---'---_.;..~-'---,....,,....--TTK I PYT ' - - - . . . , - - - ' - ' - - - : - - dsRNA:PK ' - - - ' - - - MAPKK IMEK-1
'---~~---- MOS
Figurc 4. Phylngenciic rclalionship. ol' 381mman scrinc/threonine kinase scquenccs. Thc sequcnces were ohlaincd frnm lhc Protein Kim1se Dala Bank of S, Hanks et a/ (Thc .. Salk lnstitulel and .lhc EMßL Gcnh;rnk. Swjssprot
·and I'IR dalahascs. 3!1 hlllmrn scrineWu'eonii1e kin;rse sequcnces wcrc aligncd ovcr 1hc cnlirc calalylic Jom;iins (27). Phylogcnctic rclationships wcrc dclcnnincd hy using lhc TRi:E prt1grmn of HUSAR (Hcidelhcrg'UNIX Sc,qucncc Analysis Rcsourcc. Dculsch~s · Krchsforschungszerllrum.
Hci'delhcrg). h<tscc.l on lhc progressive alignmcnl melhnd of Fcng and Doolilllc ( 35) in a.niullijllc sc_qucncc alignmcnl.
. These data indicate that MO F5 is a gene highly conserved through cvolution from echinoderms to maminalia. The scqucnccs of thc catalytic domains of 38 human serinc/
thrconinc kinascs werc uscd tö dctcrmine thcir phylogenetic relationships; As shown in Fig .. 4 pJ4<d<2_relatcd molecules fall into. a number of sub-familics. For exainp'le, the MOlS- protein is most closcly rclatcd to the PCTAIRE-subgroupand to CDKI-3 .. which share thc highly conserved PSTAIREc motif: Coinparing MO 15- with the CDK-family and PCTAIREcsubgroup~protcins wc find a hoinology betwcen 39 and 47%.
Human mul Xt'litipus M0/5 mRNA .flww dil•ergiug pallt'l'll.f
~~l t'Xprt•.\'sion in adult ti.f.mes. Using a IJNAfragmcnt of MOI5 which ovcrlapswith its kinasc domainas a prohcwc pcrformcd a Northcrn lilöl analysis öf RNAs fn~m a varicty nf human tissucs. Transcfipts nf LR kh wcrc dcicctcd inal.l tissucs tcstcd ( Jiig. 5a.hl. Vc1;y hi!!-h Ievels ill' lratlscripfs wcrc ohsl·rvcd i n-.Jilaccnla:< skclct.al musdc. k idt.Jcy; lcstis .. ovilry.
aml colon. Rt'ganling lhC lact I hat lhc catalytic dumains of protcin-kinascs arc highly conscrvcd (27) wc could not rulc
ONCOLOOYREPORTS 1: 126lJ-1275, 1994
1273
a
1 2 3 4 5 6 72.4 kB -
•
1.35 kB -
. GAPDH -
••
b
12
3 5 6 7 82.4 kb -
1.35 kb -
Actin -
out that the data presented in Figs: 5a and b nre at least partially due. to cross~hybridization. Thus, a regi(m 5' froin the kinase domain which is known
to
be more divergent withiai the family of protein kinases was chosen for the generation of a second probe. Still, MO 15 trunscripl~ wcrc . foulld in all human tissues analyzed (Fig. 5c). Coi11päring Figs. 5b andc which contain the same RNA but differ in the probe used we observc diverging patterns of hybridization under high stringency conditions. This sllggesls Lhal in hummi tissues MO 15-rclated trhnsci·ipt!; arc prcscnl. Thus, an intluence by different thermodynamic stabilities ofthe two · probes used oa:by alternative splicing ot'the MO 15-message, as shownby Levedakou et al (36), can not beruled out.In cantrast · to our data on hurnan adult tissues Xenopus . MO 1. 5transcripts were not detectcdin adult tissucs and wcre
· found to accumulalc only during oogcncsis (J 8.). Wc therefore allalyzed RNA from various Xenopus tlssues to
a 2.4 kb
~·;. I
. . :.:=:).i:~.J.;.::!:~f
.. .
c·
1 2 3 4 5 6 7 82.4 kb -
•••
1.35 kb -
ACtin -
Figurc 5. Nm'thern analysis of MO 15 expression in human tissues. Each lane
~f the mulliple tissueNorthern blots'(Ciontech, USA) contained 2jlg human poly(A)' RNA. ('.I) Lanes 1-8: heart (lane I), brain (lane 2), placenta (lane 3), lung (laue 4), Ii ver (hmc 5); skclctalmusclc (lane 6), kiuney (lane 7) and pam:rc;as (lanc Xl. (h) ;uul (c) lancs 1-X: spieen <lanc I), thymus (lanc 2).
prqstatc <lm1c j), tcstis (ianc 4), ov;ary <lane5), small intestine (lane 6).
culun (l;anc 7) and pcl"iphcrul hluud Oanc K). PCR wasused to obtain specilic probcs of human MOlS. Blots (a) and (b) were hybridized with a probe spanning the region nt 514-666 of human MO 15 thus containing the kinasc doimtin. Blut (c) was hybridized with a probe spanning the region nt 4-262 thus lacking .thc kinasc dün1itin. Positions of sizc markers are imlicatc<L Cytnskclctal·actin- ( h.c) and glyccraldchydc'3-phosphatc dchydrugcnasc (Gi\PDII).(a) -tr;anscripts whidt wcrc hyhridi.tcd tu control for n:covcry and t\on-specilic.dcgradation.
compare the palterns of MO 15 expression in the two JiiTercnt specics. Under the same hybridization conditions as applied for the Northern blots shown in Fig, 5 we detected MO 15 Lranscripts only in Xe1wpus oocytes (Fig. 6). An identical hybridizatiön pattern was obtained with probes from Lh(! ami.rtoterminal or the kinase domain (Fig. 6a and b).
Only in oocytes we found two MO 15-related trariscripts under conditions of high resolution {Fig. 6a), whiCh differ by . approximately IOObp. As. shöwn in' Fig. 7a, RT~PCR with .RNA from Xenopus confirmed the high expression in oocytes. Howevcr, with intreased sensitivity due to additional PCR cycles (Fig. 7b), weak signals also appeareJ
in Jung, ovary, und colon. .
In view.of the fact that for a gene from two very distunt phylogenetic species the homology of MOlS is very high, COnservation of the SlfUC(UfC and functiori of p40MOIS SCCms tobe crucial for.its role in cellular physiology. Therefore, the fundamental ditTerence in the paltern of expression of this gene in adulttissues is unexpected and quite remarkable.
b
1 2 3 4 5
·
.
I
(,.
\~~..•.
·~; ~~ . '.-~{~.
'
. ·
Figurc 6. Nonhern .analysis p(M015.expressio1i itl Xeiwptis laev.is tissucs. (a) RN/\ fronl hcan Oane I ),.liver (laric 2), colon (lane 3), oocytes <lane 4), ov;ary (lane 5). lung (lane 6) .and inusde.Oaric 7} was hy.hridized witha '2P-Iabcllcd pröhc starting·at codon 76 and cnding at.codon·196 ofthe Xmupus MOI.'i S1!4UI!IlCe ( 18). (h) RNA from llocytcs o:me ).) .. ovary (lanc·2), Ctllon (lanc 3); Ii ver (lane 4) and hcart (lane S).was hyhridizcd with a np"Jabelled prohe starting ät.codon 515 and ~nding at cndon685, thus containing thc kinase Jmna.in o(thcXeiwp11s MO 15 se4uencc ( IK).
1274 KüBELTet (1/:. HUMAN ANDxeiwfm.t MOI5 mRNA
2 3 4 s. 6
b -·.- ~--··
· Figurc 7. Expression tlf Xem>tms li1cvi~ M015 in Llifli:rcnt mluli tissucs ;md oocytcs. RT-PCR wa.~ uscllto m1aly7.c thc exprcssion nf M015 inRNA. TI1c source of the RNAs for thc ·RT rcaction:i is inllicatcll abovc cach hmc, -li::hing. 2=o(x:ytcs; 3=ovary, 4:i:musdc. S=olon, O=livcr. (a) 3n cyclcs; (b)
34 cyclcs.
1-:.r/Jre.\~\·itm t~( M0/5 mRNf1 in rc'.'iting cttul i11itogc'll~illtlttd•d cells. Since MOlS clearly helongs to thc family of ccll cyclc- kinases we wonoered if the expression of MOlS correlates with the proliferative activity of cells. Lymphocytes from human periplleral blood (PBL) were.grown in presence ()f phytohemagglutinine (PHA) and interleukill 2 (lL-2)
to
induce proliferation. PHA activates the expressio11 of IL-2 and its high-äffinity receplor. which are components ·of an autocrine loop Ieuding to lymphocyle proliferatiön.
PHA was addcd to lymphocytcs in culturc at dayO and removed at day 2. RNA frmnthcsc lymphocytes collccted at days O, I (lnd 2 was analyzcd foi'MO 15 cxprcssion in Northem blot experiments (Fig, 8a, lanes 1-J ). Mter rcmovül of PHA lymphocytcs were cultivated for three üdditional days (Fig. 8a; lane 7-9) ... Furthermore, eyclohe;imidc~ an inhibitor of protein synthcsis. was uscd to study the ciTcct of de 110\-'o protein synthesis on the Ievel of MO 15 transcripts;
The results show that resting cells .express MO 15 mRNA
<Fig. 8b, lane I} and that the PHA-induced autocrine IL-2 stimulation does not change the le"el of MO 15 expression
(Fig~ 8a, lanes 7-9). Suhsequcnt activaiion of lymphocytes with IL-2 docs not aller thc Ievel of MOlS exprcssion eilher (Fig. 8a, lanes 4~6). Thus, the mRNA expression of hml1an MO 15 does llot seem to be assoCiated with the mitotic activity ofeclls. Furthermore cydoheximidc docs not inlluencc
MO
15 mRNA cxprcssinn in lymphocytcs (Fig. 8h. ·tanes
1~4).a
1 2
3 4 5 6
2.4kb
-
~··
- - -- -·
1.35kb-
Discussion
_The pa:Uern of expressionof MOlS in human tissues is -- totally different from tlle expression of the hm11ologol)s gene in Xl'twplls: No MO 15 ttanscripts could he detccted in adult tissucs from XeiiOfJIIS such as adult liver. s-pleen. testis or skelctal muscle. Xell(ipus MOT5 mRNA is accumulated d~ring oogcnesis, bccm}les dcadet1yhitcd during meiotic inaturation ünd is degraded after ·tllc mid-hlastula-trünsitil.m statc of cmhryogcnesis. Shuttlcwörth et al ( 18) draw the conclusion that thc. function of Xenopus p4()Min~ correlates with processes before or during matUr..Uion of oocytes. Thus, p40r;.1111.~ would eilher control arrcst ofllleiosis .or negatively triggcr the events Ieuding to thc<activation ofMPF. In contrast. human MO 15 appcars to be expressed ubiquitously in .adulttissues. Duc to this diverging pattern of cxpression of human andXenopu.v MOI5 we would Iike to.suggest. that MO 15 has functions in.thc maminalim1 ccll which eithcr are not neeucd in amphibia or Laken over hy othcr genes in these specics.
lnterestingly, such differences in exprcssion also seem to apply to other genes: c-myb was found in a broad speetrum of - tissues from Xetw[ms (intestinc. Ii ver. heart .. Jung; ovary.
kidney. spieen, oviduct. testis andblood) (28),whilc human c-myb-mRNA seems tobe restricted tocells of hematopoietic origin (29). Also, mouse c-myb mRNA was deteeted by Northern blot hybridization only in lymphopoictic,tissues (splccn änd thymus) (30). c-mos transeripts are detectable by Nm1hern analysis in testis, ovary and brain of Xello[ms (31 ).
Using Northem blots mos inRNAwas foundin testis and ovary .of mouse and f urthermore in brain. kidney, plaeenta.
mammary gland, heart and lung hy applying the
St
nuclease protection assay (32). Various groupsapplied techniques of differenf sensitivity to evaluate the lnRNA cxpression of c-·• mos and c-myh, therefore. it is hard toestimate if the pattems of expression diverge in a similar fashion as shown for MOlS.
Many groups have shown that XeiiOfJ/IS imd starfi:o;h
· M015 is thc catalytic suhunityfaprotein ki11asc that activates pJ4nk! .( 18.19,2 I ) .. Meyerson ei al (33) con1pared the distribution of cdk-related protein kinase~mRNAs in human tissues. ln this · study. cdc2~transcripts · were only dctccted in human lung ünd placcnta. but nonc in hcart. brain.
livcr. skelctal musde. kidney and pancreas. 1t i~ surprisiilg
b
7 8 9 1
23 4
• ·-
-t ... • ... • - ... -
~gure 8. lrifluelice of PHA. IL-2 and cyclotreximide on MO 15 mRNA expression in, human peripheral blpod lymphocytes. Northeni blot analysis or RNA .lrom human lymphocytcs with il MO 15-spccitic pmhc. Each hmc cnntaincd 7 ~tg nf total. RNA. The blol' was hybridi7.cd with a pmhe spanningthe region nt
~ 14·(>flh nf humnn MOH th1111 ct>ntuininll the kinuNc dnnmin. Sit.c nmrkcrs nrc indicatcd. ln 1 MOl :1 mRNA. CXJ1rcssinn in: rcstinlllytilphocytes (hmc I).
ly111phncytc11 Mtinllthtll:d with PIIA li>r I duy Cl um: ~)und 2 dnyM llunc ;\ ), in lymplu~~:yh:N i.timlllnh:d withi'UA fnr ;' dnyN und then .:ulturcd withnut PIIA fnr I
<hntc 7), 211une K)und .\ !Junc .,.,mldillonut lhtyH. M(JI~ mKNA CliiWNSlnn in: IY111Ilhlli.'Y1~·~~ NtlmuhttL'I.fwlth;PIIA l'nr ·' dnyM und nftcf NlllllYIII nr PIIA culturcd with 11.~2 fnr I llunc 41. 2 IIane :'i) nr 3 d:1ys <hmc 6). (hl MOI5 lnRNi\ l'XJ1rcssinn in: rcstinl! lyml>hncyll'K (lurn.' I l.lymJ'Ihncytcll slimulittcd with I'HA för I day (lane ·2>. stimulatcd with I'IIA fnr 2 dnys (lunc ~~ und stimillutcd fnr ·1 duy with J>liA mtd .:ultun:d l·udditinnul duy \Vlth cyclnhcximldc und
PHA !lane 4 ). ·
ONCOLOGY REPORTS 1: 1269-1275, 1994 ·.
1275
tlllll mRNA c:oding l'ur p4()MIII~ lhc p.lHUlivc t'cgululllr ul'
p34~•11.·~·uc:tlvily 111 pt'cscnl ln llssucs whh:h m·c miNsing h11
targetp34c.~.;~ .. Meyerson et a/.(33) prescnta broad spcctrum . of expression data of various cdc-related genes. Uke MOI5 .menibers oflhis liunily, suchns cdk2, PSSAL~E •. PCTAIRE-1
·. ''"d
PCTAIRE-3 ~>huw u llroad mnge nfcxpressiOit in human . tlssues; Possibly, therel'ore p40Mut~ uctivatcs ulrgcls othcr than p34cd.::!, This has been shown already for p33~dk2 (21 ),whosetranscripts show a pattem of expression which is very similar. to MO 15.
. Our datu indicutc thutthe cxprcssion of human MO 15 does Mt correlate with the meiotic or mitotic acti vity of .cells.
Despite a very high ideritity of human und Xenopus MOlS- proteins their function in those phylogencticaHy distunt species seems to be of fundamental ditrerence~
Acknowledgements
This work was supported by thc 'Hessischc Vereinigung zur Förderung der Jugeodgcsundheitspllcge' and thc Deutsche Forschungsgemeinschart Ru242-l L
References
I. Norbury C :md Nursc P: Animah:dl cyclcs and lhcii· conlrill.
Annu Rev .B iucl\em.61 : 441-4 70; ll.l92.
2. Dmett:1 G, Luctl· F. Westendorf J;ßrizucla Ld~utit:nnan J and Be:.tch D:·cdc2 protein .kinase is cumpli!xed withbotl) cydinA and B: evidence for proteolytic inactivation of MPF. Cell 56:
82l)~838, 198l.l.
3; Murray AW :md Kirseimer MW: Cydin synthesis drives ihe early embryonic cell cydt!; Nature 339: 275-2!10, llJIN. · 4. Peter M •. Nakugawu J. Dmee M; .Labhe JC and Nigg EA: /n vitro disassembly of the nuclear.lamina and M phasecspecilic phosphorylation of lamins by cdc2 kiilase. Cell 61: 5lJ 1-602, 1990.
5. Solomon MJ, Glutzer M, Lee TH, Philippe M and Kirschncr MW:
Cydin activation of p34 ctlc2. Cell 63: 1013-1024,. 19lJO. · 6. Sherr CJ: Mammalian G1 cyclins. Cell73: 105l.l-1065, ll.l93.
7. Evans T, Rosenthai ET, Youngbloin J, Distel D and Hunt T:
Cyclin: a protein. specilied by maternalmRNA in sea urehin eggs that is destroyed at each cleavage division. Cell 33: 3!!9-
396,1983. . .
. !!. HunterT ;md Pines J: Cydins and eai1eer:Cel166: 1071-10]4, ll.ll.l I. .
lJ. Feathcrstoi1e C and Russe I P: Fissilin ye:tst· p I 07'":, .• mitutic
inhillit~r is a tyrosine/serinc kinase. Nature 349: 808-8 t I, 1991.
10. Parker LL, Atherton-Fess.lerS aild Piwniea-Wonns H: pl07w'"1 . i.s a duul-specjficity kinase lhal phösphoi·ylates p34'·dd on .
· tyrosine 15. Proc Nall Acüd.Sci l:JSAIW: 291.7-2l.l21, ll.l92. · II.McGowan CH:tnd Russcll P: Human Wccl kinasc inhihits ccll
divisJlln by phosplluryhtting p34"'k! exdusively un Tyrl5.
EMBO J 12: 75-!15, ll.ll.l3.
12. Miliar JBA and Russell P: The cdc25 Mcphase imlucer: An
· .uitconventional protein phosphatase. Ccll68: 407-410, 1. 992.
13. Gould KL, Moreno S, Owen DJ. Sazer S und Nurse P:
Phosphorylation at Thr 167 is re4uired forSchi:.i;,,.ucdwmmyces tmmbt• p34"'1L'! functiun. EMBO J Hl: 3297-330lJ, lW I.
~4. Fleig <l:JN. Gould KL and Nurse P: A dumimmtneg:ttivc allde of p34"'1c! shuws :tltcred phusplwmnino acid contenl :tnd . se4uesters p56"1<:1.1 cydin. Mol Ccll ßit1l 12: 2295-2301, 1992.
1.5. Krek W. and Nigg EA: Differential phosphorylation of vertebntte p34<dd kinase at theG.JS· and G~/M transitions of the cell cyeh:: idcntilieation of majur phosj1hmylation sitcs. EMBO J .10; 305-316; ll)lJI.
16. SoJi.,mmr MJ, Lee T :md Kirschncr MW: Rolc of phosphorylatiun in· p34cddactivation: ldentitication uf an activating kinase. Mol Ce11Biol.3: 13~27, llJ92.
17; Lorca T; Labbe JC, Devault A, FeMtuet D, {::apony)P.
Cavadore JC, J.:e Bouffant F ilnd Doree M: Dephosphorylation of cdc2 on Threonine.161 isTe4uired.l(1r kinast: ii1aetiy:ttionand normal anaphase. EMBO J .II: 2381~2390, 19l);!. ·
IK. Shutll.:wurth J, Oudfrey R lind Culnmn A: A cdc:2~n:lutcd
prlitcin kinuKc invulvl:d ln. nc~&utlv.c rcj&\lhttiu.n nl' tncit.ltl~.·
mutumtiun ul" Xt•IW/IIIJ oucyl~ll. HMIJO J 'J: ~233·3240, IW<I. ,._.
~9; Fesquet D, Labbe JC, Democourt J, Capony JP, Oula.11 S, Olr.snl F.
Lorca T, Shuuleworth J,Doree Mund Cavudore JC: TheMOIS gene llncudes the catalytic Kublinil of u protein kinase. that activates cdc2 · and. other. cycliri-dependentkinases (CDKs) through phosphorylation of Thr 161 and its homologues. EMBO J 12: 3111<H21, 1993. .
20. Solomon MJ, Harper JW and ShuttleWorth J;. CAK; the p34cokl activatinff kinase, .contains a protein "identical or closely related to p4oMo 5. EMBO J 12: 3133-3142. 1993. . ..
2LPoon RYC, Yamashita K, Adaplczewski JP, Hunt T and Shuttle.wm1h J:.The cdc2-reluted protein p40M015 is the catalytic
~ubunit of a protein kinase that can activate p33•-.ikl anil p34'....,2•
EMBOJ 12: 3123-3132, 1993. " . .·. . .·
22. Hollrich U, Br!iuninger A; Stn!bhurdt K and Rübsamen- Waigmam1 H.: Two additional proteiiHyrosine kiriases expressed in human lung: Fourth member of the tibrobl~st growth factor receptor Jamily und an intracellular protem;
tyrosine kinase. Proc Natl Acad Sci USA 88~ 1041J-10415, 19l) l.
23. Holtrieb U, Wolf G, Br!iuningcr A, Karn T,. B,öhme 8, Rübsamen-Wuigmann Hand Strebhardt K: lnduction .and down- rcgulation ilf PLK. a human serine/threonine kinase expressed inproliferating cells and tumors. Proc NatiAcad Sei l:JSA 91:
1736-1740, 1994. . . .
24. Brliu1lingcr A,. Hollrich 1), StrcbhardtK and Rübsamen- Waigmann H: lsolatilm and cha.racterization ufa humall gene that cncodes a · new subclass. ·ur pi'lJtein tyrosine kinases. Gene
I 10: 205-211, 19lJ2. . . .. .
25. Böhme B, Hollrich U, Wolf G: Luziüs H, Grzeschik .HK.
Strebhardl K and Rübsamen-W:tigmann H: PCR mediated detcctioil uf a ne.w hum:m reccpwr-tyrosine-kinase, HEK2.
Oncogcnc H: 2H57~2H62, 1993. . . . ...
26. Karn T. HoltriCh l:J, Bräuninger A. Blihme ß; Woll G,
· Rübsamen-Waigmann H and Strebhatdt K:. Structi.Jre.
" exprcssion and chromosomal mapping of TKT from man and muuse: a new subclass of receptor tyrosine kina~es with a factor
" Vlll"like domain. Oi1eogene l!: 3433-3440, 1993. .
27. H:mks SK; Quinn AM and Hunter T: The protein kinase family:
cOJ1served l'eatures and deduccd phylogeny of the calalytic domains. Science 241:42-52, ll)88.
28. Amaravadi Land King MW: Characterization andexpression of the Xt'näpus e-Myb homolog. Oncogene 9:971"974, 1994 ... · . 29. Westin EH, Gallo RC,Arya SK, EvaA,Souza LM, Baluda MA,
Aaronson SA.and Wong-Staai"F: Differentialexpression of the imll' gene in human hematopoietic cells.' Pmc Natl Acad Sei l:JSA 79: 21l.l4-219l!, 1982.
30. Mushinski JF, Potter M, Bauer SR and Premkuin;tr Reddy E:
.· DNA rearr.litgement and altered RNA expression of the c-myb oncugcne in mouse plasmacytoid lymphosarc()mas. Science 220: 795" 7l.ll!; Jl)l!3.
31. Sagula N, Osk:trssun M, Cupcland T, ßrumbaugh land Vande Woudc GF: Ftinctiun uf c~mos ·proto-oncogene. pniduct in meiotic maturation in XeJiotnu oocytes. Nature 335: 519"525,
llJ88. .
32. Propst F, Rosenberg MP, lyer A, Kaul K andVan~ WoudeGF:
c-mo.\· prill<HIIlcogene RNA transcripts .in mousetissues:
. Structi,lral features; developmental· regulatiun and localization in spccitic eell types. Mol Cell Biol7: 1629~ 1637,.1987,
33. Meyerson M, Endcrs GH, Wu CL, Su LK. Gorka C, Netso11
<;.
Hurlow E and Tsai LH: A family of human cdc2~relateil protem kinases. EMBO J II: 2909~2917, 1992. .
34. Lee MG and Nurse P: Complementätion used to clone utiuman homologuc ofthe·tission yeast cell cycle control gene cdc2.
Nature 327: 31-35; ll.ll!7.
35.Fcilg D~F unll Doolilllc RF: Pmgressive se1.1UCI1cc aligmncnt as
<I prcrCliUisite lll correc.t phylugcnetic trees; J Mol Evul 25: 351-
360, 1987. . . . ·.
36~ Levedakuu EN~ He M, Bapti.st EW,Craven RJ, Canc~·WG.
Wclcsh PL, Simmons A, Naylor SL, Leaeh RJ, Lewts ~B.
ßowcuck A and Liu ET:. Twu novcl human serine/threonmc kinascs wilh homuhigics to thc cell·cycle regulating Xt'I!OI''~s MO 15 and NIMA ki11ases: cloning• und e.har.lcterizatiun ol the•r . · expres~ion pattem. Oncogen~ 9: 1977-1988,.1994.
37. Wu L, Yee A .• Liu L. Carboi)arocHall D. VenkatesanN. Tolo VT
· • and Hall FL: Molecular cloniilg of ihe human CAK ~ .gen~
eilcoding a i:yclin~dependenl kinase-activating klnase.
· Oilcogene 9: 208lJ'-2096; 1994.
I
!1
