Overexpression of kin17 protein forms intranuclear foci in mammalian cells

Biqn'himie (1097) 79, 5Vg-6t)(~

© Soot&6 franqaise de bi~cltimie e| hitdogic m~lOculail~' t Elsc~ tel, Pro'b,

Overexpression of kinl7 protein |barns intranuc|ear loci in mammalian cells P K a n n o u c h e , G Pinon-Lataillade, P Mauffrey, C Faucher, D S F Biard. JF A n g u l o :~' Lahoratoire ,/¢' Gdm;tique de ht Radiosensibilitd, lkdpartement de Rruliobinlogie et de Radi~qJath~do,.,,ie, Direetiou des Sciences d, Viv, nt. Centre d'l~,tmles tit, Fontettay-~tttx-Roses, CI:'A. 60,68, ,vemte du GdmYa/ Ix.¢ let~'. BP 6. 92265 Folltenov-att.r-Roses t'edex. Front'e

(Received 4 June 1997: accepted 13 November 1997) Summary - - Wc used antibodies ;.ig;.|inslE colt RecA prolein to identify in inotlse cells a 45-kDa DNA-binding protein called kin 17. which has an active zhac finger and a nuclear Iocalisalion signal. Kin l7 protein produced in E t'oli binds i~referentially to lhe curved DNA of it bacterial promoter in vivo and in Htro, suggesting a transcriptional regulation activity. The fact thai in rodent cells kin 17 proiein Ic~cls increase after gamma-irradiation suggests its participation in a celhdar response to ionism~ radiation. We raised polyclomd antibodies against tile whole kin 17 protein and against its derived synthetic peptides. We report the detection of kin 17 pn~lein and of truncated flu'ms of the protein by Western blot or by immunocytochemistry after transient overexpression in cuhured human cells. Our resttlts indicale that the cl'os~-l'e~ctivity with the anli-RecA antibodies i,,, dtt¢ to ~.ulamigcnic determinant located in the core of kml7 protein, betw:een residues 129 and 22,";. The kinl7 protein is located in the nucleus and is concentrated in small nuclear dot-like structures throughout the nucleoplasm. The RecA homologous region seems to play an essential role in the localisation of kin 17 protein since the deletion of this particular region dramatically changes the form and the distrihtaion of the inlranuclear fool. We hypothesise that these dot-like structmes reflect nuclear metabolism compartmentalization.

kinl7 protein / ReeA protein / nuclear loci / mammalian cells/inducible ~ystems / Zn finger / truncated proteins introduction The celluhn' response to gcnott)xic agenls Ilas been extensively studied in E colt hactcria leading lO the molecuhn' characterisation of the SOS system, a set of 25 inducible genes co-ordintlled by a reguhttory protein c:tllcd. RecA [ I(1, I11, 2ql. RecA is a mLdtifunctit~nal prnteitt which pl:tys crtlo cial roles in recombination ~,|lttl intllagc|tc~is. Considering the structural conservation of DNA as support fi~l' the genetic inlbrmation and the essential role of RecA protein in these vital DNA transactions, we postuhtted that limctional domains of ReeA are represented in mammalian pro.. reins inwllved in DNA interactions 121. Several mammalian pa>teins displaying functiomtl or structural similarities to E c'oli RecA protein have been identified and are now being characterised I1, 8, 221. These RecA-related proteins participate in the formation of a multiprotein complex c,'tlled the recombinosome l I 1 I. They play a role in important cellular functions, specifically Rad51 protein has been shown to have an essential role during early development 112, 28l. We investigated whether .'mtibodies c,'m detect the similarities of bacterial and ma.mnalian DNA-interacling proteins. Monospecific anti-RecA antibodies allowed us tt~ detect and identify in mouse cells u 45-kDa basic protein, called kin17. Its amino-tertninal region has an active zinc ;i:Corresi,oi~deilce and reiiriills

finger (from aa 28 to 50) and a bipartite nuclear localisation signal (NLS, from aa 235 to 285) able to drive a fusion protein NLS-~-galactosidase into the cell nucleus II81. Ctmsidering the basic nature and Ihe DNA-bindin~ domains of kin l7 protein, we have proposed its parlicipation i11 DNA transactions 1271. Indeed, kin J7 prolein I~roduced in li colt binds to curved DNA of the E colt hgl t)pt?lOll ill i,ivo aild in vitrn supl~ortiug tile idea that kin l7 protein may be in, volved in regul;tling gene expressiou m mouse celh t l~, 261. Neverlheless, the precise biochemical activily of kin 17 protein remains to be determined. Interestingly, the level ol kin17 protein increases after y-irradiation of rat cultured cells. This obserwttion may indicate its participatiorl in a cellular response to ionising radiation 151. We flu'ther examined the ntolecuhtr basis of the cross~reo activity between kin l7 and anti-ReeA antibodies. Using computer analysis we previously showed that ki,~17 and RecA proteins have a short 39-residue homologous region located in the core ol' kin 17 protein (a:t 163 to 201 ). in a helix-turn-helix region supposed to interact with DNA 131. The homologous region is located in the carboxy-terminal end of RecA protein (aa 308-347) corresponding to a RecAconserved functional domain implicated in protein-DNA int e r a c t i o n and s u p p o s e d t o be a g a t e w a y for genetic recombination ] 15, 25 I. We r.'tised polyclonal antibodies in rabbits against kin 17 protein mtd used them in Western blot or in imlnunocyto o chemistry experiments. We report here the overexpression of

eglO Table L Main pta~pertiesof the expression vectors used for the transfcctions of HeLa cells. Plasm~ m~me IKTMVKinI "Y~ ~M~gialT~ P~rdlVKin I:~AItR ~M~Kin!7ACT a

l)eleted Kill17 eDNA ,fragment

Deleted residl~es

Mr (Da)

From nt 38 to 235 From nt 412 to 705 Front nt 854 to 1034

From aa O to 70 From aa 129 to 228 Froth aa 281 to 391

44 693 36 '-)38 33 709 32 407

aKiatT, ghH 7AZF and Kinl 7ACT were isolated from plasmids pKGC 102 [41, pKGC 103 and pKGC 104 [ 181 respectively. the ~ Kinl7 eDNA after transient transfection cff cultured human cellg This alk~wed us m confirm the specificity of the a w . i b o d i e s ~ t o determine the subcellular distribution of kin 17 ptot¢i~ We demonstrated the preferential nuclear iocalisation of kinl7 ~ i n in human tm~,~l~cted cells. We showed that the epitope wl'deh generates the cross-reactivity with the anti-RecA m~ibodies is indeed located in the tore of 'kin 17. We have a l ~ maplx'd t~her antigenic determinants by e×l~'essing a truncated form of kini7 Im-,tein. We observed lbr the first time that ramruled kin 171~3teir~s have different cellular localisations, One of ~ truncated proteins was di~ribnted in abnormal nuclear struclUr~,' w ~ h may deform the nuclear n~vhology, suggesting an in,nov,ant role of kinl7 protein in DNA tnetabolism.

Materials and methods Cell calt.~ at~d trmt~fecti¢m HeLa cells (kindly provided by Dr E May) were g~x~wnin Dulhec~ eo's nw~'lified Eitgle medium (DM~M) supplemented with I(~¢~¢ fetal calf sel'u~'~ {FCSI, peni¢ilih~ (I1]0 U/roLl and streptontycin i ItR).gtmL), Cells were transl~¢ted by calcium phosphate p~ecipio latium {171, They were washed 16 h later with phosphate barfer saline (PBS) and then incubated lot 32 h in DMEM I()r,I FCS and prt_~:ess~dfiw intmn,ostainin~, or t\~r Western hh~ts, Pht:~midtv~lSt~¢lioO A~I plasmids u ~ in this sludy were constructed Ih~m the vector ~MVDT21 which allo~vs a high level expression of the trunsgene l?l, We in~n~.~t the open reading frame tURF) of mouse Kint7 eDNA 114]in the Xkol restriction site of pCMVDT2 I, We u,~d two previously rept~lcd ginl7 eDNAs that have each a deletion: from a n t i . i d e s 38 to 235 or t~)trl nucleotides 8,~.1tu 10341181, These cDNAs c~xte for truncated peoteins lacking the Zn-finger (aa 6 m 71)) or the Cqerminal end (an 281 to 391) with a calculated M,. of 938 ~ and 32 407 Da, respectively, We have called them here kinlT,~ZF (deleted zinc finger) and kinlTACT (deleted carboxy terminal end), We intrc,duced Kin176ZF and gi,17ACr cDNAs into I~MVDT21 as ~ r i h e d above obtaining pCMVKinl7AZF ~nd pCMVKia 17&CT, respectively, To obtain aaxaher deletkm mutant, we removed the nttcleotides 412 to 7115 t ' r ~ Kin/7 eDNA, The truncated protein lacked the 99 amia'o a c ~ hetw~-enaa 129to 228, a region containing the sequence ~gt~', to RecA pit,rein, ~ i s protein was call~ kinl7AHR ( d e ~ ~ o l o g t ~ s region), The gin,17~HR cDNAs were geweraled as !'olk~s: a.)I ~ R amplification of the 5' region of gin 17eDNA from pcD2KinI7 (nucleotides 1-41 I, 1411 using the palmers: 5"-

AAGCTGCTGCAGCAGCTTATCGGG-3' and 5'-.G,_.Q.TA_C~TTTACACAAGCCCTCTCGCC-3'; b) amplification of tile 3' region of Kinl7 eDNA (nucleotides 706-13521 using the primers: 5'~ A G T G C A C T G A A G C T G C T G G G G - 3 ' and 5'-ATFTACCCAACTATTCACTA-3'. The two products were ligated at the Kpnl site (sequence is underlincdL We verified thb, com~struct by sequencing and used it to obtain the plasmid pCMVKin 17AHR The plasmids used in this work are presented in table I and the structure of the encoded proteins is schematically depicted in figure I. Snhcelhdar.ti'actiomuion of Hel~8 cells Cytosolic and nuclear fractions (named C and N) were obtained essentially as described by Stein et a11231. Briefly, the cells were scraped off, centrifuged and resuspended in buffer A (10 mM HEPES, pH 7.9, I mM EDTA, 60 mM KCI, 0.5% Nonidet P-40, I mM dithiothreitol, I mM phenylmethylsulphonyl fluoride). After incubation on ice for 5 rain, nuclei were sedimented at 1200 g for 5 ntiu at 4°C, The supernatants were recovered and labelled as cytosol, Nuclear pellets were waslled once in buffer A without Nonidet P-40 and suspended in buffer B (2511 mM Tris-HCL pll 7.8, 6(} mM KCI, I mM dithiothreitoL !mM phenylmethylsulpho~ nyl fluoride L Nnelei were !ysed by three cycles of freezing/thawing, Both tire nuclear and eytosol l'ractioas were cleared by cenlrilhgation (70110 g. I0 nfin, 4°C). Pnmq..~bhd~ili~ati,m HeLa cells ha~'¢sted as de~ribcd above were lysed ia bulli~r C (51) mM Tris-HCI, pH 8.0. 0.5 M NaCL I mM EDTA, 2 mM DTI" and 0.5% Triton X~I(XI)and then sonicated twice for 6 s. Aliquots were centrifuged at 20 000 g for 30 rain at 4°C generating a supentatant containing the soluble proteins (fraetioq S) and the pellet (fraction P). AnHl~dies and Western blot analysi.~ Tile production of antibodies against R~A protein (pAbanti-RecA) has been previously de~rilx~ [31. We have al~ obtained antibodies against a synthetic peptide corresponding to the residues 165 to 179 of the mou,~ kinl7 protein which correspond to a part of the RecA homologous regkm and named peptide HR. The produced serum was then calkxl pAbanti,HR. Rt,v:entlywe have obtained the pAb2064 directed against the whole kiul7 protein ([61; Kannouche eta/. submired). The,~ ,~ra were u~d in immunoblotting experiments after SDS-E~.GE separation of proteins perfi)rmed as described 16]. hmmmofluoi~.scence and im,;unocytochemistry HeLa cells grown on coverslips were fixed 48 h after DNA translitction with methanol/acetone (3:7) for 10 nrin at -20°C and rehydrated in PBS tot 15 rain at room temperature.

601 ZF

~gnkinl7

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~

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AHR

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ACT

,

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i)

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20

27

I')

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~{!

293

: ..........................

81~

i,)0

120

341i

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sequence position

|,'l[~ I. Schematic represelnatiL,l of wild type and truncated forms of int+tme kin l7 proleill. Aluhlo acid sequences are linearly represented. The naales are ir~dicated on 111oleft an,~ltile alllillO acids of each pl'Olein ¢.)11tile right. The sequences wcr,2 d l ' a ~ A , n tO scale as indicated by tilt? length of the horizontal line at Ihc bollonl. The .,'ine finger domain tZF. aa 28 to 50). Ihe RecA homologous region (HR. aa 163 to 201~ and tile bipartite imclear loealisation signal (N I.S, aa 235 to 285) are diN'~layed in hatched dark boxes. The deleted residues are numbered according to their positions in the kin l7 protein sequence and are shown as gaps.

btdiPl'ct ilt[lllftn¢!]llloivsCt,lL~(, The coverslips were incubated for 60 lilts at 37"C with the pAb2064 or the pAbanti-RecA dilated I: 1 0 0 in PBS, 3g, bovine serum albumin (BSA). Cells w.'kshed with PBS were incub;ued lbr 45 rain at room temper.'aure with Cyl~12 conjugated af|iniP,rc ,goat anti-rabbit igG (H + L) (diluted h5(10 in PBS, 3t)~ BSA after the purch;iser's instructions, Jackson hnnmnoResearch laboratories, Incl. Alter washing three times liw 5 rain with PBS. cells were stained with DAPI and mounted on glass slides with glycelgel d)ako),

hmmm~u3'tochemistl3" Cells trealed with 3¢h Iv/'.,,) I1.,O_~for 10 rain were farther incubated Ibr l0 ntin with PBS 5% normal goal serttnl, After rinsing with PBS. cells were incubated with pAb2064 or the pAbalni-RecA (I:lt)O) (or tile t~slleClive pre-inuntlrle ser.'O Ibr 2 Ii at room temperature. Cells washed with PBS were incabaled Ibr 45 rain wilh the biotinylatcd goat anti-mbbil IgG ( 1:2001. Alicr washing, cells were ineuh.'ttcd with VceItmlainreagellt Ior 30 n|in as described hy |he ,,upplier I Veetastai, AI].C I~lite Kit, Vector 14tboraturics. USA). The complexes were revealed u s i n g 3,3'-diantinobcnzidine letrahydrochloride (Polysciences, 0.5 rag/luLl ;rod I).01% H20., in TBS. Samples were nmlmtcd in imo proved Aqtt:tnlount (Gurr Laboratories, BDII). (?ells were ob~rved and pllotographed with a Zeiss Axiophol2 Photomleroscope eqniplx~dwith phase contrast and el:~iflunrescenceoptics using 20 x. and 100 × Plan-Neofluar lenses. Images were recorded on Kodak films. Alternatively we used an on-chip thermoeleca'onically cooled charge-coupled device (CCD) camera (Coolview, Plmtonic Science. UK) which was controlled by a PC computer After eye scansing of the preparations° representative images obtained using filtet:sfor rhodamine, fluorescein and DAPI were saved in a grey scale and then pseudocolored using Visiolabl000 software (Biocom, France),

Results

and

discussion

Subcellular ~fistribution ql'kh; 17 protein e~ln'e.~sedin human cells. Analysis o]'celhtktr [}'acthms by Western bh~ts in order to determine the subcellular distribution of kin l7 protein, HeLa cells were transfecled with the p C M V K i n l 7

in which the Kin / 7 e D N A was under |he control of the CM V promoter. We observed that about 12~/} t)l' the cells expressed kin l7 protein 48 h after transfeetion. The eytosol (C) and nuclear (N) fractions were analysed by Western blot using the pAb2064 serum or the pAbanti-RecA adsorbed on k i n l 7 protein immobilised on nitrocellulose as described 16]. Both antibodies allowed us to detect a major band with a M,. o f 45 000 Da only in nuclear fractions (fig 2. lanes 4 and 8). We failed to detect the 45 000 Da band corresponding to kin l7 protein in the loaded extracts flom HeLa transreeled with the vector alone indicating that under out' cxperinlenlal conditions we are unable to detect Ihc endogenous kinl7 protein (fig 2, lanes I, 2, 5 and 6~, The antibody directed against a sy,lhelic peplide corresponding to tile RccA honlologotts region (residues 165-179 of kin 17 protein) Colll'it'nlcd IItese resnhs. Ftlrlilerulore, prcinuntmc scra (Col'respomling Io pAb2(164 :rod to pAbanti-RceAI were unable to reveal any siglml under the experimental conditions described above (data not shown). Considering: i) that the observed Mr o f kin17 protein corresponds to the e;dctdated molecular mass 141; it) that the detected band comigrates with kin 17 protci, p)oduced in E eoli (not shown); and iii) the absence of sign;tl in HeLa cells extracts lransfected with pCMVDT21, we conclude that these antibodies detect the mouse kin 17 protein in h u m a n cells by Western blots.

Pr
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ct~

c~

c~

....

1

~'~

3 4

pAb2064

~

5

6

?

~

45kDa

8

pAbanti-RecA

I'lll 2, Prefer~miuldetectitmof kin 17 protein iu nuclei. HeLa celb, translated with pCMVKinl7 were t~lclionated and analysed by ~ t e r n blot, Westen| detection of kiul7 protein in the nucleu~ ffPac~ion N) or eytosol (fractinnC) of HeLa celia,.Proteins of t'racIkm~,N and C were analy~d by immanohlottingusing pAh2064 or pAb~nd-RecAas primary antihtxlies followed by doukt:y antirt~b~it lgG antibodycoupled to pen~xidase(Amershaml,The comple,~es were visualised by enhanced chenaih|miniscence (Au~ham), L~mesI, 2, 5 and 6 co.spend to fr,'~ctionsfrom cells lr~nslrodtedwith th~ vector ~lone, limited alllouuts of kin 17 protein revealed several small nu~ clear dotolike stlal¢lure~ throughout the nucleoplasm sug~ posting that kinl7 protein is concentrated at discrete sites (fi~ 3A, panels AI and A3I, This agrees with the nuclear accumulation of the eudt~genous kin l7 protein in prolio feralin~ cullurcd n'louse cells l Kannoucl|c vtal, snhmined 1. In a small fr,,tctinn of tr'ansfocied cells, pAhantioReeA stained ~ tmcleolardike structure flip 3A, pane! A2). The ~ t ~ t i o n of intranuclear t ~ i w~ts furlher confirmed by iuo divot immm~.)fluo~seence using pAb2064 revealed with a Cyr~2dabdled secondary antib~ly, Nuclear DNA was c{~,mterst~ined using DAPI (fig 3A, panels A3, A4). Cells expressing high amounts of k i n l 7 protein presented similar dotdike structures but also clusters thereof which funned brighter specks or even bigger struclures of immunoreactive material (fig 3A, panel A4), It must noted that tun~t~rigenichuman epithelial cells H 1299 and immoMali,~,d mouse BALB/c 3T3 fibroblasts displayed a su~elinlar distribution of kin 17 protein very similar to that de~fibed above alter transfection of HeLa cells. We tkailed to ob~rve any nuclear staining when: i) cells were incubated with t ~ corresl'Kntding preimmnne serum under the s~une conditions; and ii) mock transl~cted cells were anao ly,~3 by im~nt~yt(~hemistry (not shown). These data indicated that in our conditions, we are unable to detect endogemms kini7 protein by immunocytochemistry. ConsiSting that: i) immunoblots revealed a 45-kDa prorein only in nuclear extracts as compared to cytoplasmic

extracts: it) nuclei of transfected cells displayed a strong signal in inmmnocytochemistry or indirect immunofluorescence; and iii) two independent antibodies that recognise kiul7 pro :,:in produced the same naclear staining, we conelude that kin l7 protein is mainly Iocalised in multiple discrete loci in the nucleus of transfected cells. Several other proteins form intraJmclear foci 19, 13, 14, 211. Recently, it has been shown that a~Rad51 protein, an analogue of the bacterial RecA protein, is located in nuclear feel-like structures of S-phase cells suggesting a role of H,Rad51 protein in DNA replication I241. Furthermore. the number of tt~Rad5 i tinct increases alter DNA-damage [ 13 I. These results raise the possibility that the spots of kin 17 protein are a part of a nuclear compartment of the spatial and structural organisation of the genome. Further w o r k will be required to test whether the nuclear amounts of kin l7 protein increase after U V-irradiation, Delelion ~l'the ~hm~ain .~h.red by kin 17 am/RccA proteins dramatically affects the mwh'ar Iocali,wtthm o]the truncated protein We sought to determine wbether the homologous region between kinl7 and RecA proteins is responsible for the cross-reactivity between kin l7 protein and anti-RecA antiht~lies, We constructed the Kin 17AHR mutant cDNA (DHR from deleted for the RecA homologous region), encoding kin 17AHR protein which lacks the kin 17 protein core fi'om aa 129 to 228. HeLa cells transfcctcd with tile plasmid pCMV Kin 17AHR were analysed by imnmnocytochemislry. We observed a strung signal only in tile uucleus o f transI~¢ted cells using the pAh2(164 antibodies (lip 3B, panels B I and B3). The staining pattern was significardly different l'|",~lltl the pr¢~ioasly observed coloration after ovcrexpression of kin l7 proleia. Indeed, the kin l7AHR protein seenls Io Ibrm intram~clear aggregates, suggesting that the deletion of the protein coJe enhances the binding of kinl7 protein to a nuclear component, perhaps to DNA. We reasoned that if this is the case, biochemical approaches should confirm the Rirmation of intranuclear aggregates of kin l7AHR protein observed by immunocytoehemistry. We first determined the Mr of kinl7AHR protein l~'Oduced in the transfected HeLa cells by Western blotting using pAb2064. Protein extracts prepared from nuclear and cytoplasmic fractions presented a single band of about 34 000 Da Mr only in the nuclear ~raction (fig 4A. hme 2). We failed to detect any signal in the eytosolic fraction (fig 4A, lane I ) indicating that the deletion of the homologous region does not affect the subcelh|lar distribution of kin 17AHR protein. The apparent Mr correlates well with the calculated molecular mass of kinl7AHR protein, pAntiRecA or antibodies raised against a peptide corresponding to the RecA homologous region (anti-peptideHR) were unable to detect kinl7AHR protein (fig 4A, lanes 3-6). Furthermore, the fact that we did not detect kinl7AHR protein using anti-RecA antibody by immunocytochemistry

603

A U At

A2

BI

B2

II ii // A3

A4

B3

Fig 3. Kin l7 protein f~wlns nuclear It,¢i whereas kinl7AHR prolein forms intranuclcar aggreg.ate~,. HcLa cell,, transt'cctcd with pCM VKin 17AHR or pCM VKi~I17 were analysed by immunocytochemistry. A. Immunodetection of kin 17 protein in the nucleus of transl~:ctcd Hel.a cells. Transfccted cells gn~w'n on coverslil-,s were fixed and procesmd lot immtmocytochcmislry IAI and A2) or indirect immtmo|Ju~,tescence (A2t and A4) .'is described in M~lle~'itJLs"alld methods. Kinl7 protein was revealed using pAb20fi4 (A|) t)l"pAb~mti-RecA ~A2~ as F,rimary alltibodies lifllowed by a biolinyhtted go:a ~mti-rabbil IgG coupled to peroxidasc. The co0q)lcxes were re,~ealcd with DAB. M~LBnL /ic,ttions of 1000 ×
th:~t

indicates that pAbam i-RecA recognises a kin 17 protein epitope which is lacking in kin l7AHR protein and suggest the cross-,'eactivily could well be due to the homologous region (fig 3B, panel B2). Secoadly, we transfected pCMVKinl7 or pCMVKinI7AHR into HeLa cells. The expressed proteins were extracted at high salt concentration ,'rod centrifuged. Soluble proteins were recovel'ed in the supernataut (a fi'action labelled S) and cellular debris was recovered with bigger aggregates in the pellet (fraction P). As expected, we detected a large proportion of kin 17 protein in li'actiou S by Western blots (fig 4B, lane I). In contrast, kin l7AHR protein was mainly detected in fraction P, indicating its association with intranuelear components of high molecular mass (fig 4B, lane 4!. It should be noted that the kin 17AHR-containing aggregates were solubilised only alter incubation of

fi'action P with 8 M utva (data not shown) confirming a strortg interaction between kin 17A_HR prolein and a nuclear component. Since overexpression of kinl7AHR completely prevented foci lbrmation, we conclude that the residues between amino acids 129 to 228 could play an important role in the biochemical activity of kin 17 protein, Proteins lacking the Zn finger domai, or the C-termimd end disphty a sttbcelhdar distribution simiho" to that ~f kin 17 protein We have determined the distribution of kin l7AZE a protein in which the zinc finger is deleted. HeLa cells transfected with pCMVKin 17 or pCMV Kin 17AZF were incubalcd for 48 h and kinl7 and kinl7AZF proteins detecled using antibodies pA°

6t)4

A

/// C N

C N

C N

34 kDa

I

2

3

4

5

6

B

and kin 17:~ZFplx)teinsonly in the nuclear fractions (fig 2 and fig 5A, lanes 2 and 6), The observed Mr of kinl7AZF protein is very close to the calculated molecular mass 1181. hnmunocytochemical detection of Kin 17AZF pt~oteinsrevealed a staining pattern similar to that obtmned tbr kinl7 (fig 5B, panels B 1 and B2). Indirect immunofluorescenceshowed nuclear discrete foci (data not shown). These data suggest that the N-termhml region plays a secondary role in the subcellular Iocalisation of kin 17 protein, When expressing kin l7ACT, a protein truncated at the C-terminal end, the pAbanti-RecA antibody revealed a 33 000 Da major band in traction N and a weaker signal in fraction C as judged by Western blot (fig 5A, lanes 7 and 8), lmmunocytochemistry showed the abovementioned puttern of nuclear loci but also a more uniform staining through the nucleoplasm, In a small number of cells ,dight cytoplasmic staining was also observed (fig 5B, panel B4), Surprisingly, the antibudy pAb2064 Ikfiled to reveal any signal by Western blot or immunocytochemistry using the same experimental conditions in several independent experiments (fig 5A, lanes 3 and 4, Iig 5B. panel B3). These data indicate that antibody pAh20(~ recognises an epitope located between aa 281 to 391, at the C-terminus of kinl7 protein. Moreover, the detection of a fraction of kin l7ACT in the cytoplasm suggests that the C-terminal region is necessary for the complete nuclear Iocalisation of kin 17 protein. Conclusion

S P

S P tp,

g, 12

34

Fig 4. Biochemical analysis of kin l7AHR protein overexpressed in Itel~ ~.'dls,A, pAhanti,RecAfails to detect kin 17AHR protein in Western blot, HeLa cells transfected with pCMVKinlTAHR were fraetionatedas ~sctil~,d in Mnterhds a,d methtuls, Fractions N and C were aaaly,~d by immanohlotting using pAh2(~i4,pAI~mi-RecAor pAbanti-HRas primary antibodiesand donkey antirabbit IgG antihxly coupledto I~roxidase as a~we, B, Kin 17AHR ptolein has a decrea~d solubility, HeLa cells transiently transfecttM with OCMVKinlT&HRor pCMVKinl7 were l y ~ ~nhigh NaCI ~mcentration, Pn~leinswere separated by centrifug~aioninto ~dnhl¢ ft~tion (St and pellet (Pt, Equivalentamountsof pta)teins wer~ anal),st:dby immnnuhh~tingusing pAh2064,

btmti-RecA t~r pAb2064, As expected, Western blot revealed two major bands of 45 and 37 kDa ct)rtesl~mding to kin 17

We have shown that a.tibodies raised against the whole kin 17 protein or against its derived synthetic peptides specifically rccoguise epitopes of kinl7 protein in its native and denatured form as judged by Western blot anti by immunocytochenfical analysis. Thcrelbre. it is unlikely that tile lack of antigen-antiserum reactivity observed lbr tile truncated forms of kinl7 protein is due to the conlbr,uational changes and suggest that the recognised epitopes are linear. The cross-reactivity between kinl7 and anti-RecA antibodies is due to an epitope located in the core of kinl7 protein, between aa 129 and 228. Interestingly. another major antigenic determinant is located between residues 281 and 391 of kinl7 protein. Taken together these data indicate the hydrophilic nature of the core and the C-terminal part of this protein. Tile obtained antibodies revealed tile preferential nuclear Iocalisation of kinl7 protein in mammalian cells and is in agreement with previous observations. Indeed, the human kinl7 protein has been detected in nucleated proliferating epithelial keratinocytes 161 and the rat kin l7 protein is relocalised in the nucleus after )'-irradiation 15I. Taken together, these observations indicate that the subcellular Iocalisation of kinl7 protein is dependent of the physiological state of the cells. A bipartite NLS of mouse kin l7 protein seems to be the peptide signal responsible for the active transport into the nucleus1181. The expression system described here will be used to precisely define the

605

A BI N

CN

CN

CN

37 kDa 33 kDa

1

2

3

pAb2064

4

5

B2~

6

7

8

pAbanti-RecA

//M B3

B4

Fig 5. lntracelhdar distribution of kin 17AZFand kin 17ACTproteins. HeLa cells traust'ccted with pCMVKin 17AZFor pCMVKin 17ACTwere analysed by Western blot and immunocytochemistry as described in legends to figures 2 and 3, respectively. A. pAb2064 does not recognise kin l7ACT protein. Cytosol (C) ard nuclear (N) fr',tctions fi'om cells were almlysed by immunoblotting using pAb2064 or pAbanti-RecA as primary antibodies and visnalisc-das descrihed in the legend to figure 2. B. KinI7ACT protein, mainly nuclear, is present in the cytoplasm of a few Iransfected cells. Cells were fixed and processed for immunncytochemistry as described in the legend to figure to 3. The complexes were revealed by DAB. Kin 17AZFpnaein was detected using pAb21)64 (BI) or pAbanti-RecAlIB2)as prirlary antibodies. Kin l 7ACTprotein was imlntmostained using pAb2064 (B3) or i~Abanti-l~ecA(B4) :Is primary antibodies. All photographs are shown ,'l! a magnific;aion of 8{}0 X.

functional properties of the region containing the NLS by deletion analysis. The overptx)duction of kin17 protein forms small intranuclear specks. The endogenous K i n l 7 gene p e r se presents a similar intranuclear distribution when its expression is enhanced three-fold (Kannouche et al, 1997, submitted). These observed patterns are similar to that of other nuclear proteins such as cyclin A, PCNA, RPA, CAF-I, HsRad51 and BRCAI. These nuclear foci reflect the functional compartmentalisation of important nuclear transactions like replication, repair, transcription or RNA splicing 113, 14, 20, 21,241. Since it has been suggested that kinl7 protein may play a role like a transcriptional regulator 1261, it is tempting to specuhtte that the foci lormed by kin l7 protein are likely rehtted to the transcriptional process. However, the possibility that these nuclear foci reflect another type of DNA metabolic activity cannot be completely discarded. Overexpression of kin l7 protein in human cells perturbs cell division leading to low proliferation rates (Biard et al,

1997. submitted). Moreover, kinl7AHR protein binds tightly to a nuclear structure of high molecular mass for° ruing large intranuelear aggregates with a shape differen! from those tbrmcd by the wild type kinl7 protein. These aggregates modify the nuclear morphology of transfected cells (Kannouche, unpublished results). This observation supports the idea that the so-called homologous region is involved in the regulation of the interaction between kinl7 protein ,'rod a nuclear component, probably chromatin or DNA. Interestingly, RecA protein deleted for the homoloo gnus region binds tightly to double-strand DNA as compared with the wild type suggesting that this part of the protein plays a role in regulating RecA-DNA interactions It61,

The truncated proteins kin I 7AZF and kin 17ACT present a subcellular distribution similar to that of the wild type kinl7 protein. Surprisingly, in a small fraction oi' transfected cells, kin l7ACT protein has it cytoplasmic localisation suggesting that the C-terminal end of kin17 protein

m a y ¢ o n i r i b u t e t o d r i v e t h e p r o t e i n i n t o t h e n u c l e u s . T-~ken together the~ results sirongly suggest the partieipution of k i n l 7 p r o t e i n in a n u c l e a r D N A t r a n s a c t i o n . T h e s t r o n g b i n d i n g o f k i n 1 7 A H R p r o t e i n to u n u c l e a r c o m p o n e n t o p e n s the ~sibilily to e~ly i~ntify kinl 7 partners and could be a n iiriptwlanl l o o l t o d e f i n e its f u i ~ i ~ a l properties.

Acllaow~nts P K~cl~ benefited from fellowships f~om the I N S T N a n d CEA~ =rhis w o r k w a s nlad~ possible by funds provided by Itte contrit~l,~ A R C nr till~i a n d 1157. We are grateful to Dos T ' l a m c h e n k o lind R Devtwet for providing us with p K G C I 0 3 and p K G C I I i 4 ~a~nlids, We thank I)r~ E M a y a n d JC Btmrdon for giving us r ~ M V D T 2 I, I,Ve thank Dr R M a u n o n r y ion a n t i , i d l e s , advice and frnltful d i ~ u s s i o n s on iniunocytt~chemical detection.

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