i I ! 4 ( I t}92) i - 17 ~ Iq~}2 Elsevier Science Publishers B.V. All righls rcse~vcd I}31}4-41~}X/~2/$i}5.|11~

Biochimh'(~ ct B i o p h y s i ( ' . . q ( ' t . ,

B B A C A N 87243

The Rel family: models for transcriptional regulation and oncogenic transformation Henry R. Bose, Jr. l)('p~#r/t~¢'t~t ¢~fM&'robiofcJgy cuad l'/t¢' ("¢'//R¢'s~'arch hasoilts1r. Unil'¢'~:~}ty ¢~f '/'~.~ (at ..luxtin...l,.stitt. I:¥ ( USA

(Received 22 N o v e m b e r I~)~)I}

Conlenls I,

Inl.lducli~n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I

II,

The wrc! t m c ~ e n e and ils llvln:ili~ptfieli¢laD!el cell . . . . . . . . . . . . . . . . . . . . . . .

2

III, Slruclnre and expression ~I'wn'/and il.~ccIlulaI'hornolog, . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

IV.

Rehiliortsllil'J helwecn N F - k B and the Rcl proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

V.

T h e NF-kB Iamily of transcriplitm

5

VI.

Bit~chcmical s l r u c l u l e and I'unclion of N F . k B

VII,

Structural a n d functional relationship belween IkB and Rel-associatcd pp4{) . . . . . . . . . . . . . . .

.............................................

VIII, Regulation t)f R e l / N f - k B Iranscl'iplion faclor.s

.....................................

....................................

IX.

Dorsal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

X,

T r a n s h ) r m a t i t m I)y Ihc v,rel tract)gone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. B, C. D. .I,

7

9 lip

Aclivalion of the Iransforllliilg potential t)f the c-n'l prolt}-t)ncogcnc v-rd sequences which ctmtribule It) its It'ansfornfing aclivity . . . . . . . . . . . . . . . . . . . . . . . . . Suhct:llular localitm and Iransforn)ation by v-rd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Models for Iransformalion by the v.rd oncogenc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

('onclusitn) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgcmenls References

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I. Introduction Many tumorigenic viruses contain transformationspecific genes called oncogenes [ 17,18]. The oncogenes of the acutely transforming retroviruses have been Correspondence to: H.R. Bose, Jr., Dept. of Microbiology and the Cell Research Institute, University of Texas and Austin, Austin. TX 78712-11195, USA.

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It} I() II

II II 14 14 14

transduced during the cv()luti()n of these viruses through recombination events between the viral genome alld host sequences. The nontransforming cellular genes are termed proto.oncogenes. "Fhc viral oneogene products are very homologous with their cellular counterparts but in general have sustained structural alterations that account tot their translormo ing potential. The elevated expression of a protooncogcne, as a result of a genetic rearrangement in its

vicinity, can al.~ initiate the events which lead to tumor development [ 19]. Normal tissues are composed predominantly of differentiated cells which are no longer capable of cell division. Differentiated tissues, however, maintain a limited number of committed stem cells which may be induced to proliferate to provide for tissue regeneration. Thie~ cells respond to external signals (i.e.. growth factors, hormones) through a network of intracellular mes~nge~ which ultimately transmit the external signal to the nucleus of the cell. Neoplastic diseases develop as the result of the uncontrolled proliferation of these undifl~rcntiated stem cells. The known protooncogenes appear to be involved in the transmission of signals ibr cells to divide a n d / o r to difl~rentiate (reviewed ill Rot'. 15q), Various prot~++on¢o~enes have been shown to encode growth factors [41},177]. growtl~ factor receptors (with protein kinase domains) [39], G proteins [76], cytoplasmic protein kinases [126] and transcription factors [22]. The viral oncogenes represent similar signal transducers which now fail to res ~ n d to the normal control mechanisms. The study of viral oncogencs and their cellular homologs has identified many of the genes and their structural alterations which are responsible for the induction of cancer. The analyses of these genes is alm~ facilitating the dissection of signals involved in the regulation of cell division and differentiation. in most cases the biochemical activity of the oncogene/proto-oncogene product and its subcellular Iotation have pr~wided important clues into how Ihe proto-oneogene functions in signal transduction, One of the protoooncosenes that has been difficult to define a function for in the signal transduction pathway is tile cm,t proto-onco~ene, the cellular homolog of avian v.nq, The nucleotide sequences of c- and v-n.I did not identify any known biochemical activity [2~,155,1781, Furthern~re, unlike most onc~gene products, v-nq could ~ detected in l~th the cytoplasm and the nucleus of the transt'~twmed cell [49,58,1~,175] and v-tel may transform cells irres~ctive of its subceUular Ioea.. tion [591, Recently, the sequence of NF-kB {nuclear factorkappa binding), a eucaryotie tran~ription fitctor, was shown to be similar to those of c- and v-nq within an amino-terminal domain [54,911. NF,.kB transcription complexes are dimeric proteins consisting of one or two m e m ~ r s of a family of pn~teins related to the c-Rel proto-oncogenc, The viral oncogene, v,~rl, carried by avian retieuh~endotheliosis virus (REV-T) was the t'i~t member el" the Rcl family to be identified. REV-T is the most virulent of all acutely transforming ~tro,~'iruscs and induces the malignant transformation of lymphoid and myeioid tells [121. Alterations in the c-re/ locus have a l ~ been reported in patients with large ~11 iymphoma, suggesting the involvement of tel

in the pathogcnesis of this human neoplastic disease [11}5]. The Rei family of transcription factors participates in the regulation of the expression of a number of cellular and viral genes (reviewed in Rcfs, 5, %) and the c-tel proto-oncogene product binds to and activates the transcription of human immunodeficicncy virus (HIV-I) [9,98,118]. The tel gone has been highly conserved in evolution and one family member, dorsal, has been identified in Drosophih~ mehmogaster. Dor~al participates in the control of dorsal/ventral polarity during early Drosophih~ mo~Tdzogem'sis [156]. II. l'he v,cel oncogene and ils hematopoietic tarilet cell

The virus which ca~ries tile vend oncogenc, reticuIoendotlldiosis virus (REV°T), is a member of a small group of distinct avian retroviruses [87,107,l{1~] and is the only acutely tr,msfiwnling member el' this l'anl|ly, REV-T is replication-defective and co-replicates with a helper viru,~ called reticuloendotheliosis associated virus (REV-A, ReI', 74), REV-T is the most virulent of all retroviruses and REV-T (REV-A) induces a l'atal lymphoma in gallifiwm birds within 7-111 days after infection [23,143]. REV-A facilitates the pathology of this disease by suppressing the immune system of birds infected with REV-T (reviewed in Ref. 16111. Tile nature of the cells transfiwmed by REV-T is determined, ill part, by the cell killing activity of its co-replicating helper virus 111,13]. Tumors from REV-T ~I~EV-A) infected bird,,, contain transformed lymphoid cells which generally do not comain immunoglobulin chain gent rearrangements. These cells express mycloid or T cell surface markers. Birds infected with REV-T co.replicating with a less cytopathic, genetically related helper virus (chicken s~'ncytial virus) develop tumors which contain mature IgM positive B cells with diftbrent degrees of V~ diversification. REV-T transforms cells in vitro and these transformants are phenotypically similar to the cells transformed in animals [74]. Unlike most cells transformed in vitro by retroviruses, v-nq converts a normal cell to a tumorigenic state [99]. REV-T transfi~rmed spleen cell lines which do not contain helper virus sequences are tumorigenic and induce a lethal lymphoma when injected into histocompatihle birds. These features of the Rel system provide the opportunity to identify the molecular events which lead to transfiwmation and immortalization of lymphoid cells in vitro and an insight into the mechanism which leads to a tumorigenic cell. In vitro REV-T derived transformed spleen cells can be divided into two groups of committed B cells according to the status of their immunoglobulin chain gene rearrangements [34,182,183]. The cells without immunoglobulin chain gene rearrangements are princi-

pally stem ceUs wifich express B ccU markers [183], During prolonged in viln~ propagation, these cells fail to undergo immunoglobulin chain gone rearrangements, indicating that thcsc cell lines havc bccn frozen in differentiation as a result of the expression of v-rd. The second group ,ff cells transfi)rmcd by REV-T in vitro have functional immunoglobulin chain genc rearrangements and most of the cell lines transformed by v-tel synthesize lgM [182]. During prolongcd in vitro propagation many of these cell lines continue to undergo immtlnoglobulin chain gene rearrangements and diversification [182]. The majority of the spleen cells transt'ormcd in vitro by REV-T gives rise to immortal cell lines [14,t~6,qt)] and a number of REV-T transformed cells have been serially propagated over ll) years Jl82], REV-T (REV-A) also induces Ihymonlas when injected directly into the thymus o1' young chickens (Evans and Bose, unpublished data). Both B and 'I" cells are transformed ill the IllylI1LIsand tllcs¢ cells ~ll'C tumorigenic° REV-T transfi~rmcd cells obtained from the lhymus fi~|'m colonies in sol'! agat ~ but atlcnlpls to

establish immortal cell lines have been unsuccessful. When bone marrow cells were infected with a replication-competent avian retrovirus which expresses the v-re/oncogene transformation also occurs [I 15]. Some of the clones express not only lymphoid and myeloid markers but also erythroeyte markers suggesting that a novel cell type may be transformed by this construct which would b c a very early hematopoietic progenitor cell. Unlike the majority of the cells transformed by REV-T (REV-A), these cells undergo senescence [I 15]. The infection of fibroblast cultures by the replication-competent members of the rcticulocndothcliosis virus group [1{~3,1~4] including REV-A [73] induces a biphasic response. !nfcctcd fibroldasts initially exhibit an extensive cytopathic effect which is dependent on the ratio of infectious virus/cell. When cells are infected with REV-T (REV-A), morphologically transliwmed cells emerge from the chronically infected cultures [46,113]. TransGwmed fibroblasts do not immortalize and eventually the cultures senesce. REV-T, like the other acutely transfiwming avian retroviruses which transform fibroblasts, induces s,'lrcomas in experimentally infected birds [113]. The ability of REV-T to transfiwm fibroblasts has been debated [14,44,58,73, 113]. Faihlre to observe fibroblast transformation was likely due to the cytopathicily induced in the cultures by REV-A. Wh,:n fibroblasts were infected with a retrovirus expressing v-rel in the absence o| REV-A, fibroblast transformation was readily observed [115]. Though the helper virus REV-A replicates in mouse fibroblasts and canine cell lines, efforts to transform mammalian cells by P,EV-T or the v-tel oncogene have met with failure, in murine cells the induction of v-tel expression temporally correlates with cell killing [52,1401.

III. Strtzctul'e and expression of v.rel and its celiui.ar bomolo~ The v-rd oncogcnc was acquired by REV-A from a turkey c-tel sequence [29,176,178]. Though several OllCOgellCS have been acquired by rctroviruscs infccting different vertebrate species, v-rel was apparently only acquired once by a virus during evolution. The v-rel oncogene is distinct from other known viral oncogencs [ 155,176,178]. Upon the transduction of the v-rel oncegent multiple deletions occurred within the REV-A gcnome. REV-T contains a small truncation of the gag gene and nearly complete deletions of both the pol and era' genes [31,127]. The v-tel oncogenc was inscrled into envelope sequences [127] and contains 11 amino acids encoded by REV-A ent' sequences in place of the first two amino acids of the c-r,'i protci;~ (Fig. I; Rcfs, 29, 178). The v-re/ protein contains 1 alilino ;~cids derived fronl out-of-franlc e t l l ' sequcncc,~ at its carboxy-end which replace 118 amino acids of the carboxy-tcrnlinus of c-re/. In addition, v-rei contain.,, numerous amino acid substitutions and three small in-frame deletions [178]. The v-tel gene is expressed from the strong promoter/enhancer element.~ of the REV-T long terminal repeat (LTR) into a 3.t) kb subgenomic message which utilizes the REV-A ent' splice donor and accepter sites [69,71)]. The rcmowd of structural genes from REV-A was required to producc a transforming virus [32,111]. Viral constructs contain° ing intact gag and pol sequences express reduced levels of v-re~ transcripts and fail to transtorm spleen cells. The v-tel oncogenc encodes a 59 kDa protcir which is weakly phosphorylated on scrinc and thrcouinc residues (op5t)'"*"t; Refs. 49, 58, 71, 148, Its7). The turkey c-re/ gcnc (which gave rise to v=rel) is very sitnilar to the chicken gent and the encoded proteins are approx. 95% identical [29,17~]. The c-tel proto-oncogene spans more than 3(} kbp of chicken

V and C - r e l C o m o a r i s o n

p75 c-rel NHz

oC@OH

p59 v-rel

NH ~ -

-C('X3H Rel Homology Domain

~

- Sequences unique to V and C-tel

~

- Sequences in c.rel responsible for tran~aclwat,on

l:ig. I. Structural and futlctioital tiomam,,, in Ihu viral Ir~ln~fornlJrlg protein and it~ cellular horntdtJgy.

DNA and is expressed predominantly as a 4.0 kb tran~ript [33.69.7()]. This mRNA contains a short G / C rich 5' noncoding region, a 1.8 kb coding sequence and an A / T rich 3' untranslated region. Chicken c-tel is expressed at highest levels in cells of lymphoid origin [69.7t)] and at much reduced levels in avian fibroblasts [114], Avian c.rel encodes a 75 kDa protein (p75 ''''~) which is vet)' weakly phosphorylated in REV-T transformed cells [102,149]. A 2.6 kb c-tel transcript is also pre~nt at modest levels in avian lymphoid cells [33,114] and is the predominant tel transcript detected in the ova of prepubescent hens, suggesting that alternate splicing and/or polyadcnylation of the c-rel mRNA m~y occur in different tissues [114], The promoter which lies upstream of the chicken c#'t'l gene lacks the TATA and CAAT sequences geno erally ¢onset~'ed in euca~otic promoter elements [30,(~5]~ The cm't promoter, like many housekeeping gene~ and some oncogenes is CC rich, Different initiation sites for corel transcription are used in fibrobla~t and spleen cells [30], In spleen cells multiple initiation sites are used with two of them preferentially employed, a c o m m o n feature of non-TATA promoters. The c,rel promoter is relatively weak and is less than l(}0-fold as active as the LTR of REV-T. The binding sites for several transcription factors, including SP! [16], Krox-24 [94], HIP-I [109] and NF-kB [97] reside within 07 bp of the major c.rd transcription initiation site [65], The c.rel promoter is repressed in transfcctcd fibroblasts overexprcssing c-rd indicating that it may he autoregulated [65], Cells co-expressing c-~vl from a retrovirus vcch:, and a reporter gcllc under the coP,Ire[ of the c-n,t promoter demmastratcd a 10-2[bfldd decrease in the expression of the reporter gone. Autorcg-

ulation may explain why c-ret transcripts are filr less abundant in avian fibroblasts when compared to norreal and transformed avian lymphoid cells [69]. Mutations in the consensus NF-kB binding site do not affect the level of transcription from the c-tel promoter [30]. The suppression of the C')r'£'l promoter by c-re/ in fibrob/aMs is, therefore, indirect and not mediated through the consensus NF-kB site located upstream of c-ret. IV. Relationship between NFkB and the Rel proteins The relationship between NF-kB aim the ~,1 proteins was established when the nucleotide sequence of the p50 subunit (p50) of NF-kB was defined (Rcfs. 54, 91; reviewed in ReI~. 5(,,57). The nuclear lorm of NF°kB with DNA binding activity is a heterodimcr comB~sed of a 511 and 65 kDa subunit [81,82,180]. This protein binds a decameric DNA sequence (5'-GGGACTPFCC-3') called the kB sequence [L)7]. The eDNA o! thv NF-kB DNA binding subunit encodes a 105 kDa precursor which contains p50 within its aminoqcrminal region [54,91]. This precursor contains a stretch of approx. 350 amino acids near the aminoterminus which is h!ghly conserved (51% identity) with the amino-terminal region of c-re/and the dorsal protein of Dro~ophila. The NF-kB t}5 kDa subunit, which contains the transact(vat(on domain, also contains an aminoqerminal region of 32i} amino acids which shares extensive sequence similarity to c-rd and to a lesser extent with the p5|) DNA binding subunit of NF-kB [I 22]. This conserved region, the Rcl homology domain (Fig. 2), provides fi)r a number of activities relevant for the assembly of an active transcription fitctor. This

NH

CO0~

D~O$ NF~kB

~]]

, ~uo~

uniquo to V aria G.,el

tran$~¢bvatiOn

- Ankyhn repeat

Fig. 2. Homoh~gy in the .,,tructurdl and functional domains in the c.rel proto-oncogene and the NF-kB precursor to p105.

I I - 320 Amino Acid Residues 21 i.trca|ed at N = lemlinus ?,} DNA binding II)omam U

4~ Dlmeri.,atltm Domain 5} Nuclear Transloeation Signal

(b~ Consensus Protein Kinase A site: Are Are X Set Tl lkB binding Domain PKA

NIl,.

~

>.715~

_ _

.

.

.

. (7~,

001t

liiistlng in P4e~lNi:kli

Fig, 3, The [uni.'tiOllill elelliellls which reside in the Rcl htullohlgy domiihl,

domain contains the I)NA binding region and is involved in the formation of hetc|'odimers between different Rel I'amily n|embers 154,91,122,137]. The dimcrization dornain of NF-kB p5(} (KBF-1 ) [103] and &m~al [79] is functionally separated from the DNA binding domain. The precise mapping of the sequences involved in DNA binding and dimerization of the other Rel-related proteins has not been completed and these domains may overlap. The c- and v-wl proteins can oligomerize or dilnerize with the p51) subunit of NF-kB in vitro [93,137]. The Rel proteins have no obvious homology with any previously characterized DNAbinding motifs suggesting that the Rel family of proteins use a novel DNA binding and dimerization domain. The sequences required for DNA binding and dimerization by Rel proteins do not contain leucinc zippers. Zn 2' fingers or homed.domains characteristic of most eucaryotic transcription fact ws. Proteins which inhibit the binding of R e l / N F , kB transcription factors to DNA also interact within the Rel homology domain I92,1221. The general features of the Rel family of transcriptional activators are illustrated in Fig. 3. Rel proteins have a short amino-terminal region of variable length which is not conserved among family members. This is followed by the Rel homology domain which contains the DNA binding and dimerization domains. Also present within the Rel homology domain is a negative acting element which inhibits the transactivation sequences [27,128]. The amino-terminal regulatory and the carboxy-terminal transactivation domains are likely to cooperate in modulating the function of these transcription factors, in addition, the Rel homology domain contains a stretch of basic amino acids located at its extreme carboxy-end which functions in nuclear translocation [29,59]. Approx. 24 residues amino-terminal to this nuclear translocation signal is a consensus site for serine phosphorylation {Arg-Arg-Pro-Ser) by

protein kinase A (PKA)[117]. This sequence is conserved in all Rel proteins with the exception of the recently identified p49 DNA binding subunit of NF-kB [137]. Tile carboxy-termirmi regions of the Rel proteins arc not conserved among family members. In general this region is rich in serine, proline and glutamine rcsiducs and has an overall negative charge. The carboxy-region contaios the domain for transcriptional activation [27,86,128] as well as a domain which mainrains the protein in the cytoplasm [29]. in addition to being structurally related to NF-kB, the tel proteins functionally interact with NF-kB to regulate gene expression. Both v-Rel and c-Rel proreins bind to kB sites from either the HIV-I LTR or the major histoconlpatibility complex (MHC} class i promoter [%77,85,91]. In T cells, NF-kB activity can be induced by exposing the cells to phorbol esters or as a result of the expression of the HIV-I tat protein [21,142]. In phorbol ester stimulated T cells NF'-kB ente|'s the nucleus within minutes while c,rel appears after s,v,',c t:latl hours of stimulattion [! 12]. At the present time c-pv/appears to bc it positively acting transcription fitctor. On the other hand. cells co-expressing tax (the transactivation protein encoded for by HTLV) atnd v-tel, v-re/binds to the kB enhancer and inhibits NF-kB activated transcription from the IL-2 receptor a-chain promoter and the HIV-! LTR [9,77]. Mutant v-rel genes which are unable to bind DNA arc unable to inhibit tax induced NF-kB activation [10]. V. The NF-kB family of transcription factors

NF-kB is a pleiotropic nuclear transactivating factor initially idemified as a protein which binds to the B enhancer of the murine &- light chain gem:. This protein initiates immunoglobulin chain gene transcription [141]. NF-kB was originally believed to be expressed only in mature B cells [141], but was subsequently detected in other cell types following their stimulation with lipopolysaccharide or phorbol esters [142]. Later it was shown that nuclei of some T cell [35] and monocytes [63] also constitutively express NF-kB activity. Therefore, NF-kB is not a cell-type specific transcription factor. In many cell types NF-kB is present in an inactive cymsolic form which can bc induced following exposure of these cells to a wide variety of activating agents including cytokincs [1(}4,11(1,124]: T ceil mitogcn.,, [21,63,119,142] and bacterial lipopolysaccharidc [142]. Exposure of cells to detrimental agents such as inhibitors of protein synthesis [147], agents that damage DNA [153,154], and infection by a number of DNA and RNA containing viruses [9,01),72,98,13t~,147.109, 170] activates NF-kB. Several viral transactivating I'aetors also induce NF-kB [9,98,136,147,1t~9,171}]. The human T cell leukemia virus (HTLV-1} transactivating

protein Tax constitutively activates NF-kB [9,98] and, therefore, may contribute to the development of leukemia in these patients. Activation of NF-kB by these various agents leads to immune, inflammatory, o r stress responses (reviewed in Ref. 5). There is also a good correlation between the proliferative state of T cells and activation of NF-kB. since NF-kB is involved in the induction of the expression of the ~ subunit of the T cell receptor [2!,98] as well as'the T ceil growth hctor, intcrleukin-2 [75], Induction of NF-kB binding activity occurs in the absence of protein synthcsig mdi/~hting that NF-kB is regulated l~}sttranslationally [142], The presence of an inactive ~t.+,s, lic forn~ which can be activated without de novo protein synthesis suggests that it might serve as a second messenger to rapidly transduce signals received at the plasma membrane (reviewed in Ref, ~¢~), The inactive form of NF.kB is present in the eytog)l of unstimulated cells and can bc activated to bind DNA in vitro by treatment of cytosolic extracts obtained from uninduccd cells with tormamide or ~)dium deoxyehohtte (DOC)[3], The inactive cytosolic

Q

F~, 4.. ~ t ~l lJl~ ~:livath~n of R~I/NF-kB, RcI/NF-kB Iranscript i ~ |~bws ~ u ~ t ~ ~s ~'~.~ndar~ m~z.~ng~zrs. These tran~ription ~a~s are ~qucst~zred in the c~'toplasm of the cell hy an inh~Jb~%- ~ [ ~ . l ¢ . IkB. Wh~zn the ¢¢11r~cei~'es an exog~:nous signal to ~d~de a k i n ~ bcvt~ncs actix-aled ~,'hich pht~phot~'lates IkB, IkB ~ t ~ i a l ~ c s them lhe Rel/NF-kB c~nplcx and the complex moves h~ the n~¢h.~l,Ls,where it palrlkip~les in the activation of genes which ¢ ~ t a i n kB elcn~nts in Iheir pn)rm)tcrs.

TABLE ! Cell.hzr emt ,.'ind gem,s which contain a kB-rel~led .w~pr'm'e h~ their i~romolt,r / t,nhatwt'r ¢lt'mc;ffs

Gent family lmmunorcceplors Igu light chain /3 chain*T cell receptor Interlcukin 2 ~ chain receptor Class ! MHC H-2k h /3-microglobulins Cytokines ~ointerfcron interlc~kin 2 inteHeukin o IUlllol II¢crosis l'~lclof ~ IUlllOr necrosis |actor ~

Reference 97, 141 83 q. ~8 7, 82 82 48. 75 1(~1, 145 14,1 I II1

(iMoI'SF (IoCSF Virt~es adeaovirus

13~.~ 121

Cyl¢llllCgillovffU~ I!1%1 Olhel:~ my¢ prolo-on¢ogen¢ serum amycloid A precursor vimcnlin

I,If~ 11~

I?~

4I 4~ Ittl

fiwm is bound to an inhibitor protein (IkB), which was initially characterized as a protein that specifically inhints DNA binding by NF-kB [31. Treatment with dissociating agents like DOC disrupts this NF-kB:IkB complex resloring DNA binding aclivlty. Fig. 4 illustrates a proFa~sed model for ~ctivation of NF-kB. A large number of cellular and viral genes contain kB-rclated sequences within their promoter/enhancer elements, These sequences compete with the consensus kB sequence for NF-kB binding in vitro. Examples of cellular genes which contain a kB-related sequence are listed in Table I. There are over 12 factors which have been identified which bind kB or related motifs. These have different apparent molecular weights and some display different patterns of induction. Moreover, additional NF-kB family members arc still being identified [25,120,135,137], The relationship :~f al! these factors to one another will only be resolved when cDNAs encoding ~hese various factors have been isolated and analyzed, At the presem time transcription factors which hind kB-related sequences can be divided into two groups: (I) proteins which have homology in their DNA binding and dimerization domains and form heterodimers in vitro (pS0, p65 NF-kB [4,141], v and c-rel [54,91], dorsal [156], KBFI [81,82,180], H2TF [6,7] REL B [135] etc,) and (2) kB binding proteins which share no sequence similarities to Rei/NF-kB (MBF-I, PRDI I-BFI [8,43]). This second group of kB-binding proteins contain Zn z+ fingers. There are two classes of

Rei-related proteins; those which are processed like p50 and p49 and others which are not processed such as c-re/, NF-kB, p65, REL B and Dorsal. Several of the kB-binding protein complexes described in the literature may be essentially identical or formed as the result of the dimerization with an incorrectly processed form of one of the subunits. The inappropriate selection of a partner during dimerization may also lead to assembly of a complex which binds DNA in vitro but may play no role in the cell, Until a physiological role has been established for these various kB-binding proteins their classification remains speculative. VI. Biochemical structure and function of NF-kI|

The p511 subunit of the nuclear form of NF-kB was demonstrated to possess DNA binding activity in vivo by ultraviolet cross-linking to DNA and in vitro by pcrl'orming DNA binding cxp~tmlcnt, . . . . s with puril'icd 1150 [4,81~]. The ~5 kDa protein which co-purifies with 11511was initially reported not to bind DNA based on elcctrophoretic mobility shift assays and ultraviolet crosslinking experiments [4,89,171]. The sequence of the eDNA encoding the p65 subunit has been determined and the amino-terminal region 1321) residues) shares homology with the DNA binding domain of p50 [1221. The DNA binding specificity of the pSO homodirect and the p50:p65 hctcrodimcr was distinguished when artificial palindromic kB motifs were employed in binding competition expcrimcn,s 1172]. i'ile NF-kB hcterodimcr contacts the pcntame~tc half sites of kB motifs by one p511 and one p65 molecu.le./'he p51)-p65 heterodimer binds preferentially to motils in which the half sites are of a slightly different sequence. By contrast, p51) dimers bind more efl'icicntly to palindromic motifs with identical sequences in each half site. Therel'orc, contrary to previous assumptions, p65 does have a DNA binding region that in cells becomes activated through oligomcrizjfion with p51). pi~5 can also bind as a homodimcr to DNA in vitro, although a protein: DNA complex with the mobility equivalent to a p65 dimcr has not been detected in nuclear extracts from cells [91,172]. The subunit containing the transactivation dom',.~i.n of NF-kB is p65. Initially, it was presumed that p50 does not contain a transactivation domain since cells co-transfected with plasmids which express p50 and a reporter gene downstream of a kB sequence show no elevat~:d e~prcssion of the reporter sequence [91]. Furthermore, expression of the a-chain gent of the IL-2 receptor requires the presence of p50:p65 heterodimers [68]. When p50 homodimcrs bind to the kB sequence found in the enhancer of MHC genes in plasmid constructs containing a reporter gene, the reporter gene is transcribed fD. Baltimore, personal communication). Apparently, the binding of p50 ho'

"

modimers to this specific kB sequence activates transcription through a different mechanism than those involved in normal kB transcriptional activation. The p51) subunit of NF-kB is synthesized in tile form of a large precursor (p105) [54] which is protcolytically processed [44]. The carboxy-terminal region of the precursor contains seven ankyrin repeats. Ankyrin repeats are Iound in a human erythrocyte protein called ankyrin as well as several tissue differentiation and cell cycle control proteins [106]. Ankyrin repeats may serve to hold the p105 precursor in the cytoplasm by binding it to cytoskeletal elements. These repeats are also present in proteins with lkB-like activity (see below). The pl[)5 precursor does not have DNA binding activity mid a carhoxy-terminal trtmcation of this precursor is required for it to bind DNA [54,91]. Since p50 can dimerizc with itself or with p65, cells may initially synlhesizc pS0 as a precursor which hicks DNA binding ttctivily. °f'o prevent the tk)rrmltion of p5()homodimcrs, which woukl lack transactivating activity when bound tO IllOSt kB motifs, one could anticipalc that the pllJ5 precursor is only proteolytically processed in stimulated cells. The carboxy-terminal region of p105 encodes an ikB which specifically interferes with the DNA binding activity of p50 h~modimers (D. Balti* more, !. Verma, personal communication). This 70 kDa on,rein called lkB-y is produced fi'om an alternatively spliced transcript rather than as a protcolytic processing product of p105 (i. Verma, personal communication). Recently, a second DNA binding subunit which heterodimerizes with NF-kB p65 was identified [137]. This alternate DNA binding subunit is synthesized as a 1011 kDa protein which is closely related to the pill5 pn'ecursor. Like pi()5 it must be processed to generate the 49 kDa l'orm with DNA binding activity. The 49 kDa protein can also fl~rm heterodimers with other tel-related proteins in vitro. The inactive cytoplasmic form of NF-kB contains p51)' p(~5 and an inhibitory subunit, lkB [2,21t]. Purification of lkB from cell extracts revealed two forms, the major form of 35-37 kDa (lkB-a) and a minor tbrm of 411-45 kDa (lkB-/3) [55,18 i ]. The physiological significance of these alternative li)rms is not known. Both fl~rms inhibit the DNA binding of NF-kB complexes, lkB proteins specifically inhibit p50:pfi5 heterodimcrs from binding DNA [4] while they do not interfere with p5(I homodimer DNA binding or the DNA binding of unrelated transcription factors. The IkB molecules binds to and presumably covers the region encomp~tssing the DNA binding domain of p65 located near the amino-terminus [122]. The p65 subunit of NF*kB is, therefore, required ti)r the inhibition of NF, kB by IkB. The addition of IkB to NF-kB complexes bound to DNA results in the release of the transcription complex from the kB sequence in vitro [181]. The binding of lkB to p65 may result in a conformational change in

the pSO:po5 complex and reduce its affinity for the kB motif. Alternatively, lkB may inhibit a DNA bound NF-kB complex because of the high dissociation rate of the complex from a kB site. VII. Structural and functional relationship between lkB and Rel.associated pp40

Like NF-kB, the products of v-rel {ppS9 ,'~''t) and c.rel (p75 ¢'"'~) are principally k~'alized in the cytoplasm of unstimulated cells. In the cytosol of transformed ~vian lymphocytes pp59 '~'''~ and p75 ~''''~ are ass~ciated wilh a distinct set of cellular proteins (pp4(), p7~1, p115 ~nd p1241 [31~,37,1112,11t~,150,t68], In REV:r transo formed lymphoid cells, two cytosolic and one nuclear complex have helen identified [3¢~], The vast majority (75~7~) of pp59 ~''''~ ill the eytosol is complexed with a heavily phosph~rylated form (serine, threonine)of a 411 kDa cellular protein, (~cl filtration arid fast pressure liquid chromatography indicates that this complex has a mass of at)pros= 4(X) kDa [168]. The remaining cytosoli¢ ppS~ '-''''t is associated with two high molecuhtr weight proteins (p115, pl241. The 711 kDa protein present in both of the cytosolic complexes is the constitutive form of a heat sht~k protein (p7lV'") [!112]. The 124 kDa protein is the avian hornpipe of NF.kB pill5 the precursor for the pSll subunit [28]. Until the identi~ ..... ,,~ all of the proteins is known, the function of multiple Rel complexes in REV-T transti~rmed cells will not be understt~l. Approx. 10g; of the ppS~}'~'''~ expressed in the cell can be detected in the nucleus with a weakly phospht~iated h~rm of pp,.llt [3hi. The v°nq product i.~ expressed in h~w abu~dance and acct~unts fl~r tkIXB¢;~, ~ff the methionine labeled proteins in transfi~rmed cells [1tt2]. It is a relatively st'd~lc p|'~ rein with a halt'life of approx. 8 h in the~e complexes In normal avian lymphoid cells p75 ' ''~ also resides in t~a~ cyto~lic ~,'~m~plexes: one complex which contains p ~ ) and the second ctmaplex containing t7115 and pl24 [371, in unstimulated avian lymphoid cells p75c-n'/is only dctccled in the cyt~plasm [37, I I(~,14~}]. c.nq and pp4(J are expressed at significantly lower leeds (Itl°fidd) in normal lymphoid cells relative to the level of ppSgc.~el detected in REV-T t,ansfi~rmed cells

[371, The similarity of si~c between IkB and pl'~ltl and the detergent ~nsitiv¢ astoria|ion ~f pp411with v.~,l and IkB ~ith NF-kB suggested that Ihcs~ proteins were ~imilar 137,1112,1511,1~], Furthermore, c-nq containing c~plex¢~ in c',ut~s~thc extn~cts can be activated to bind DNA by d~ ~¢c~ents, suggesting that an IkB-like protein may ~gulate the c-nq protein in viwt~[72, 112]. It is now es.'tabli~ed that the avian Rel-a~sociated protein, pp4(l. is functh)nally and antigcnically related to murine and human lkB [38.92]. Immunt~fffinity purified pp4(i in-

hibits the in vitro DNA binding activity of tile NF-kB protein complexes as well as c-ret oligomers. The 40 kDa protein isolated from v-re/containing complexes is antigenetically and functionally related to lkB-/~ [87]. The tryptic maps of human lkB-/3 and avian pp4(I indicate that these proteins are highly related, perhaps identical proteins. Radiolabeled lkB-/~ can be immunoprecipitated with antiserum specific for pp40 and this antiserum also abolishes the ability of lkB-~ to prevent NF-kB from binding DNA. Antiserum specific for pp4(I doe.,, not precipitate or inhibit the activity of IkB-t~, The ability of pp40 to inhibit Rel and NF-kB DNA l~inding is mt~dulated by phosphorylation [92]. In tile case of NF-kB, phosphorylation of lkB-~e by the protein kim~se C pathway activates DNA binding by NF-kB in vitro 155]. Phosphorylation of purified pp41) in vitro by Wotcin kinase A activates DNA binding by c-s~,l or NF~kB i~21, There are some flmctional differences between pp4l| (IkB-/3) and lkBn~. Birth pp40 and IkB~ bind to Rel and NF-kB and prevent their binding to DNA in vitn~ [38,92], while IkBo~ is specific h~r the ph5 subunit of NF-kB and thercfi~re does not regulate DNA binding by the v- or c-nq proteins 14,122]. Furthermore, the lkB.~ which is present in the cyloplasm of cells c~mlplexed with NF-kB is posltlh||ed to dissociate heft}re tile nuclear transits:alton of NF-kB. I}v co{l{rasi, pp4t~ {IkB-/J) has been detected in the nuclc~ of REV=I" transfl~rnled lymphoid ceils ¢omplcxed ~i;ll wrd [3hi. M~retwer, experiments using antiserum npecific lbr pp4lJ (IkB-/~) indicate thai c-rd ix ct~mp~,:xed with IkB-/:t in nuclear extracts of stimulated WEtll cells. and this complex binds kB motil~ [g2]. The nuclet~lidc sequence of the cl)NA cnct~tling avian pp4|)(IkB-/3) has recently been defined !3Sl. 'lhc protein predicted from the open reading frame can be divided into three domains (Fig. 41. °l'hc amintHerlllihal domain has a consensus pht~sphory,hllion site fl~r casein kinase II and a consensus tyrosinc phosphoryhllion site that is related to the binding domain fl~r phosphatidylin~sitol-3-kin~asc° The middle p~rli~n ~|' the protein consists o|' I'ivc anky~in repeats. Four of the five ankyrin repeats (repeals I, 2. 4, 5) present in pp4() are responsible fi~r binding this inhibitor to Rel/NF-kB but the carboxy-tcrminal region of pp4() is also required to inhibit DNA binding [781. These ankyrin repeats and the carb~xy-terminal region of pp4() apparently fi~rm a structure which associates with the Rel homology domain to inhibit DNA binding activity. The ('OOH-terminal region of ikB/pp40 contains sequences (PEST) that have been implicated in rapid protein turn over [129]. A eDNA clone (MAD-3) encoding a protein that is induced rapidly upon monocyte adhesion has also been idenlified [67]. The size of its encoded product (35-37 kDal, presence of ankyrin repeats, potential protein

CK II silo Tyr site '~

PEST

I

II

III

IV

V

ill

MAD

t

-3

t

CK II site

PKC site

~

- Ankynn Repeats

~

.PEST Sequence I

POlt~nh;dPllo'.~ptlolVlallOn Silo

I;i~, 5, The ~li|lfltll¢ illld I'uncliol|al dol|mins of IlrOlcJll~ ~vilh IkB activily,

kinasc C phosphtu'ylation site and ability to inhibit DNA binding by NF-kI-I suggesls that this gene encodes an IkB-likc activity. The amino acid sequence of the two tryptic peptides derived 1"1'o!11purified rabbit IkB-a+are identical to peptides of the protein encoded tiu° by MAD-3, indicating that MAD-3 encodes an IkB 13Sl. Conlparison of the predicted amino acid setluenccs of pp4lJ and MAD-3 showed a similarity over a large portion of the proteins lFig. 5). This similarity boggles at the amino-terminal dorn,'|in encompassing the conscnsu~ pht~sphorylation sites and extends beyond the ankyrin repca's into the PEST sequences. The protein kinasc (" (PK(') phosphoryh|tio|l site present in the carboxy-tcn'minai rcgk)n of MAD-3 is absent in pp411, indicating that the i~roducts of pp4() and MAD-3 may be regulated by different kinase pathways. Like pp40 Ihe MAD-3 protein interferes with both NF-kB and c.tel DNA binding (I. Ver|na, personal communication) indicating that it encodes the human honnolog of IkB-B.

VIII. Regulation of Rel/NF-kB transcriptkm factors Si||cc NF-kB is a ubiquitously expressed transcription factor which participates in lhe regulation of many different cellular genes, it is not surprising that its activity is rcguh|tcd at several different levels. Subcellular location is one major mechanism whicll operates to rcguh|tc the activity of this family of transcription factors. Inactive complexes are sequestered in the cytopla,;m preventing their translocation to the nucleus [2]. The regulation of nuclear translocation for these transcription factors involves the binding of an lkB-like inhibitory molecule to the Rei homology domain. These molecules cover the DNA binding and dimerization

domains and may also mask lhe nuclear targeting signal [92,122]. The ankyrin repeats found in lkB molecules could serve to attach these inactive transcription complexes to cytoskeletai elements ill tile cytoplasm of the cell. When ikB is phosphorylatcd in vitro it dissociates from NF-kB [55,146]. It has been proposed that the translocation of NF-kB to the nucleus occurs as a result of a change in the phosphorylalion of IkB resulting in its dissociation from the complex [55,146]. Consistent with this model when REV-T transformed cells are exposed to Zn -~~ the synthesis of pp40 is transiently repressed and the c-rel protein translocates to tile nucleus [161]. The existence of multiple inhibitory molecules may prtwide the cell with greater control over tile function of a set of ubiquilous!y expressed transcription factors. The potential to assemble multiple I~el-family complexes Ihat arc con. Irollcd by a family o1' inhibilors mighl also allow for a graded response to exogenous stimulalory signals. The subcellular location of Rel family members is also t°cgulated by sequences which arc intrinsic to each transcription factor. Deletion of the first 19 N-terminal amino acids (Hannink, personal communication) or deletion of !03 C-terminal residues results in the nuclear translocation of the c-eel protein in avian fibroblasts or lymphoid cells [29]. The nuclear translocation of amino-terminal truncated c-tel is likely duc to the rctritwal of sequences essential for the efficient or stable binding of pp40 since it binds to the aminoterminus of c-tel [37,92,116,150]. The subccllular location of c-tel must also bc reguh|tcd by sequences intrinsic to the fi|ctor as well as by pp4() since there is a significant population of c-reJ molecules in tratlsti)rnlcd and norrnal lymphoid cells which are not conlplexed with pp40 yet re|nain cytosolic [37,411]. in t|ninfected avian lymphoid cells, the majority (611%) of p75 ~'°''''/ exists in a cytosolic complex which does not contain pp41l [37]. The carboxy-terminus of c-tel also appears to function to hold the protein in the cytosol [29]. Unlike the carboxy-terminus of NF-kB pll)5 or the lkB molecules, the c-tel protein does not contain ankyrin repeats or other recognizable elements which could serve to attach the factor to cytoskeletal elemc1~ts, Ti~e rmtive conformation of c-rel is unknown but it is likely that the carboxy-terminus of c-tel is folded in a fashion which would sterically mask the nuclear translocation signal located in the carboxy-terminal end of the Rel homology domain. It has also been suggested that the conformation of the c.rei protein may be such that the carboxy-cnd may prevent the phosphorylation of a seeine residue (Ser-266) within the PKA recognition nu~tif adjacent Io its nuclear targeting signal [117]. The insertion of two amino acids into the PKA consensus .~equence results in a shift in the localization of the c-rel protein from the cytoplasm to the nucleus in avian fibroblasts. Evidence that the serine residue in the

I0 PKA site is phosphorylated in cells, however, was not demtmstrated. In addition to regulation at the level of subccllular location, the transcriptional activity of the Rel/NF-kB proteins can also be modulated by their relative affinity tiw the same or different kB-relatcd motifs. Rel and NF-kB pa~teins can bind the same DNA sequence in vitro and competition between proteins that bind to the kB mquence in the class i MHC H-2K" enhancer has been relx~rted [82]. The sequence specific binding sites and affinities for different kB-related sequences by various kB binding proteins is currently being analyzed. The relative affinities of the different kB binding proteins for specific sequences present in promoter/ enhancer elements in the vicinity of Rei/NFokB reguo lazed genes would provide the cell with a great deal of transcriptkmal control over gene~ regulated by tl~is filmily of transcription factors. Tl~e transcriptional regulation of mo~t eucaryotic genes is governed by the activitiesof a number of different transcription factors acting in concert. The synergistic interaction of Rel/ NFokB faetor.~ with unrelated transcription factors adds an additional element in the control of gone expression, IX. I)o~al

The Rel family of transcription factors has been highly ~.xmserved in evolution and l)rosophih~ contains a related gene called dorsal [156]. The ~hu:~almaternal morphogen was initially identified on the basis of a genetic lesion (reviewed in Ret\ (~2), Null mutations within &~,sal ( d i ) result in the abrogation of normal dorso-ventral polarization. The dorsal protein is piv. oral in the establishment of proper polarity within the early D~.~ol)hila embryo [ 123,15¢q. In unfertilized eggs. the p~tein is detected in unifi~rm amounts throu#tout the ¢¢~vtt~ ' ~1. Within ~0 rain alter fertilit~ation, the dors¢d pn~tein selectively, enters the nuclei of ventral tells, while remaining cytosolic within dorsal cells 1130,133,157,158]. At this t~int, the relative levels of protein within ventral and dol~al regions are nearly equivalent. Within 1511rain after fertilization, the ventral regions contain an overall higher level of the d ~ a l pn~tein, Do~d is m~t~lulated thnmgh at least 16 maternally active genes and null mutations in It| of tilese genes inactivates dorsal function [I.~2]. With mutations in an~* of the~ l|t d~r~il-group genes, the dorsal protein is expn:~,~cd at nurmal l,~cl:,,b~ l r.-~-:~;~,s sequestered within the t~l~,~t~! [ 13lk133, !57]. Proper dorso-ventral ffolari~atkm ~ q u i ~ s the nuclear mmslocation of the d~,~d protein. The DNA ~quencc recognized by dorsal is similar .vet di.,ainct them the con.~nsus kB enhancer sequence [~]. While NF-kB appears to be a transcription activa-

tor, the dorsal protein has been implicated ill botl~ the activation and repression of specific genes. Genetic analyses have identified at least four genes regulated by dorsal [80,131,132,151]. The presence of dorsal within the nucleus directly or indirectly initiates expression of both twist and snail, which regulate the differentiation of mcsodcrm within ventral regions [24.151.165.166]. However, do~:~ai is also thought to repress the expression of zerkmdh (z('n) and decal~'ittaplegic (dpp)within the ventral region [ 132,134]. These genes control the differentiation of dorsal amnioserosa and epidermis, respectively [125,174]. Mutations in dop:~al, or in other dorsal.group genes, result in the failure to activate twist and snail [1651 while .ten and dpp remain expressed i, an unrestricted filshion within both the &~P:~a/ and ventral regions of the embryos [132]. Interestingly, the dorsal protein appears tO bind long-range repression elements willliil the pronloter of :e, [7% X. Transformation by the v-tel oncogene

A-A, Ac#t'adon of the oun,~Jbrming Iu~tential el the c-tel I~tWto-otl('ogeate Avian lymphoid cells transfiwmed by REV-T contain 8-10-lbld higher levels of v-n,I message and protein than c°rd [69], A gene dosage effect is not, how. ever, adequate to explain wily v-M is a strongly transIbrming protein since the elevated expression of the c-re/ proto-oncogene is not sufficient to activate its trans!~wmin~ potential [29]. When expressed at levels equivalent to v-rd in transfectcd spleen cells, c-rd transh~rms ceils with a frequency only IC~ t)l' Ihal of the v-n'/ oncogen~ [6{~1. The specific changes in c-rd which are required I~ activate its Iranslbrming potential are not known, The deletion of e-M sequences during its transduction into REV-A as well as the subsequent mutations introduced during reverse transcription of the recombinant provirus are also impel tant for activation of v-rel transhwming potential. Changes in sequences in the amino-terminus of c-rel are required to activate the transfi)rming potential of c-rd [50,1(~2]. A recombinant virus containing c-re/with the amino-termilrlal alterations present in v-m/ allows this construct to transfi~rm avian spleen cells in vitro with the same efficiency as wild type v-rel [64]. Only a small percent of those transfiwmants, however, give rise to immortal cell lines (5~i) [64.162]. The mutations in the middle of the wild type v-rd gene when in.,orporated into c-r~q together with the amino-terminal alteration results in a recombinant rctrovirus capable of transforming and immortalizing spleen cells with an efficiency equivalent to REV-T [64]. The c-rel protein also contains ! 18 carboxy-terminal amino acids which were deleted during the acquisition

I! of the ret sequence by REV-T [29,178]. This carboxyterminal domain contains the transactivating elements of c-tel, as well as a cytoplasmic retention domain [27,29,86]. The fusion of this sequence to the DNAbinding domains of yeast or heterolcgous mammalian transcription factors results in a hybrid protein with transactivating activity [27,861. This c-rel transactivation domain is not required for translormation of cells by the v-tel protein. Its presence, however, does not suppress the transforming ability of v-rei since a v-rel fusion protein containing the 118 amino acids at the carboxy-terminus of c-rel is also transforming [64]. X-B. r.rel sz,quem'es whk'h contribute to its tran.~j'ormhtg actir'ity The v-:vl protein contains two regions which arc required for its full transl'ormntion potential [51),51,59]. The first domain consists of the amino-terminal 300 amino acids which includes REV-A cm' sequences as ~vell as the Rel homology domain. The II amino acids encoded for by REV-A cm' sequences at its amino° terminus are required for wild type v°rel transfonlfing activity. Replacement of these era' sequences with a methionine residue reduces the transforming efficiency ot' v-re/[15,50]. The era' sequences encoded within ,v-rel contain point mutations relative to the corresponding sequences in the era' region of REV-A. These envelope mutations also enhance the transforming activity of v-tel [15] thouglJ they are not required since c-rei proteins with only carboxy-terminai deletions are also strongly transforming [86]. In addition to the era' sequences, any alteration of the Rel homology domain in the amino-terminal region of v-tel abolishes the transforming activity of the v-tel protein [64]. The structural integrity of the PKA recognition signal in the Rei homology domain is critical tor v-rel transforming activity [117]. Substitution of the conserved serine residue within the recognition sequence with acidic amino acids (Asp or Glu) significantly reduces transformation and transcriptional repression by v,tel. These results suggest that phosphorylation at this consensus PKA site could have a negative effect on v-rel transforming activity. Large deletions of the sequences in the carboxy-terminal region of v-tel also a~olish its transforming ability. At the present time the specific contributions of the changes in the amino- or carboxy-end of v-rel to its transforming potential have not been defined. X.C. Subcelhdar iocathm and tran.~formation by t,.rel In REV-T transformed lymphoid cells approx. 91)% of the protein is cytoplasmic and 11)% can be detected in the nuclear fraction of metabolically labeled cells [36,58,59,148,175]. While subcellular location is a major

mechnnism used ~,~ regulate the transcriptional activity of NF-kB and dorml, the v-tel protein may transform cells irrespective of its subcellular location [59]. Constructs which express v-rei proteins in which the nuclear targeting signals were mutated transform spleen cells with an efficiency equivalent to wild type [59]. Thc subcellular distribution of the v-re/ protein in REV-T transformed lymphoid cells and avian fibroblasts are distinctly different. The v-tel protein in transformed lymphoid cells is principally cytosolic [59,148,175]. In REV-T (REV-A) infected but morphologically unaltered fibroblasts, the majority of the v-rel protein is nuclear based on immunofluorescence [58,113]. Upon transformation the vast majority of the v.tel protein becomes localized to the cytosol as in transformed lymphoid cells [I 13,115]. Fibrobhist.,. infected by REV-T (REV~A) express only half the level of the v-tel protein detected in the transformed lymphoid cells and its half lift is also considerably shorter them in lymphoid cells [1131. The v.tel protein also influences the expression of the tel associated proteins [93]. in MSB-I cells, a cell line transformed by the Marek's di.,~ease virus (a herpes virus), modest levels of the c-tel protein are expressed [26,102]. The c-rel protein expres.,~ed in these cel!~ is not complexed with pp40, p115 or pl24. Introduction of the v-tel gene into MSB-I cells resuhs in the synthesis of the rel-associated proteins which then form protein complexes like those found in REV-T transformed lymphoid cells [93]. This obsetwation may explain the difference in the subcellular distribution of the v.rel protein in REV-T infected ver~.;us morphologically transformed fibroblasts. Though ;Man fibroblasts express the Rcl associated protein:t, the level of pp4() (IkB) is significantly lower in fii,roblasls relative to normal lymphoid cells [I 13]. lnitia!iy, pp59r-tel would be expressed in great excess twcr pp40 and would, therefore, be principally a nuclear protein. The presence of pp59 v'r''! could lead to the increased expression of pp40, resulting in a shift to a predominantly cytosoiic location in morphologically transformed fibroblasts. in avian fibroblasts and unstimulated REV-T transformed lymphoid cells, the c-rel protein can only be detccled in the cytoplasm. The c.rel protein can be detected, however, as a kB-binding protein in nuclear extracts of phorbol ester stimulated Jurkat cells [11)]. Likewise, the exposure of REV-T transformed lymphoid cells to Zn 2+ induces the nuclear translocation of the c-rel protein [161]. In Zn"+-stimulated cells the synthesis of pp40 is transiently depressed and the translocation of the c.rel protein to the nucleus accompanies the dissociation of pp40 from the c-rel protein. X.D. Models for transfi)rmation hy the r.rel om'ogene The mechanism by which the v.rel oncogene transforms avian lymphoid or myeloid cells is still poorly

12 and inhibits, rather than activates genes which contain kB elements in their promoters [10,77,128]. Transformation-defective v-rel mutants which fail to bind DNA are unable to repress expression from promoters containing kB sequences [10,117]. There are at least three plausible mechanisms by which v-re/could alter the expression of genes resulting in cell transformation. Model one suggests that the

understood, Although v.rel was initially reported to encode a transcriptional activator [52,86], it does not contain the carboxy-terminal transcriptional activating domain of the ¢.rei protein. The v-tel protein does, however, contain the DNA binding and dimerization domains of the c-~,l protein and mutations introduced in this region abolishes the transforming ability of v-re/. The v.re/oncogene product binds to kB sites in DNA A

e~

~

~

)

CylopIRsm

F~.. b. l ~ i h l e mechani.~,x b~• which the v.r~,l oncogene transforms cells. (A) Modal I: general suppression by v-rel at kB sites. The v.rel protein, ~h~h h~k~ a tran.s,'~¢tivaling domain, ~.'~uld dimerize and oc~:up~, kB sites pre~,enting the activation of these genes by fnnctional Rd/NF-kB family m~mb~rs. (B) M(~el 2" Sl~cific suppression of cell c~'cle regulator. The v-rel protein when occup.ving kB could block the tr~n.~ripti~m ~ a g~ne imxdved in the ~.'xmtrol of cell ~:~;cle regulation. (C) M(~Iel 3: sequestration of inhibitory molecular by v-rel which are r ~ . x p ~ i ~ for r~l~nlion tff EB binding proteins in the cy.to~d. The v-re! protein could bind to and sequester IkB molecules allowing the inappropriate nuclear translocation of ReI/NF-kB transcription complexes.

13 v-tel protein could IIomodimerize or heterodimerizc wilh the pS0 subunit of NF-kB, forming inactive transcription complexes in the transformed cell (Fig. 6A). The v-tel protein could also dimeriz¢ with c-re/al|ering the specificity or regulation of the c-re/ transcription complex. The high level of v-tel expression in REV-T transfi~rmed cells relative to e-re/and pS0 would lead to the v-tel protein being present in virtually all the Rel/NF-kB dimers. These inactive complexes could occupy kB sites in the vicinity of genes whose transcription is regulated, in part, by Rel/NF-kB transcription factors. When bound lo DNA these inactive complexes could slerically hinder the binding of functional Rel-related transcription filclors to these occupied kB sties. This would reduce or abolish the transaclivation ~,I' the genes under Rel/NF-kB conlrol. Alternatively, lhc v-rd protein could helerodimerize with other Rel fainily members n'esullit]ig in the formation oI' a transcriplion coIlllllex which IIIIi~ II~ incapable of I~inding ill kl] sequences. It] support of this model is lhe reccnl obscrvalion lhal KBF-I inlllalllS, which cannot l'fiml DNA but do dinlcrize wilh c-tel, iilhibil !~el/NF-kl] Iranseriplional aclivalion [103]. These KI3F-I nlulanls I'unction as donlinant negative regulators. The binding of v-rel or v-rt, I/pSI) (NF-kB) complexes could also actively lea,.t to the altered expression of gent(s) critical lk~r the control o1"cell proliferation (Fig. 6B). NF-kB is known Its transcriptionally activate a number of lymphokine genes and their receptors. It is

very likely thal in an REV:I" transformed cell, v-re/ is acting in bolh a passive and aclive manner. in addition to tile DNA binding and dimerization domains, v-rd has retained tile binding domain for inhibitory pn~teins (ikB) wllicll seques|er tile c-Rel/ NF-kB transcription complexes it] tile cytosol [37,102,147,168]. An alternative, but not mutually exclusive model, is that v-rd sequesters these inhibitory molecules thus allowing tile inappropriate activation of Rel-related transcription Iktetors (Fig. 6C). These transcription factors could then enter the nucleus and cause aberrant gene expression leading to transformalion. This is an attractive possibility since v.rel is expressed at levels 10-101M'old greater than that of c-re/ and the inhibitor of c-rei (pp40) is expressed ;it levels essentially equivalent Its that of c-re/. ('onsistent with this model arc the ol~servations that v-re/ has h¢cn shown to activate spccil'ic genes. In nmusc t'ihrohlasls the expression of v-re/ weakly elevated the expression of a nu/nbcr of rept~rler genes [52]. In MSI]-I cells the introduction of v-re/ leads Its tile elevated synthesis ()I lhe Rel-associaled prolcins pp4(}, p115 and p124 [93]. Though il is unknown :il lhe present lime whether lhes¢ genes conlain kB binding sites in their pr()moler elements, these observations suggest that v-w/ may alter lhe proper regulation of other kB binding proteins [2]. Alternatively, v-tel when bound to kB sequences may inhibit the expression of a gen¢ critical to cell cycle control. The down regulation of a repressor

y

$ Fig, 6. (conlinued).

@

14

I~, v.rei could allow the cxprcssion of a lymphokine and/or its receptor. All of these models propose that gent expression from promoter elements containing Rel/NF-kB sites is altered in v-rel transibrmed cells. The genes, whose proper mnction is compromiscd in cells transformed by REV-T. remain to be identified. It should bc noted. however, that mutations which map outside the Rel homoh)gy domain also severely interfere with the transforming activity of v-rel. Thus, v-tel must also have ocher biochemical properties which have not been defined at the present time the:;, contribute to its transh)rming ability, Understanding the mo~ccular basis for tl~etransfi~rmat,on of avian lymphoid cells by vonq is likely to pr~widc fundamental insights into the transfi~rmation process in certain 6~rm:sof human lymphoma. The c-re/ protoooncogcnc has hccn mapped to chromosonlc ! 13 [26], a site associated with rcarrangemcms ?~p12o2~ or amplifications in non-Hodgkin's ly~pl~oma. Cell lines derived from a diffuse large cell lymphoma con~ain chimeric tel tran~ripts. The amino°terminal half of tile c-re/ coding region which contains the Rcl homolo~ domain was fused to the coding regions of an unrelated gone that lbrmcd the carhoxy half of the protein [I{)5].The translation of this aberrant tran,scriptwould yield a wl-rclatcd protein containing the D N A binding ~nd dimcrization domains. The transcriptional activating domain as well as a cytoplasmic retention sequence a! the car~xy-cnd of c.r('l would bc absent in this c-rrl fusion protein, These changes arc strikinglysimilar to the alterations in the avian c,r(q sequence which occurred durin~ ils Iransduction h~, REV-A to pr~Mucc RtW°T. Activalio. of Ihe c-w/ h~us has also bccn reported ill avian B cells in aqan Icuktt'~isvirus induced neoplasias Is41. Xl, i~oa¢luskm The Rel l~mily of transcription factors regulate the [ran~lctiv~lfion of genes by binding to a 10 hp kB DNA ~ t i f in the enhancer and promoter elements of a wide '~'arietyof genes, Presently, most or the genes known to ~Icti~'ated by Rel/NF-kB encode immuno-modulatory t~tokines, cell surface receptors which function in immune regulation or acute phase response proteins. The ReI~ homok~nj domain, which is hvca|ed near the amino-terminus of these proteins, contains the D N A binding and dimcrization domain, as well as a nuclear ~ranslocation signed linked to a consensus PKA phosph~yl~thm site, This domain al.,o c~)ntains the binding sites for IkB proteins, The v- and c-tel proteins can olig~n~criee or heler~Jimcrize with the DNA binding s,.'~nits of NF-kB, Rei family mem|~rs exist in an inactive form complcxcd with inhibitory molecules (IKl~s) in the cytoplasm of many different cell types.

These inhibitors prevent DNA binding and are apparently responsible for the cytoplasmic retention of these complexes. DNA binding by Rel/NF-kB requires release from these inhibitory molecules. Unlike most inducible transcription factors, the nuclear translocation and activation of Rel factors is induced following exposure of cells to a number of different activating agents including cytokines, mitogcns and agents which induce cell damage, The only known Rel family member with transforming activity is the v-rel oncogcnc of the acutely transforming retrovirus, avian reticuloendotheliosis virus (REV-T), REV-T transforms and immortalizes avian T and B lymphocytes, as well as monocytes. The v-wl oncogcnc is a truncated version of the c-re~ proto. oncogenc which is transcriptionally silent duc to the deletion of the carl~xy-tcrnfinal c-wl transcriptional aclivation d( mdlil during transducfion by REV-A. The v.tvl protein contains the D N A binding and directionlion domains of the c-M pro|tin and mutation in this region abolishes the transl'ormhlg ability of v-wl. The v.wl pr~luct binds to kB sites in D N A and inhibits rather than activates the genes under Rcl/NF-kB control.Transformation-defective v.rel mutants arc unable In repress expression from promoters containing kB sequences. Models arc proposed In explain how the v-tel oncogenc transforms cells hascd on the slrucIoral and functional rehltionship between the c-r(q protein and NF-kB. At'knowledgemenls I would like to thank William Bargmann, Rose Evans, Ananda Krishnan and Bob Storms for discus,qon and helpful suggestions on this manuscript. I would al~ like to thank David Baltimore, "l~)m Gilmore and Inder Verma fiw communicating their unpublished observations. This work was supported by NIH grant Ca 331~2 and Ca 21~16~)from the National Cancer Institute. References I Andcl~m, K,V, and NusMein.Volhard. (', t I~)Xh)I,i Gamch~gencsis and The F,arl~ Emhr~,o, t(iail, J., ed.), pp. 177-Ig4. Ahm R. Lis,,,, Nc~ York, ..'b llaeucrle. P,A, an~l llaltmlore, l). (l'lSl~l Science .4..s., 54(l-~54h. .~ Baeuerle. P,A, and ll;dtimt~re, D. (lqXN)Cell 53. 211-217. 4 I~acuerle, P,A, al!d Ilaithnorc, I), (I~;S~H Genes I)ev, 3, Ihl~L)I(1~)8, 5 Bacucrle, P,A, (I~)~Jl) Bit,:him. Biophys. Acla 1072. h3-81). h Baldwin, Jr,, A,S. and Sharp, P,A. (1~}87) Mol. Cell, Biol. 7, 305-313, ? Baldwin, Jr,, A.S. and Sharp, P.A. (11~88) Proc. Natl. Acad. Sci. USA ~5, 723-727. 8 Baldwin, Jr., A.S., LeClair, K.P., Singh, It. and Sharp. P.A. ( l ~ } ) Mol. Cell Biol. I0, 1406-1414.

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The Rel family: models for transcriptional regulation and oncogenic transformation.

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