The IκB proteins: members of a multifunctional family

~EVIEWS

38 Ji, H. etai. (19)3) Ce!173, 1007-1018 39 Oliver, S.G. et aL (1992) aattoe357, 38--46 40 Marschalek, R., Brechner, T., Amon-B/Shm, E. and l)ingemlann, T. (1989) Science 2,H, 1493--lq96 41 Reiter, W-l)., Palm, P. and Yeats, S. (1989) Nucleic Acids Res. 17, 1907-191-i. 42 Sprinzl, M., Dank, N., Nook. S. and Sch6n, A. ( 1991 ) Nucleic Acids Res. 19, 2127-2171

O.~. VOYTAS IS IN THE DEPARTMENT OF ZOOLOGY AND GENETICS, IOWA STATE UNIVERSITY,2 2 0 8 MOLECULARBIOLOGY BUILDIN~ AMES~ 1,4 50011, USA; J.D. BOEKE !$ IN THE DEPARTMENT OF MOLECULAR BIOLOGY AND GENETIC,%JOHNS HOPKINS UNIVERSITYSCHOOLOFMEDICINE, 725 NORTH WOLFE

ST, BALTIMOP~MD 21205, USA.

The IKB proteins: members of a multifunctionalfamily

M a n y eukaryotic transcription factors play key roles in cellular decisions involving growth and differentiation. Members of the Rel/NF-KB family of transcription factors (herein termed Rel proteins) have been extensively studied because of their important part THOMASD. GILMOREAND PATRICEJ. MORIN in the regulation of both cellular genes involved in growth and development (often in cells o f the intorune The lgB proteius bind to Rel/NF-gB transcription factors system) and of viral genes (reviewed in Ref. I). The and modulate their activities. Although originaUy described ability of IKB proteins to modulate the activity of only as cytoplasmic inhibitors of Rel/NF.g B transcription eukaryotic ReI/NF-KB transcription factors is an exciting complexes, it is now clear that lgB proteins also have other and complex system for studying the regulation of tran- functions. scription factors by post-translational mechanisms. The Rel family includes cellular proteins from insects to mammals, and one retroviml oncoprotein (v-Rel). All in the nucleus. The subcellular location of Rel transcripRel proteins are related through a highly conserved tion complexes is controlled, at least in part, through amino-terminal domain of approximately 300 amino interaction with the IKB family of proteins (reviewed in acids, the Rel homology (RH) domain, which contains Ref. 2). IKB was initially named and defined as a factor the DNA-binding, dimerization and nuclear Iocalizatkm that inhibited the NF-KB transcription complex (a functions (Fig. I ). One class of Rel proteins, whose mem- pS0-RelA heterodimer); that is, direct assc)ciation of a bers include pi05 and pl00, has carboxy-terminal single molecule of IKB with NF-KB caused NF-KB to l)e resequences that contain multiple copies of ankyrin tained in the cytoplasm and inhibited its DNA-binding acrepeats (see l')elow); when synthesized in vgm, these Rel tiviW in vitro3. However, it is now clear that IKB proteins proteins bind DNA only weakly, and can undergo pro- can affect the activiW of Rei proteins in other ways, and that IKB proteins are themseh'es regulated at multiple levels. teolysis in v k ~ t() remove the carboxy..temlinal sequences. After proteolysis, the m:tture proteins (pSO and p52, respectively), which essentially contain only the RH IgB proteins: structures and interactions with ael domain, can bind DNA avidly in t,gm. A second class of proteins The known IKB proteins include the vertebrate Rel proteins (c-Rel, RelA, RelB, Dorsal, Daf, v-Rel) has carboxy-temlinal sequences that contain transcriptional proteins IKB(x, IKB[3, Bcl-3, pl05, pl00, IKB~/, possibly activation domains, and these proteins do not not sE ANKYRIHq undergo proteolysis. Mature r--- REPEATS Gene Protein Rel proteins of both classes CLASS I N R ~ I p501.105 I itllllNllU,c,,ll bind to specific target sites NFKB2 pS2/p 1O0 I--DNA-blndlng "1 (KB sites) as homodimers ~.~ I" B-binding H (e.g. p502) and as heteroJ-OlmerlHtlon--J dimers (e.g. p50-RelA) and have distinct DNA-binding CLASS 'i C-rel c-Rel I NN~NNN~RH~NNNNN~ I specificities that depend on relA ReIA {p65) I-- TcXT"~! the compositkm of the relB RelB dimer. Although most Rel dorsal Dorsal complexes are involved in daf Daf transcriptional activation, /qGInl certain others, such as p502 and Dorsal>, can act as The two classes of Rel proteins. Both classes contain the Rel homology domain (RH), which repressors of transcription. contains sequences needed for DNA binding, dimerization, IKB binding and nuclear targeting (N). A second distinctive fea- Class I Rei proteins have carboxy-tenninal sequences that contain seven ANK repeats (shaded) and ture of Rel proteins is fllat an acidic sequence (ACID) located between the last two ANK repeats: the ANK repeats are their subcellular location may removed by a cellular protease. Class II Rel proteins do not have carboxy-temfinal ANK repeats and vary: Rel proteins may be are not cleaved: in addition, their cadx)xy-terminal sequences contain transcriptional activation kx:alized in the cytoplasm or domains (TX ACT). TIG DECEMBER e l,)t)3 El,,,:vier 5t'ience Publisller ~, Ltd ( | 'K) o l t ~ - 0=)2=, 93 $g)().()1}

1993 rot. 9 xo. 12

~tEVIEWS

sequence (TPLH), but even this may diverge among ANK repeats, and sequences outside of this core are 105 p105 extremely variable. Individual ANK repeats are usually more conserved between different IKB proteins than are ANK repeats within a given IKB molecule (Fig. 3). 8 65 I. Although the detailed (proposed) three-dimensional structure of the ANK repeats is not I~eo~ 37 I Ileal "o'° I known, Michaely and Bennett ~ have proposed a I ~:el~ 43 ? model in which a 33 amino acid ANK repeat is composed of an interrupted Bcl-3 47 I IHI (x-helix (residues 4--11), a short turn, and a I~-sheet cactus sa (z go.c) I "°'° IRF I (residues 16--23) that is 53 (maternal) aligned anti-parallel to the pEST (x-helix. The variable amino acids at both ends of this ,.-ram main structural ANK element Structure of IKB proteins, showing the general structures of known IKB proteins and their may form k)ops that connect approximate molecular weights (MW). IKB(xhas also been called different names in different the ANK repeats, and collecspecies: p~0 in chicken; ECi-0 in pig: RL/IF-1 in rat; and blAD-3 in human. In each IKB protein, a tively the ANK repeats of a centrally located region of ANK repeats (small hatched and filled boxes) is flanked by variable amino-terminal and carboxy-ternfinai domains (open boxes). ANK repeats that are most closely given IKB probably form a related between IKB proteins are shown as identical boxes. ACID, sequences rich in glutamic higher-order structure that :rod aspattic acids: PRO, proline-rich sequences; PRO/SER, proline- and serine-rich sequences; can make direct contacts PEST, sequences found in re:my proteins targeted for ,'apid degradation. with interacting Rel complexes, It will he of great interest to understand the molecul:u' structure of tile ANK repeats and how spedIKBS, and the product of the Dmsopbikl gene ficity is conferred by these repeating units. cact.s (Fig. 2). IKBoL, IKB[3, Bcl-3 and Cactus are each Little is known about the sequence reqttirements the sole product ()f a dedicated gene pl05 and pl00 for interactions betv,'een tile IKB and Rel proteins. are full-length NF-KB precursc)r proteins that have However, in at least one case it is known that an IKB several of the activities ascribed to IKBs (see below) requires intact ANK repeats if it is to ass(~.'iate with and, therefore, are also included here. IKB~ contains sequences from the carboxy terminus of pl05 and the RH domain: mutations within the core "I])LH is encoded by a subgenomic mRNA from pl05,, s() sequence of ANK repeats 1, 2, 4 or 5 of chicken IKBtx far, this protein has only been detected in mouse abolished its ability to interact with Rel protein,,;('. IKB0I lymphoid cells ~, IKBS, which consists of the carboxyproteins rnake contact with at least two sites within terminal sequences of NF-KB pl00, has been predicted the RH domain: the Rel nuclear localizing sequence to exist on the basis of analogy with IKB~/, but has not (NLS) and a conserved RXTXRXRXXCsequence known to yet been detected in cells. be inw)lved in DNA binding "~-II. However, sequences IKB proteins are structuraUy related in that they all carboxy-terminal to the Rel homology domain probably contain multiple copies of a 33 amino acid sequence also have a role in stabilizing ReI-IKB complexes in t,it~). known as an ankyrin or cell-cycle repeat (herein called For example, chicken c-Rel and Dorsal proteins that have the ANK repeat) located within their central regions. deletions only of carboxy-terminal sequences (outside ANK repeats mediate protein-protein interactions and the RH domain) enter the nucleus of tissue culture have been found in a number of proteins, including cells 12.t3, suggesting that they can no longer bind their human erythrocyte ankyrin, yeast and vertebrate transcognate IKB proteins. Furthen'nore, the carboxy-terminal cription factors (e.g, GABP~, SWI4 and SWI6), and prosequences of RelA are needed for full IKB0~-rnediated inteins related to Dn>sophila Notch (reviewed in Ref. 5). hibition of DNA-binding by RelA (Ref. 10). Finally, it is Apart from Bci-3 and Cactus, all IKB proteins have an likely that IKBOLpreferentially interacts with a Rel dimer acidic region located carboxy-terminal to the last full rather than a monomer "7, and that the IKB0z monomer ANK repeat. Outside of the ANK repeat sequences and contacts both p50 and RelA in the heterotrimer ~. the acidic region, the IKB proteins have no obvious similarities. Activities of IKIIproteins In addition, the ANK repeats are themselves quite Several disparate activities have been ascribed variable. In general, they contain a core amino acid to IKB proteins (Table 1). IKB proteins can be Protein

MW (kDa)

PROTEASE

~1

TIt; DECEMBER 1993 VOL.9

ANKVRIN _ _ ~

REPEATS

No. 12

~'~EVIEWS

distinguished on the basis of their abilities to bind particuhr Rel complexes and modulate their activities.

Consensus (Hum A n k y r i n ) REPEAT

Sequestering ql'Rel pmteiu complexes iu the crtoplasm Interaction of IKB0t with either hetercxtimeric or homcxtimeric complexes that contain RelA or c-Rel causes k~.'alizalion of these complexes to the cytoplasm of cells (reviewed in Refs 2, 14). On the other hand, I.,.,Bcx appears to have quite low affinity for pS0 hornodimers, and substantial amounts of IKBtx are needed to retain such homtxtimers in the cytoplasm s. h(Bet may cause c}¢oplasmic localization of Rei complexes by masking the NLS of the Rel proteins7.S; for example, the acidic sequences located carboxy-temfinal to the IKB0t ANK repeats may cover the basic residues of the Rel protein NL";. However, the possibility that IKBot interacts with a cytoplasmic-anchoring structure cannot be excluded, It is important to note that the c}¢oplasmic retention is mutual: free IKB0t or Rel complexes not associated with an h(B are usually found in the nucleus -.s.i~. It is likely that h(Bet enters the nucleus by passive diffusion through nuclear pores s, whereas Rel proteins have specific nuclear targeting signals. Although less is known atx)ut IKB~/and IKBI], they can also cause specific Rel conlplexes to be retained in the cytoplasm% However, binding of an IKB protein to a Rel complex does not always result in cytoplasmic retention; for example, p522-Bcl-3 and pS0a-Bcl-3 complexes are generally located in the cell nucleus m,l'~. It is unclear whether the pS0 NLS is required for interaction of the protein with Bcl-3 (Refs 17, 18). It is likely that an intramolecular interaction between IKB and the RH domain occurs with the NF-KB p105 and pl00 proteins. The simplest evidence supporting this proposition is that while p105 is located in the cytoplasm of cells, the processed pSO (which lacks the cadmxy-terminal ANK domain) is found in the nucleus. Deletion of acidic sequences that lie between the last two ANK repeats ()f p105 also alk)ws the protein t() enter the nucleus; this also suggests that there is an ionic interaction between the ANK repeat domain and the pS0 NLS (Refs 9, 19). Lastly, heterodinlers that contain p105 or plO0 can sequester other Rel proteins (e.g. c-Rel, RelA) in the cytoplastn, and can inhibit the DNA-binding activities of these proteins 2°-a2 (Fig. 4); the nature of these protein-protein interactions is not known.

....

I

Hum IKB~ Hum Bcl-3 Hum IKBy Hum pi00 Cactus

DGDSFLHLAIIEEEKALTKEVIRQVKGDLAFLM DGDTPLHIAVVQGNLPAVHRLVNLFQQGGRELD NGDSVLHLAIIHLHSQLVRDLLEVTSGLISDDI NGDTPLHLAIIHGQTSVIEQIVYVIHHAQDLGV DGDTPLHLACISGSVDWAALIRMAPHPCLLNI

Consensus

DGDTPLHLAIIHG---OV--LO

REPEAT

...........

II

Hum IgB~ LQQTPLHLAVITNQPEIAEALLGAGCDPELRDF Hum Bcl-3 LRQTPLHLAVITTLPSVVRLLVTAGASPMALDR Hum IKB~ LYQTPLHLAVITKQEDWEDLLRAGADLSLLDR Hum pl00 LHQTPLHLAVIGTQTSVVSFLLRVGADPALLDR Cactus VAQTPLHLAALTAQPNIMRILLLAGAEPTVRDR .................................................. Consensus REPEAT

L-QTPLHLAVIT-QP-W--LL-AGADP-LLDR

III

Hum IKB~ RGNTPLHLACEQGCLASVGVLTQSCTTPHLHSI Hum Bcl-3 HGQTAAHLACEHRSPTCLRALLDSAAPGTLDLE Hum IwBy LGNSVLHLAAKEGHDKVLSILLKHKKAALLLDH Hum p100 HGDSAMHLALRAGAGAPELLRALLQSGAPAVPQ Cactus HGNTALHLSCIAGEKQCVRALTEKFGATEIHEA ................................................... Consensus REPEAT

H G N T A L H L A C - - G . . . . . . O L . . . . . . . . L---

IV

Hum IKB~ NGHTCLHLASIHGYLGIVELLVSLGADVNAQEP Hum Bcl-3 DGLTALHVAVNTECQETVQLLLERGADIDAVDI Hum IKBy DGLNAIHLAMMSNSLPCLLLLVAAGADVNAQEQ Hum pl00 EGLYPVHLAVRARSPECLDLLVDSGAEVEATER Cactus DGERCVHLAAEAGHIDILRILVSHGADINAREG .................................................. Consensus REPEAT

DGL--OHLA ........ L-LLV--GADVNA-E-

V

Hum IWB~ NGRTALHLAVDLQNPDLVSLLLKCGADVNRVTY Hum Bcl-3 SGRSPLIHAVENNSLSMVQLLLQHGANVNAQMY Hum IwB~ SGRTALHLAVEHDNISLAGCLLLEGDAHVDSTT Hum p100 GGRTALHLATEMEELGLVTHLVTKLRANVNAGT Cactus SGRTPLHIAIEGCNEDLANFLLDECEKLNLETA .................................................. Consensus REPEAT

SGRTALHLAVE--N--LV--LL--G---N--T-

VI

Hum Bcl-3 SGSSALHSASGRGLLPLVRTLVRSGADSSLKNC Hum IKBy DGTTPLHIAAGRGSTRLAALLKAAGADPLVENF Hum p 1 0 0 AGNTPLHLAAGLGYPTLTRLLLKAGADIHAENE Cactus AGLTAYQFACIMNKSF~4QNILEKRGAETVTPPD .......................................... : ....... Consensus REPEAT

-,-,-,,-,-,-,---,---,---,,,

.... N-

VII

Hum Bcl-3 HNDTPLMVARSRRVIDILRGKATRPASTSQPDP Hum IKBy PGTTPLDMATSWQVFDILNGKPYEPEFTSDDLL Hum pl00 RGHTPLDLTCSTKVKTLLLNRRQNTMDRPLTPP .................................................. Consensus

-G-TPLD-A-S--V-DIL-GK---P--TS---P

t'IGE

hzbibition oJ'DNA binding in t,itm In most instances, interaction of an IKB protein with a Rel complex prevents the Rel complex from binding DNA that contains KB sites. Each IKB has a unique spectrum of activity for inhibiting DNA binding by Rel homodimers and heterodimers. In all IKB proteins, with the exceptions of Cactus and Bcl-3, acidic sequences located carboxy-terminal to the last complete ANK repeat are necessary for this inhibitory activity".m.23; at least in the case of v-Rel, inhibition of DNA binding by IKB(x may occur by direct masking of sequences important for DNA binding I I. Interestingly, Hatada et ai.'-3 have recently shown that deletion of three amino acids from the first full ANK repeat of IKBs' converts its characteristic inhibitory activity to that of IKBcx. TIt; DECEMBER

-G-TPLH-AA--GH---V--LL--GA--N

Ankyrin-like repeats of IKB proteins. Comparison of tile amino acid sequences of the ANK repeats of the human IKB proteins and the Drosopbila cactus protein. Tile ANK repeats are phased as in Ref. 5. and the c(msensus ANK repeat of human erythmcyte ankyrin is shown at the top. A given residue is considered consensus if it is present in the nlaiority ,')f proteins. Note that sequences within a given repeat are more highly related in different IKB molecules than are different repeats within a given IKB. O. any hydrophobic residue. However, interaction of a given h
1993 VOL.9 X(). 12 i2 c

~EVIEWS transcription in several ways. First, p502-Bcl-3 and p522-Bcl-3 complexes can bind to KB sites DNA~blnding in vitro and in vivo, and can Transcriptional Cytoplasmic Removal lgB activate transcription in tranactivation protein retentiona Inhibitions Enhancement from DNA sient transfection experiments in some cell types u'.~-9. While RelA ReD, p105 the ANK repeats in Bcl-3 p50 p50 c-Rel c-Rel are needed for its interaction with p52, it appears that sep52 p52 plO0 quences both amino-terminal RelA RelA and carboxy-terminal to the c-Rel C-Rel ANK repeat domain mediate transcriptional activation: the GAL4--IKBa p50z RelA-R RelA-R RelA2 Bcl-3 sequences flanking p50-RelA c-Rel-R c-Rel-R p522 these repeats are rich in RelB-R pSO--ReiB proline and serine and can acRelA-R RelA-R RelA2 tivate transcription when fused p50-RelA c-Rel-R c-Rel-R to heterologous DNA-binding domains m,3°. In addition, Bd-3 GAL4-1KBy c-Rel-, 3' c-Rel2 can activate transcription inp50z directly, alleviating the represRelA2 sion caused by p50 z by removing it from KB sites and allowing 8 p52-ReiB p50, activating complexes such as pS0--RelA p50-RelA p50-RelA to bind to these sites27. Bcl-3 (Nuclear) p502 p50z Bcl-3-p52a p52z Bcl-3-pS0, Furthermore, we have GAL4-BcI-3 shown that fusion proteins containing the DNA-binding Cactus Dorsal Dorsal domain of GAL4 and sequences from IgBa and IKBy can efaR represents any vertebrate Rel protein. ficiently activate transcription from reporter plasmids carrying GALl-binding sites in both verIKB-mediated dissociation of ReI-NF-KB complexes tel:'atc and yeast cells t~,30. In the case of IKBtx, it .[n,m DNA is likely that activation is achieved by GAL4-IKBoLIn addition to their ability to inhibit the initiation mediated recrt, imlent of Rel proteins to the DNA. On of DNA binding by Rel complexes, certain IKBs (for the other hand, IKB',/(the carboxy-terminal segment of example, IKBcx and Bcl-3) can remove Rel complexes p105) appears to contain a genuine, acidic activation pre-formed on DNA in t,itm a~,'~'. It is thought that this domain located between the last two ANK repeats; provides a mechanism by which a cell can attenuate however, it is not known whether this activation a Rei-mediated effect on transcription; as shown in domain can if,notion in vivo as part of full-length NF-KB Fig. 4a, the removal of an activating p50-RelA complex p105 or in a complex with other proteins. from DNA by IKBcx can silence NF-KB target genes. Removal of a DNA-bound Rel complex by an IKB has Regulation of ReI-IKB interactions not been conclusively demonstrated in t,u,o; however, A variety of agents and cellular stimuli can 'induce' Franzoso et al. 27 have shown that cotransfection of a NF-KB activity, causing dissociation of IKB from a Bcl-3 expression plasmid can inhibit p50a-mediated ReI-NF-KB complex and allowing translocation of the transcriptional repression from promoters that contain complex to the nucleus (reviewed in Ref. 1). Many of KB sites (Fig. 4c). these stimuli are activators of protein kinases. This has led to the commonly proposed model for regulation of IKB-mediated enbancement of DNA bindJnR b I, Rei IgBa (Fig. 4a), wherein phosphorylation of IKB0t leads proteins to it:.~ dissociation from a ReI-NF-KB heterodimer, Curiously, certain IKBs can enhance DNA binding unmasking of the Rel protein NLS, translocation of the by Rel complexes in vitro. A bacterially expressed IKB0t NF-gB complex to the nucleus, and rapid degradation protein has been shown to increase the DNA-binding of the free IKBcx protein by a cytoplasmic protease. activities of p522 and p502 (Ref. 28). The mechanism by Interestingly, the NF-KB p50-RelA heterodimer can which certain IKB proteins enhance DNA ~,:inding by induce expression of the IKBa gene through KB sites Rel proteins and the biological relevance ca this effect upmeam of the gene, thereby establishing a regulatory are unknown. loop wherein newly synthesized IKB can remove NF-KB from its target DNA sites and cause inactivation and Transcriptional activation by IKB proteins cytoplasmic sequestration of the NF-KB complex3t-34. Recently, IKB proteins have been shown to activate However, many aspects of this model remain to be T~mm 1. Prope.rties o f l g B proteins

T|G DECEMBER1993 VOL,9 NO. 12

-J30

r~EVIEWS

a)

.

•

b)

•

•

• SIGNAL

ACTIVATION OF IkB KINASE AND/OR PROTEASE

• SIGNAL

ACTIVATION OF p105 PROTEASE

~ '~) DEGRADATION

t

~k "

~ . ~ ~ Q U I L I ~

mRNA

( NI:ICRI 1 d x ~ d . . . . . . . . .

"~.

~

,~rlER IZATION ~

l PROTEI N /I ) SYNTHESIS " (p105)

~EOLYSIS

plO5

DNA-DINDING~ INHIBITION C ~ FlY UCL-3 Bcl-3

il(O

RelA

p52

p50

TRANSACTIVATION Fly N- AND C-TERMINAL REGIONSOF BCL-3

p105

FIG~ Models for modulationof the activityRel/NF-KBtranscriptionfactorsby IKBproteinsand their regulation.(a) Exampleshown is for regulation of the NF-KB RelA-p50 heterodimer by IKBa, but may also apply to IKB~and IKB'y.Each pS0 and RelA molecule contains sequences important for DNA binding and nuclear translocation.,these sequences appear to be directly covered by IKBa, and the trimericcomplex may be furtherstabilizedby carboxy-terminalsequences of RelAthat are involvedin transcriptionactivation.Afterreceivingan appropriate signal, nuclear NF-KB DNA-bindingactivityis induced by a signal transduction pathway that involvescellular kinase(s) and protease(s). IKBa is degraded by a chymotrypsin-likeprotease, either while stillcomplexedwith NF-KB,or after phosphorylationof IKBand its release from NF-KB;alternatively,the induced kinase may activate the IKBa-specificprotease. The active NF-KBcomplex is then free to translocate to the nucleus where it can activate target genes, including those that encode IKBtxand p105. Newlyformed IKBacan enter the nucleus by diffusionand remove the NF-KBcomplex from DNA. (b) Exampleshown is for p105 regulationof RelA.The plOS--RelAheterodimeris retained in the cytoplasm through intramolecularmasking of the nuclear targetingsignals. Afteractivationof a pl05-specific protease, the carboxy-terminalANK repeats of p105 are removed and the p50--RelAheterodimer can wanslocateto the nucleus. (c) Bcl-3, a nuclear protein, has two activities. Bcl-3 can remove inactive p50z homodimers from DNA, allowing these sites to be occupied and activated by other Rel complexes. In addition, the p52z-Bcl-3and p502-Bcl-3complexestan bind to KBsites and activatetranscriptionby means of the amino- and carboxy-terminaltranscriptionactivationdomains in Bcl-3.The type of interactionthat occurs between Bcl-3and p502or p52, presumably differsdepending on whether Bcl-3 is involved in removalof the complex from DNA or transcriptionalactivation:this could depend on modificationsof the proteins or on the type of KBsite involved. proven. Moreover, recent evidence35 suggests that while still complexed with NF-KB, IgB0~ may be a target for a cellular chymotrypsin-like protease, and it may be this IKB(x-specific protease that is activated by the induced kinase activity. Furthermore, it is unlikely that the model proposed for regulation of IgBtx can describe satisfactorily all IRB-Rel protein interactions (compare parts a, b and c, Fig. 4). One of the first findings that suggested that association of IKB with NF-KB might be regulated by phosphorylation was the rapid appearance of nuclear NF-KB

DNA-binding activity in cells that had been treated with phorbol esters, which are inducers of protein kinase C (PKC) (Ref. 36). In addition, treatment of cytoplasmic extracts of unstimulated pre-B cells with PKC or protein kinase A (PKA) can unleash KB site-binding activity37, and purified IKBs can be phosphorylated in vitt, by PKC, PKA and heine-regulated EIF-2 kinase 2q'38. However, the precise effect of phosphorylation on IKB proteins is not always clear and may vary for each type of IKB. For example, Ghosh and Baltimore38 reported that phosphorylation of IKBtx by PKC abolishes its ability

TIG DECEMBER 1993 VOL.9

m

NO. 12

~EVIEWS to inhibit the DNA-binding activity of purified NF-KB, but that treatment of IKB0t with PKA has no effect. Link et al. 3'~ reported that IKBoLand IKBI3 purified from placenta are both inactivated by treatment with either PKC or PKA. In contrast, Kerr et al. 2~ found that bacterially expressed chicken IKB0t is inactivated by phosphot3'lation by PKA in vitro, but is unaffected by phosphorylation with PKC. Finally, Bcl-3 and IKB[3 appear to require phosphot3,1ation to be active as inhibitors of DNA binding by Rei complexes r'.3'~. Description of the regulation of any IKB by phosphot3'lation awaits identification of the kinase(s) that act on that IKB in t,it~J, the chamcterizatkm of the sites of phosphotTlati,m, and detemaination of the effect of mutations at these sites. In vertebrate systems, it has not yet been sh, w
Although there is no firm evidence that IKB proteins have a causal role in an}' of these malignancies, one might suspect that they are inw)lved. In most of these cases, it is likely that changes in the structure or level of production of IKB proteins would increase expression of genes regulated by Rel complexes. IgB proteins in primitive eulLaryotes To date, Drosophila melanogaster is the most primitive organism known to contain a ReI-IKB system (i.e. Dorsal-cactus). Evidence that GAL4-IKBcx activates transcription in vertebrate cells by recruitment of Rel proteins to DNA and that GAL.-t-IKBcxcan activate gene transcription and inhibit growth of Saccharomyces cerevisiae suggests that Rel-like proteins exist in yeast30. In additkm, NF-KB-binding sites are necessary for transcriptiona! activity of the HIV-1 LTR in Scbizosaccharom l,ces p o m b e " . The ease of genetic analysis and manipulation in S. cerevisiae and S. p o m b e means that identification of Rel or IKB proteins in these systems is likely to be important in defining the pathway of activation of NF-KB-Iike complexes.

Concludingremarks Interaction of an IKB protein with a Rel complex can have diverse consequences; these effects probably depend on the specific Rel and IKB proteins involved and perhaps on the modification of these proteins. The next challenge will be to determine what IKB-Rel complex interactions actually occur iu rive, which in vitro activities of IKB are relevant in vi{,o, which enzymes act on IKB proteins to modify their activities in t,it,o, and whether IKB proteins interact with proteins other than Rel proteins.

IKB proteins and cancer In several instances, re:m'angement of sequences th;tt encode IKB proteins has been associated with lymphoid re:align:moles, t14:19 tr:mslocations that map near Acknowledgements the start site of transcription of the human BCi,-3 gone \Vu thank C. Li, I"., l,oeclfler and tnembers of our labhave been fcmnd both in human chronic lyrnpl'i(~cytic cmlton3' for critical readings of the article. We also thank P. l{aetlel'le, A. Baldwin..It'. tl. Bose. ,It', R. Ill'ave. C. G01inas. let,kemi:ns :rod in a small nurnl~ur of other human lymphoid maIigrmncies (Ref, 43; T, McKeith:m, pers, S. Ghosh. T. McKeith:m, C. Scheidereit, I!. Siebenlist, R. htuward ;,nd 1. Vernla fi~t"c(mmltmicaling results befi~re conllllUn.). These tr.'msl¢~catkms generally resuh ill publicaticm. Wc~l'k in T.I),G,'s lahon'atc~n3' is supl')orted by the overexpression of BCZ-3 in cell lines derived fl'om these National Cancer Institute :rod the ,Mas~:Ichusetts Divisicm of cancers; increased production of the protein could the American Cancer Society. T.I).G. is supported in part decrease the number of inhibitory pSO, homodimers by an American Cancer Society Faculty Research Award. I~ot,nd to DNA or could increase the number of P.I.M. is supported by it graduate scholarship from the Natural p~O,-Bcl-3 or pCi2,-Bcl-3 activation complexes. Sciences anti Engineering Res-arch Council of Canada. Inactivation of IKB sequences may also be inw~lved in human malignancies. Dalla-Favera and co-workers References have identified several rearrangements of the NFKB2 I Baeuerle, P.A. ( 1991) Biocbem. Biopl~.l's. Acta 1072, 63--80 gone, which encodes pl00, in lmrnart lymphoid tumors; 2 Schmitz, M.L, Henkel, T. and Baeuerre, P.A. (1991) "li~t,nds this locus maps to hurrmn clmm't(~some 10 and is Cell Biol. 1, 130-l-tO also rearnmged in the HUT78 T cell line+0.j~. All these 3 Baeuerle, P.A. :rod Baltimore, !). (1988) Sck,nce 242, ,VFKB2 rearrangernents restllt in 3' truncations that re~40-546 move sequences encoding the plO0 carboxy-terminal 4 Inoue, J-l., Kerr, L.I)., Kakizuka, A. and Vet'ma, I.M. ( 1992l ANK repeats. One of these truncated proteins is constiO.,ll68, 1109-1120 tutively located in the nuclett:¢ of cells, binds DNA more 5 Midlaely,P. anti Bennett, V. ( 1992} Ti.t,nds Cell Biol. 2, 127-129 avidly than full-length pl00 and readily |'()rills activation6 lnoue, J-l. et ai. (1992) Pl~oc. A?ttlAcad. &i. USA 89, competent tOtal'flexes with RelA. .t333---t337 Finally, in transformed chicken lyml3hoid cells, the 7 Beg, A.A. et al. (1992) Ge~tes De,'. 6. 1899-1913 v-Rel oncoprotein of the avian Rev-T retrovims is largely 8 Zahel, U., Henkel, T., dos Santos Silva, M. and Baeuerle, found in it cytoplasmic complex with IKB0c, p105 and P.A. (1993) ~IIBO.L 12, 201-211 pl00. Even though IKBtx affects chicken c-Rel and v-Rel in 9 Henkel, T. et al. (1992) Cell68, 1121-1133 it very similar way ill t'illO, s o m e evidence suggests that in 10 Ganchi, P.A., Sun, S-C.. Greene, W.C. and Ballard, D.W. vit'o, v-Re[ is less inhibited by IKBcxthan is c-Rel (RoE .~6). (1992) Mol. Biol. Cell3, 1339-1352 Tit.; I)EtZEMBER 1993 VOL.9 XeX 12

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r~EVIEWS 11 Kumar, S. and G~21inas.C. ( 19931 Proc. NatlAcad. Sci. USA 12 13 14 15

16 17

18 19 20 21

22 23 24

25 26 27 28 29 30

90, 8962--8966 Capobianc¢~, A.J., Simmons, I).L. and Gilmore, T.D. (1990) Oncogene 5. 257-265 Rushlow, C.A.. Hart, K., M:mley,J.L and Levine, M. (1989) CeI159, 116%1177 Nolan, G.P. and B:dtimore, 1). (1992) Curt'. Opiti. GelleL Det,. 2, 211-22(} Morin, P.J. and Gilmore, T.I). (1992) Nucleic Acids Res. 20. 2455--2458 BouTs, V. et al. (1993) Ce1172. 729-739 Nolan, G.P. et al. (19)3) Mol. Cell. Biol. 13, 3557-3566 Inoue, J-l., Takaham, T.. Akizawa. T. and Hino. O. ( 19931 Oncogene 8, 2067-2073 Blank, V., Kourilsky, P. and lsrai31,A. (1991) F~llBOJ. 10, 4159-4167 Rice, N.R., MacKichan, M.L and Isra61, A. (1992) Cell71, 243-253 Mercurio, F., DiDon:ito, J.A., Rosette, C. and Karin, M. ( 1~)31 Genes Dev. 7, 705-718 Naumann, M., Nieters, A., Hatada. E.N. and Scheidereit, C. (1993) Oncogene 8, 227%2281 Hatada, E.N., Naumann, M. and Scheidereit. C. (1993) FJIBOJ. 12, 2781-2788 Kerr, L.D. et al. ( 1991 ) Genes Dev. 5. la,6~-1476 Zabel, U. and Baeuerle. P.A. (1990) Ceil61.25%265 L6veillard,T. and Verma, I.M. (1993) Gene F_xp.3, 135-150 Franzoso, G. et ai. (1993) Fa~IBO,I. 12. 3893-3901 DuckeR, C.S. et al. (1993) Moi. Cell. Biol. 13. 131%1322 Fujita, T. et al. (1993) Genes Dev. 7, 1354-1363 Morin, P-I., Subramanian, G.S. and Gilmore, T.D. (1993)

Nucleic Acids Res. 21,215%2163 31 Sun, S.C.. Ganchi, P.A., Ballard. I).W. and Greene, W.C. (1~)3) Science259, 1912-1915 32 Brown, K. et al. (1993) Proc. Natl Acad. Sci. t~A 90.

2532-2536 33 Beg. A.A., Finco, T.S.. Nantermet. P.V. and Baldwin, A.S., Jr (1993) Moi. Cell. Biol. 13, 3301-3310 34 tie Martin, R. el al. (1993) £1*BO,L 12, 2773-2779 35 Henkel, T. el ai. ( 1993} Natto'e365, 182-185 3 6 Sen, R. and Baltimore, D. (1986) Cellq7. 921-928 3 7 Shirakawa, F. and Mizel, S.B. ( 19891 Mol. Cell. Biol. 9, 2424-2q30 38 Ghosh. S. and Baltimore, D. (1990) A2tttnre3-H, 678-682 39 Link, E. et al. (1992) ,L Biol. Chem. 267. 239---246 40 Mosialos. G. et al. ( 1991 ) Mol. Cell. Biol. 11, 5867-5877 41 Whalen, A.M. and Steward, R.L Cell Biol. (in press) 42 Shelton. C.A. and Wassemlan. S.A. ( 19931 Cell. 72. 51%525 43 Ohno, H., Takimoto, G. and McKeithan, T.W. (1990) Cell 60. 991-997 44 Neri, A. et ai. (1991) C,,1167, 107%1087 45 Fmcchiola, N.S. et ai. (1993) Oncogene8. 2839--28-t5 46 Diehl,J.A., McKinsey. T.A. and Hannink, M. (1993) Mol. Cell. Biol. 13. 1769-1778 47 Toyama, R.. Bende, S.M. and l)har. R. t 1992) Nucleic AcMs Res. 20, 2591-2596

T.D. GILMOREAND P.J. MORINARE IN THEDEPARTMENTOF BIOLOGY,BOSTONUNIVERSITY,5 CUMMINGTONSTREET~BOSTON, MA 02215, USA. I i

The human immun0deficiencyvirus integrase protein

Integration of a DNA copy of the retroviral RNA into the genome of the infected lmman cell is essential for replication of HIV (Refs 1, 2). Studies of avian, murine and human retrcwiruses have revealed that the following steps ate inwflved in the DNA integration reaction (Fig. 1). After infection of a permissive cell, the retroviral RNA is converted into double-stranded, blunt- CORNELISVINK AND RONALDH.A. PLASTERK ended DNA. This process is catalysed by reverse transcriptase (liT). A few nuclec~tides (in most cases The DNA Integration step in the replication o,cle of the two) are remcwed from both 3' ends of the linear vital human immunodeflclency virus (HIV) has been recognized DNA, which is part of a large nucleoprotein complex -'4'. as an intportant target in atttiviral strategies. There are After transfer to the nucleus of the infected cell, the two main reasons for this. First, integration of HIV DNA 3'-hyd,'oxyl ends of the viral DNA are cot, pied to phos- into the human genome is required for replication of this phates in both strands of the target DNA. These phos- retrovirus. Second, since the integration reaction does not phates are a-6 bases apart. In the resulting integration have an obvious cellular counterpart, drugs that intermediate, the two non-pairing nudeotides at the 5' speciflcaU.j, inhibit integration may not be toxic for the cell ends of the viral DNA remain free, and are presumably Here, we focus on the only protein knou,n to be required remcwed by the cellular DNA repair machinery. The for retroviral integration, the integrase (IN) proteitt single-stranded gaps that flank the integrated retrcwirus (or pr~wims) are probably also repaired by cellular enzymes. As a result, the prcwims has lost 2 bp from This subject was recently reviewed by Craigie '~. and is each end and is flanked by short, direct repeats of the therefore not addressed here. target DNA. Proviral DNA is used as a template fi~r the synthesis of viral RNAs, which can serve either as Integrase and its DNA substrates IN is the only protein known to be required for retrogenomes or as mRNAs for the production of proteins. Viral genomes and proteins are packaged into virions, viral DNA integration. The IN proteins from different retr(wiruses vax3' in size from 30 to --t6 kDa, are encoded which bud from the cell surface and can subsequently by flae 3' end of file 1~,I gene and are released fi'om a infect other cells. Selection of target sites for remwiral integration is Gag-Pol polyprotein precursor by proteolytic processing. The DNA sequences required for integraticm are not completely random with regard to the primary sequence and/or chromatin structure of the target DNA-.s. short, imperfect inverted repeats at the outer ends of TIG DECEMBER1993 VOL 9 N(). 12 I

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