LETTERS TO NATURE
`
`I-'1. Auoniczewsl-... M. xnnler, G. & Lamers. \t. 12. Eur. .r. Immun. 21. 517 626 H.991‘.-
`i5. Lu. r. 1.. Johnson. G D.. Gor:Inn.J 8. MacLennan. I. 0 Vi Jmrmin Trm‘ar13.3?-21‘.1992I.
`15. Mazda. K. in an‘.
`.r
`rmrr-on 1-II. 23-'10-2347 11992}.
`17. H21-mar‘. H.. Lanmin. I
`at Gus1au55cn.S.Eur. .r.
`.'f'l’|fl’l£lF‘, 23. t?33—;'l‘:.2 (1993:.
`I8. neuron. I’ 8. Solegeloerg H. L. J. immun. 139, 1459-1465 i19a'i'l
`I9. Mont, M at til. Nature 162, 245-24 I’ \'1993i.
`20. Reagan. A. D.. Snapper. C. M , Var. Dusen. Fl.. Paul. W. E. 8: Co"-rafl. D. H. J irrrlnun. 842.
`3B68—3H?r‘| t1989l
`21. Lup. H.. Holstelter. l-i
`. Bancr|ereau_ J. & Delespesse, G_ J lrrirrluii. 143. 2122- 2129 (19911.
`-RCKNOWLEOIGEMENTS. We thank H. Mutt.-zslaal. E. Strolling. L Egcrsdom. C. westohar and
`n Fahrenoncn rorter,nnnr.a|:issI~51.ar-ca; EL Ledermane for the B6 ES cs.» line; L Kllsdlltc ans
`22. Sherr. E._ Macy. E., it-Irtala. H.. G -V M. 8: Saudi‘. a J‘
`lrnmrm. 1.12, -131--569 H.989!
`M Koo! for Help with gene targeting; D. Conrad tor Fcer2a coht-\: and I Mccctnric-II_ fi_ Ir||l‘|oI_
`23. Saxon. it. Kurot. earner. M . Elchlc. H.. ME|x_ E. E. at. zr-eng. H. J. Jmmun. 141. 4000-4006
`‘.1991!
`P. Shaw.
`'1. Car‘sn’.'... M. E:-r.-inn and D. H. Kat: to’ cut-cal read.-rzg or the rnaru:s:rt|::. M.Ft. is a
`2-1. Street N. E et oi‘. J. tninauri. 1-“, 1629-1539 i1990'i.
`recipient ol 3 Special Felluu. award tram the |.eU:tefl'II.'| Society of ilmerica. The’. work was
`25. Campbell. K. J... Lees. .ei.. Finleielman. F. D &Conrad u. H Eur J‘ rmm-In 22. 210?—?112
`supported In part by a grant from me NIII to U H. hate. The Easel institute Ior Irnrrtiunootty
`0997:.
`was founded and I5 supported Lw Itoilnian La Roche. Basel. SWIIIEIIBFU.
`
`
`26. S r::IaIr_ N Fl‘. SIC rmnsur.-. today I, 35-36 :19a3i_
`.‘-1?. Ntstnke. L. Hour. M r. lamers. M. c Hlflifitfhnrques 1.1. 914-515 11993:
`2B. Mane. M. P . Neilser. P. & ttehler, G i\.'ucie.u: -turd:-1?;-s.11. 6722 11990;.
`29.
`lino. M . tee. w. T. 8. Conrail. U ||.J.I'fl1tTllr|'l I38. IE-15-185‘. i‘19a'l'i
`
`A mammalian protein targeted
`by G1-arresting rapaInycin-
`receptor complex
`Eric J. Brown, Mark W. Albers, Tao Bum Shin,
`Kazuo Icllikawa, Curtis T. Keith, William 5. Lane‘
`8; Stuart L. schrelberi
`
`Department of Chemistry. Howard Hughes Medical Institute and
`" Harvard Microchemistry Facility. Harvard University.
`12 Oxford Street. Cambridge. Massachusetts 02138. USA
`+ To whom correspondence should be addressed
`
`THE structurally related natural products rapamycin and Flifillo
`bind to the saute intracellular receptor. FKBPIZ. yet the resulting
`a9
`6’
`£4‘? c‘°++‘9:'>°@Q
`s<‘“.:;< er“e:< or“
`
`a
`
`-1i—FFlAP
`
`500 nM GFK-ligand
`5% 93'
`
`complexes interfere with distinct signalling pathways”. FK I11!-‘I2.-
`raparnyein inhibits progression through the Gt phase of the cell
`cycle in ostcoisarcoma". liver” and T cells” as well as in yeast”.
`and interferes with mitogenic signalling pathways that are involved
`in G1 progression°‘"'. namely with activation of the protein piflm
`(refs 5. 11-13] and cyclin-dependent kinases“"“"'. Here we isolate
`a mammalian FKBl'«-rapantycin-associated protein (l-‘ltnl-') whose
`binding to structural variants ol' rapam_\-‘cirl complexed to FKBPIZ
`correlates with the ability of these ligands to inhibit cell-cycle
`progression. Peptide sequences from purified bot-"inc I-‘RAP were
`used to isolate a human CIINA clone that is highly related to the
`DRRUTURI and DRRZXTORZ gene products from Sm:c!mro-
`my:-as ceretrirint-"“"‘“. Although it has not been previously demon-
`strated that either of the DRRI TOR ene products can bind the
`FKBP rapanlycin complex dirceti_v"' 9.
`these yeast genes have
`been genetically linked to a rapamrcin-sensitive patht-ra_1.' and are
`thought to encode lipid kinases”"“.
`
`b
`
`RBL
`
`-Jurkat
`
`ff
`+eԤq
`..<i.,«+§"‘.§*’:;-+“é+.;«~*"§"..§'i'if
`
`T
`M (K)
`
`2004-»
`
`--
`
`- -
`
`-(—-FFIAP
`
`20° _,
`153 -3-
`
`116 —:h-
`55 —r-
`
`45 _;p.
`31-1-
`
`1-: —1n-
`
`Qgggi 4i—GFl(
`
`E I I E .'.'.-E
`
`500 nM GFl<—ligand
`12.5% gal
`
`ll6%-
`97%-
`
`97*?-
`66*?‘
`45-?‘
`
`_
`.
`
`.'
`
`-
`
`-(—FH.AP
`
`2.5 pllil GFK—ligand
`5% gal
`
`CC
`”“*!gInunn
`14—‘)I-
`-u......-._..-—-"
`
`200 —1-
`I16 -1-
`
`.
`—
`
`5% gel
`
`-€-—GFK
`
`12.5% gel
`
`FlG. 1 Identification of FRAP protein in three mammalian cell lines. a.
`GFK alone or indwidual GFi-l—ligand complexes were added to MG-63
`cell lysates :2 x 10" cells per condition] to a final concentration of either
`500 nM or 2.5 pM and the mixtures incubated for 10 min at 4 C. Fusion
`protein comoiexes were recovered by glutathione-alfinity chromato-
`graphy. and the proteins detected by silver staining after 5% SD5-
`PAGE. Because of compression. FFIAF’
`IS not resolved by 12.5“-‘o SD3-
`PAGE. so both 5% and 12.5% gels are shown. The amount of FRAP that
`was retained by affinity chromatography saturated at concentrations of
`GFK—Rep greater than 500 nM [l'| these experiments and in others using
`concentrations of GFK Rap ranging from 100 nM to 5].lM (data not
`shown]. in. GFK alone or individual GFK-ligand complexes were added
`to a final concentration of 500 nM to lg.-sates prepared from either
`2 - 10‘Juri«atTIyn1phocyles or 103 rat basophilic leukaemia [RBL}ceIls
`oer condition. Lysates were treated as in a. FK8F'12. but not FKBP13 or
`FKEIP25 {ref. 23] is able to mediate the actions of rapomycin in 5.
`756
`
`cereuisiae. In addition, we found that YFK188 (ref. 24:. an FKBP12 null
`strain. could be complemented with GFK :P. K. Martin. B. Gladstone.
`G. Weiss. 0. T. Hung. S.L.S., in preparation}. Thus the GST appendage
`of the fusion protein does not preclude binding of the biologicaliy rel-
`evant target to the GFK—rapam1.rcin complex in yeast.
`METHODS. MG-63. Jurlratand RBL cells were grown in media containing
`10% FBS and lysed at 4 C in PINT bufier (150 mM NaCl. 50 mM Tris-
`HCI. pH 15. 2 mM EDTA. 2 mM EGTA. 25 mM NaF. 100 uh-‘J Na;.'u'O.t.
`25 mM 2-glycerophoephate. 0.2 mm PMSF, 1 pg rnl" leupeptin, 1 pg
`ml
`’ pepstatin A and 2 mM D'|'|'J containing 0.5% Triton X-100. Lysates
`were clarified by centrilugatlon at 25.000g. and the Triton X-100 II‘! the
`supernatant was diluted to 0.33% by adding 0.5 vol PINT buffer. GFK
`prehound to stoichiometrlc quantities of FK506. ketcu iso- or unmodified
`rapamycin was added to lysates as described. Each condition was then
`passed through a 250-pi glutathione-Sepharose column, which was
`washed with PINT buffer containing 0.5 M Nacl and 0.3% Triton X-100.
`NATURE ‘ VOL 369 - 30 JUNE 1994
`
`© 1994 Nature Publishing Group
`
`West-Ward Pharm.
`Exhibit 1006
`Page 001
`
`West-Ward Pharm.
`Exhibit 1006
`Page 001
`
`

`

`a
`
`Mm
`
`200 _}
`
`116 —}
`
`«*3‘é:2:i5’
`
`
`
`-‘(-FRAP
`
`FIG. 2 Purification of FRAP from bovine brain and CDNA cloning of
`human FR.-KP. a. Fivefd|d—enriohed bovine FRAP [S-column eluate: see
`below} was conditioned with [100 nl\i'|]. gutathione—S-transferase~
`Fl-(B912 fusion protein (GFKJ. G!-"K-FK505 or GFK-Rap. Complexes with
`fusion proteins were recovered by giutathionevafiinity chromatography
`and detected as described in Fig ‘l
`legend. We also found FRAP in
`bovine liver and thymus. 1:. Predicted translational product of the human
`FRAP cDl\:A clone. Bovine FRAP peptide sequences aligned to human
`FRAP are indicated by underlined segments in the reading frame shown
`translational stop codons were not encountered upstream of the initial-
`ing methionine. r:. Northern blot analysis of human tissue. Jurkat T cell
`and MG-63 cell poly [Al' RNA. The Jurkat/MG-B3 and multiple tissue
`Northern blots lclontechl were hybridized with "P-labelled probes
`derived from the 182 hp PCR fragment and the 5.5 lib clone ttextl.
`respectively. Hybridization to human fi—actin probe is shown as an inter-
`nal control for loading.
`METHODS. Bovine FRAP was purified by grinding 900 g of bovine brain
`in blender with 1 litre of PIP lCi.3"ii. Triton l(—10Cl. 50 rnlvi sodium phos-
`phate. pH 7.2. 2 rnlvl EDTA. 2 mM EGTA. 25 mM NaF. 100 plvl Na, V04.
`25 mM 2-glycerophosphate, 1 rnl\i"| PMSF. lug mi" leupeptin. 1 pg
`mi " pepstatin A. 1 mlvl benzamidino and 2 mlvi DTT}, The hornogenale
`was centrifuged at 25.{lOl‘.|g and the supernatant [20 g total
`protein} was loaded onto a 1 litre Séiepharose :Pharmacia]
`column. The column was then washed with PIP and eluted
`with PINP {PIP with 1M Nacli. GFH—rapamyt:in was added
`to the pooled eluate to a final concentration of 100 nM
`and recovered by glutathlone-aliinity chromatography. FRAP
`was resolved by SDS~PAGE and transferred to PVDF. Fol-
`lowing digestion with trypsin or endoproteinase Lys-C
`(Boehringer Mannheim} bFRAP peptides were micro-
`sequenced". The Jurkat T cell cDNA library [Stratagene]
`was constructed through random and ollgo cl‘l priming
`oi cytoplasmic oligo dT purified RNA iref. 25}.
`cDl'~lA
`screening. Jurliat and MG—63 RNA isolation and northern
`blotting and were performed by procedures similar to those
`previously described”. A 182 bp fragment was amplified
`from a human brain stem library lstratagenel and labelled
`by incorporation of "’P-oCTP in the course of rear-nplification
`by PCR. The sequences were analysed using BLAST [net
`25} and the University of Wisconsin GCG (ref. 2?: software.
`The human FRAP CDNA sequence has been submitted to
`Genbanli.
`
`Hm-
`”""'
`
`Hib-
`
`!.¢lr.b-
`
`LETTERS TO NATURE
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`We used two structural variants of rapamycin.
`rapamycin (S. D. Meyer and S.L_S.. manuscript in preparation}
`and 25.26-iso-rzipainycin". to identify any biologically relevant
`targets of" the FKBP rupamycin complex. Both variants bind
`tightly to human l-'KBPl2. as shown by their ability to inhibit
`rotamasc activity of the recombinant protein IK, values were
`0.2 nM for rapamycin". 2 nM for lteto-rapamycin. and 0.! nM
`for iso-raparnycin). But the variants are about two orders of
`magnitude less potent than raipamycin in preventing the progres-
`sion through Gl oi" MG-63 human ostcosarcoma cells. The
`values ol‘IC,.. thall‘-maitimal inhibitory concentration) estimated
`from dose response curves are l]_| nM. 15 nlvl and 50 nM for
`l'ap£tI"i1)'Cll1. keto- and iso-rapiirnyuin, respectively. '['husl|1c cont-
`plexcs ol‘iso- and kcto-rapamycin with l~'KBP|2 should bind to
`NATURE ' VOL 369 ‘ 30 JUNE 1994
`
`less elibctivcly than |~‘KBPi3—
`
`the FKBPIZ rapamycin target
`rnpamycin itself.
`A fusion protein oi‘ glutathione—.S'—transfi:rasc with l-'KBPl2
`(G5-"Kl was used to identify candidates for the biologically
`rclcvant targets of FKBPIZ rapamycin, MG—63 cells were lyscd
`by detergent and complexes oi‘ (JFK rapamycin. GF-K—FK506
`or GFK alone were added individually to clarified lysatc at it
`linal concentration of Sill} nM or 2.5 plvl (Fig. iii}. A protein of
`approximate relative molecular mass ZZILUOU (Mp-22llK) was
`detected in the (JFK rapamycin sample by SDS—l‘r‘\GE and
`silver staining (Fig.
`la, lane 3}. This l"KBP—rapainycin-3559:.
`lil.l.(.‘{.l protein (FRAPl was not fl2l&llI'll.!(l with (JFK FK5ll6 or
`GFK alone (Fig. la. Iiuics l and 2}. No other rapzlniyt-in-specific
`prolcins were detected by silver staining (Fig. In} or by a similar
`75?
`
`© 1994 Nature Publishing Group
`
`West-Ward Pharm.
`Exhibit 1006
`Page 002
`
`West-Ward Pharm.
`Exhibit 1006
`Page 002
`
`

`

`LETTERS TO NATURE
`
`affinity purification procedure itsing Iysates from [”S] methio-
`nine—labe]|ed cells (data not shown}. The GFK -ltctorapamycin
`and G1"-‘K—isorapamyciri complexes bound l-"RAP less cflectively
`than GFK—rapamycin: at concentrations of 500 nM. the ketc-
`and iso— complexes were unable to retain the 220K protein ( Fig.
`la. lanes 4, 5). whereas at higher concentrations ofthe complexes
`(2.5 ttM) detectable quantities of F-"RAP were retained (Fig. in.
`lanes 4. 5). This is consistent with the finding that these com-
`pounds are still strong cell-cycle inhibitors. albeit less potent
`than rapantyr.-in itself. Thus. the binding of GFK ligand com-
`plexes to l-"RAP correlates with the ability of the ligands to
`impede G1 progression in MCr—o3 cells. FRAP was also detected
`in J urkat T-lymphocyte cells and rat basophilic leukaemia cells
`{Fig lb). two mammalian cell
`lines that are also sensitive to
`rapam}'cin""1. No other rapamycin-sp-ecific bands were observed
`in each case.
`FRAP purified from bovine brain [bI—'RAP] had a similar
`specificity for CrFl(—|igand (Fig. Zrr]. Microsequcrtcing of
`bFRAP protcolytic fragments [298 amino acids in total. Fig.
`lb) led to the design of a pair of degenerate oligonuclcotidcs for
`use in the polymerase chain reaction (PCR]. A 182 bp PCR
`product allowed for the isolation of overlapping clones from a
`human Jurkat T cell JIZAP [1 cDNA library. yielding 16 kb of
`contiguous sequence. Using these cl')NA sequences as probes. a
`band migrating at approximately 8.5 kilobases was detected by
`Northern blot analysis of oligo dT purified RNA isolated from
`a variety cl’ human tissues and cell lines (Fig. Zr‘). The human
`CDNA sequence encodes an arnino—acid open reading Frame
`(ORF) and aligns with 99% identity to the bl-‘RAP peptides
`(Fig. Eh]. As N-tenninai peptide sequence from purified bovine
`FRAP was not obtained, the initiating methionine shown in Fig.
`2!:
`is unconfirmed. The predicted molecular mass of this ORF
`[-300K) is greater than that inferred by the mobility of FRAP
`during SDS -PAGE (above).
`Human FRAP is highly related to the DRR.l_.-“TORI and
`DRR2,/TOR2 gene products. Overall
`it
`is 44% identical
`to
`DRRI .-"'I‘ORl and 46% identical to [)RR2,’TOR2. The region
`of greatest homology to DRRIETORI and DRREETOR2 lies
`in the C—tennin-al 660 amino acids of human FRAP (51% and
`59% identical. respectively). In addition. this region has homo-
`logy to several known phosphatidylincsitol ltiriases (21% identity
`on average).
`including matnmalian phosphzitidylinositol
`3-
`kinasem" (I-’l3K}. a yeast Pl?-K I/P.S'34 (refs. 17 and 18) and
`PIKE (ref. 20). These similarities indicate that FRAP may also
`have phosphatidylinositol kinase activity.
`Through the introduction of minute structural changes in
`rapamycin. this study implicates I"-‘RAP as a mediator ofG| cell
`cycle progression in mammalian cells. Identification of FRAP
`as the target of FKBPIZ-rapamycin together with the earlier
`demonstration oi’ calcineurin as the target of FKBPIZ-FK506
`(ref. 2) addresses a fascinating aspect of immunophilin research.
`namely that the imrnunophilin FKBPl2 can bind two distinct
`natural products and thereby gain the ability to bind two distinct
`signalling molecules involved in cell cycle entry and progression.
`Further biochemical characterization of this unique mammalian
`protein should elucidate its role in propagating the mitogcn-
`initiatcd signals that lead to the activation of p'IElS°" and cyclin-
`Cdk complexes.
`El
`
`Received 3 May: accepted 20 May 1994.
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`ACKNOWLEDGEMENTS.
`l\-|.W.-M. and E.J.3. have made an equal contribution to this research.
`We trienk S. J. Danisrietsky and C. M. Hayward for raeraparnyciri: S. 0. Meyer for preparing
`Ivaloran. R. F. Slandaert
`for advice and assistance;
`|'-‘_ A. Seal
`for critically reading the
`manuscript: Anlana Rao and Lewis Itunl-tel tor helpful! discussions; and ‘H. Robinson, Fl. crirez
`and V. Bar:a-,- tor their assistance. this work was sunwneo by a grant mm the NIH [5.t..S.l.
`M.W..it. is a Howard Hughes Meoieai Institute p(Efl0C10|"fll fallow. EJ.a. is an NIH proooctoral
`Fellow. K.l. is a Ulfillllffs scientist sponsored by Sarikyo Co. Ltd. and $.L.3. is an investigator with
`the Howard Huyies Medical Institute.
`
`
`
`Functional dissection
`
`of the yeast cyos-Tupi.
`transcriptional
`
`co-reprossor complex
`
`Dlmltrla Tzamarlas & Itovln Strultl‘
`
`Department of Biological Chemistry and Molecular Pharmacology.
`240 Longwood Avenue. Harvard Medical School, Boston.
`Massachusetts 02115. USA
`
`DNA-ENDING repressor proteins mediate regulation of yeast genes
`by cell type (Mcn1l.io.2 and aliul). glucose (Migl) and oxygen
`(Roxll (refs I-4 respectively). An unusual feature of all these
`regulatory pathways is that transcriptional repression requires two
`physically associated proteins’ that do not bind DNA C_vc8(Sst:I6)
`and Tupl. The Cye8—Tupl complex has been proposed to be a co-
`represstrr that is recruited to target promoters by patltwayvspecific
`DNA-binding protelr$°. but the specific functions of the individual
`proteins are ttuItnr:n-i-'n. Here we show that when it
`is bound
`upstream of a functional promoter through the [ream DNA-binding
`domain. Tupi represses trariscription in the absence of Cycfl. De-
`letion analysis indicates that Tupl contains at least two non-over-
`lappittg trariscriptiorral repression regions with minimal primary
`sequence similarity. and a separable Cyc8-interaction domain.
`These Tupi domains. which do not
`include the [5-truruiduein
`Il'lDl‘lf51, are necessary and partially sulficient [or Tnpl function.
`We suggest that Tupl performs the repression function of the
`Cyefl-Tupi co-repressor complex. and that Cyc8 serves as a link
`with the pathway-specific DNA-binding proteins.
`It has been previously shown that Cyc8 can repress transcrip-
`tion in a Tupi -dependent manner when bound upstream of the
`intact (‘l"(‘I promoter through the heterologous Lcxfit DNA-
`binding domain". Similarly. a I.ertA Tupl hybrid protein confers
`a 16-fold reduction of expression from a promoter containing
`four l.exA operators upstream of the C YC! promoter (Table
`1}. Le)tA Tupl and LcrtA--Cycll also repress expression of a
`lrt'.r3 gene containing a single Lexfit operator upstream of the '1'.‘
`TATA element (Fig.
`lri). suggesting that they can inhibit basal
`transcription. Surprisingly, LexA-Tupl retains almost its entire
`‘ ‘to whom corirettlrioritlence snouio he addressed.
`
`T58
`
`© 1994 Nature Publishing Group
`
`NATURE - VOL 369 - 30 JUNE 1994
`
`West-Ward Pharm.
`Exhibit 1006
`Page 003
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`West-Ward Pharm.
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