Proc. Natl. Acad. Sci. USAVol. 91, pp. 12574-12578, December 1994Genetics

RAPT1, a mammalian homolog of yeast Tor, interacts with theFKBP12/rapamycin complex

(phosphatidylinositol 3-kinase)

M. ISABEL CHIU, HILLARY KATZ*, AND VIVIAN BERLINtMitotix, Inc., One Kendall Square, Building 600, Cambridge, MA 02139

Communicated by Fred Sherman, August 18, 1994 (received for review June 30, 1994)

ABSTRACT Rapamycin is a potent immunosuppressantthat blocks the G1/S transition in antigen-activated T cells andin yeast. The similar effects of rapamycin in animal cells andyeast suggest that the biochemical steps affected by rapamycinare conserved. Using a two-hybrid system we isolated mam-malian clones that interact with the human FK506/rapamycin-binding protein (FKBP12) in the presence of rapamycin.Specific interactors, designated RAPTI, encode overlappingsequences homologous to yeast Tor, a putative novel phospha-tidylinositol 3-kinase. A region of 133 amino acids of RAPT1is sufficient for binding to the FKBP12/rapamycin complex.The corresponding region in yeast Tor contains the serineresidue that when mutated to arginine confers resistance torapamycin. Introduction of this mutation into RAPT1 abol-ishes its interaction with the FKBP12/rapamycin complex.

Rapamycin and FK506 are structurally related immunosup-pressants that block distinct steps in T-cell activation. FK506interferes with the early induction of lymphokine gene ex-pression stimulated by the binding of antigen to the T-cellreceptor, whereas rapamycin blocks subsequent lympho-kine-induced cell division (1-4). Rapamycin specificallyblocks the cell cycle in G1 in lymphocytes (1, 5-7), certainnonlymphoid cells (8-10), and Saccharomyces cerevisiae(11). The molecular details of how these drugs act are onlypartially understood. Both drugs bind to and inhibit thepeptidyl-prolyl cis-trans isomerase activity of FK506/rapamycin-binding protein (FKBP12). However, inhibitionofpeptidyl-prolyl cis-trans isomerase activity is not sufficientfor immunosuppressive activity (1, 5, 12). Rather, theseimmunosuppressants confer gain-of-function to their cognatebinding proteins. FKBP12 complexed with FK506 binds toand inhibits the Ca2+-dependent phosphatase calcineurin(13-16). Calcineurin, in turn, regulates the nuclear localiza-tion of transcription factors required for lymphokine geneexpression (17, 18).

Genetic analyses in yeast identified a putative novel phos-phatidylinositol (PI) 3-kinase as the possible target of theFKBP12/rapamycin complex. Mutations in TORI (alsocalled DRRI) or TOR2, genes specifying different forms ofthe putative PI 3-kinase, render yeast resistant to rapamycin(11, 19-21). These studies raise the question of whether Tordirectly associates with the FKBP12/rapamycin complexand whether inhibition of Tor activity is sufficient for theantiproliferative activity of rapamycin in yeast and mamma-lian cells. The results presented here demonstrate that themammalian homolog of yeast Tor, RAPT1, interacts withFKBP12/rapamycin and defines a region of 133 amino acids,the RAPT1-binding domain, sufficient for this interaction.The serine-to-arginine mutation in Tor that confers resistanceof yeast to rapamycin when introduced into the RAPTI1-

binding domain prevents formation of the FKBP12/rapamycin/RAPT1 complex. Based on these results we con-clude that RAPT1 is a target of the FKBP12/rapamycincomplex.

MATERIALS AND METHODSComponents of the Two-Hybrid System. The yeast strain

L40 and the two-hybrid plasmids containing the lexA DNA-binding domain (pBTM116) and the VP16 activation domain(pVP16) were a gift from Stan Hollenberg (22). A PCRproduct containing the gene encoding human FKBP12 wascloned into the EcoRI andBamHI sites ofpBTM116, creatingeither an in-frame fusion between lexA and FKBP12 (plexA-hFKBP12) or the same fusion separated by six glycineresidues (plexA-G6-hFKBP12). The lexA-FKBP12 fusionsused to identify the FKBP12/rapamycin interactors are re-ferred to as "baits."

Construction of the flcbl-2 Allele. VBY567 (MATa his3-A200 trpl -901 leu2-3, 112 ade2 L YS2::(lexAop)4-HIS3URA3::(lexAop)8-lacZ gal4 gal80 fkbl-2::ADE2) was con-structed as follows: a linear fragment containing the disruptionmarker ADE2 flanked by FKBI noncoding sequences wasused to transform yeast strain L40, selecting for adenineprototrophy. Ade+ yeast transformants were tested for rapa-mycin resistance to confirm that the wild-type FKBI allele wasreplaced by ADE2.

Library Screen for Rapamycin Target Genes. VBY567strains harboring each binding-domain fusion were trans-formed with a mouse embryonic day 10.5 cDNA PCR library(22) generated by random-primed synthesis of poly(A)+RNA. The library transformants were plated onto mediumlacking tryptophan and leucine, allowed to grow for 3 days at30°C, pooled, and stored as frozen stocks in 50% (wt/vol)glycerol. An aliquot was thawed and outgrown in liquidglucose medium for one doubling and then plated ontophosphate buffer-containing (pH 7.0) plates under the con-ditions described, at 106 per 15-cm plate. Blue colonies (His+LacZ+) were picked after 4-11 days of incubation at 30°C.Each candidate was retested for growth on medium lackinghistidine (minus His) with or without rapamycin. Candidateclones that grew in the presence of rapamycin and failed togrow on medium without rapamycin were further tested forthe plasmid dependence of their growth phenotypes (23).DNA samples prepared from such candidates were used totransform Escherichia coli strain MC1066 [Rec+ AlacI-Ax74galUgalK strepAr, trpC9830 leuB6pyrF: :TnS(kanr)] carryinga leuB mutation that is complemented by yeast LEU2.PlasmidDNA samples were prepared from Leu+ bacteria andthen used to transform yeast strains to test interaction of thecandidate library clones against different baits. Nonspecific

Abbreviation: PI 3-kinase, phosphatidylinositol 3-kinase.*Present address: Vertex Pharmaceuticals, Inc., 40 Allston Street,Cambridge, MA 02139.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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interactors (those conferring His+ LacZ+ when paired withan unrelated bait fusion) were eliminated, while clones spe-cific for FKBP12 bait fusions were retained. Candidateclones that have fulfilled all the above criteria were analyzedby DNA sequencing on both strands.

Sequence Analyses. The homology between mouse Raptland yeast Tor was found by searching the National Center forBiotechnology Information sequence data bases using theBLAST network service. Subsequent sequence alignmentswere performed by the Clustal comparison method using theanalysis software MEGALIGN (DNAstar, Madison, WI).

Construction of the Serine-to-Arginine Mouse Raptl Muta-tion. The mouse Raptl serine-to-arginine mutation was con-structed by oligonucleotide mutagenesis. Coding and non-coding strand oligonucleotides containing the mutation wereGAAGAGGCAAGACGCTTGTAC and GTACAAGCGT-CTTGCCTCTTC. PCRs were performed using these oligo-nucleotides in combination with oligonucleotides GAGTT-TGAGCAGATGTTTA and the M13 universal primer, whichare sequences in the pVP16 vector, 5' and 3' of the mouseRaptl insert, respectively. pVP16 containing mouse Raptlwas used as the template for PCR. The PCR product, digestedwith BamHI and EcoRI, was cloned into the BamHI andEcoRI sites in pVP16. The resulting clone was sequenced toverify that the clone contained the serine-to-arginine muta-tion and no others.

Isolation and Characterization of Human RAPTI Clones.The human RAPTI homolog was isolated by screening 3 x106 A phage plaques of an Epstein-Barr virus-transformedB-lymphocyte cDNA library (24) with a mouse Raptl probeat 30% formamide/5x standard saline citrate (SSC)/5xDenhardt's solution/1% SDS/200 ,ug of salmon sperm DNAper ml. Ten overlapping clones of human RAPTI wereobtained.Northern Blot Analysis. The multiple tissue Northern blots

(containing 2 ,Mg of human RNA per lane) were from Clon-tech. Hybridizations were at 42°C in 5x SSPE/5x Den-hardt's solution/30% formamide/1% SDS/200 yg of dena-tured salmon sperm DNA per ml. Washes were at O.1xSSC/0.1% SDS at 550C. The blot was exposed for 5 daysprior to autoradiography. The levels of RNA loaded in eachlane were independently monitored by hybridizing the sameblots with a human G3PDH probe and were found to besimilar in all lanes, with the exception of skeletal muscle,which had 2-3 times the signal (data not shown).

RESULTS

Use of the Two-Hybrid System to Identify Proteins ThatInteract with FKBP12 via a Small Molecule Ligand. Weundertook an unbiased approach to identify proteins thatinteract with the FKBP12/rapamycin complex by using atwo-hybrid system to screen a mouse cDNA library. Protein-protein interactions in this system are detected by the ex-pression in yeast of two reporter genes, yeast HIS3 andbacterial lacZ, which contain binding sites for lexA. Trans-

activation of the reporters is dependent on the formation ofa complex between proteins fused to the lexA DNA-bindingdomain (baits) and to the VP16 activation domain (libraryplasmids). If interaction of the two hybrids occurs, the yeaststrain will be prototrophic for histidine and contain detectable,-galactosidase activity (22). For this work, we had anadditional criterion-i.e., that rapamycin mediate protein-protein interaction.To test the feasibility of using the two-hybrid system to

identify proteins that interact with FKBP12 via a smallmolecule ligand, we assessed the function ofhuman FKBP12fusion constructs in terms of their ability (i) to interact withhuman calcineurin in an FK506-dependent fashion and (ii) tomediate rapamycin toxicity. For the latter test, we firstdeleted the FKBP12 gene (FKBI), producing a variant of theL40 two-hybrid strain VBY567 resistant to rapamycin. Usingthis strain we tested the ability of the FKBP12 fusion con-structs to complement the FKBI deletion and confer rapa-mycin sensitivity.The lexA-FKBP12 fusions, either containing or lacking a

polyglycine linker, were functional by the two criteria out-lined above. lexA-FKBP12 interacted with VP16-calcineu-rin A in the presence but not in the absence of FK506 (Table1). The endogenous yeast calcineurin B may facilitate thisinteraction, given the requirement for calcineurin B inFKBP12/FK506/calcineurin complex formation (25). In thesecond test of function, the lexA-FKBP12 fusions conferredrapamycin sensitivity to the yeast strain VBY567, which isotherwise resistant to rapamycin (Table 2). Although the twolexA-FKBP12 fusion constructs conferred different levels ofdrug sensitivity, the results indicate that both are capable ofbinding rapamycin. These results together indicate the utilityof the two-hybrid system for detecting protein-protein inter-actions mediated by a small molecule.

Identification of FKBP12/Rapamycin Interactors. Twoscreens were conducted in parallel using FKBP12 baitscontaining or lacking the polyglycine linker. Clones contain-ing putative interactors were selected on medium containingrapamycin at a concentration that does not inhibit cell growthbased on the results in Table 2. Of the 30 x 106 yeasttransformants screened, 28 clones were obtained that interactwith FKBP12 in the presence but not in the absence ofrapamycin (Table 3). Twenty-one clones contained overlap-ping segments of a sequence homologous to yeast TOR,which we have designated mouse Raptl (mouse rapamycintarget). The remaining seven clones represent six uniquesequences showing no homology to yeast TOR.The rapamycin-dependent interactors were tested on me-

dium lacking histidine containing various concentrations ofrapamycin. The mouse Rapt) clones interacted with theFKBP12 baits at concentrations of rapamycin as low as 15.6ng/ml. Better interaction was observed with rapamycin at125 ng/ml in terms of lacZ expression. None of the clonesinteracted with the FKBP12 baits in the absence of rapa-mycin (Fig. 1) or in the presence ofFK506, demonstrating thestrict dependence of this interaction on rapamycin. More-

Table 1. Interaction of human FKBP12 fusions with human calcineurin in the presence of FK506Plasmid Growth without histidine Color on X-Gal

Binding domain fusion Activation domain fusion No FK506 FK506 (0.2 ,g/ml) No FK506 FK506 (0.2 ug/ml)pLexA-hFKBP12 pVP16 - - White WhitepLexA-hFKBP12 pVP16-hCNA - + White BluepLexA-G6-hFKBP12 pVP16 - - White WhitepLexA-G6-hFKBP12 pVP16-hCNA - + White BlueYeast reporter strain L40 (22) was transformed with the indicated pairs of plasmids. pLexA-hFKBP12 encodes an in-frame fusion between

the LexA DNA-binding domain and human FKBP12; pLexA-G6-hFKBP12 encodes a similar fusion containing an additional spacer of six glycineresidues at the fusionjunction. pVP16 encodes the nuclear-localized acidic activation domain; pVP16-hCNA encodes an in-frame fusion betweenthe activation domain and the full-length human calcineurin A protein. Expression of the HIS3 and lacZ reporters constitutes evidence ofinteraction. X-Gal, 5-bromo4-chloro-3-indolyl ,B-D-galactoside.

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Table 2. Ability of human FKBP12 fusions to mediate rapamycin toxicity in yeastPlasmid Growth on medium containing rapamycin, ng/ml

Binding domain fusion Activation domain fusion 15.6 31.2 62.5 125 250 500

pLexA-hFKBP12 pVP16 + + + + + +/-pLexA-G6-hFKBP12 pVP16 + + + +/- +/-pLexA-da pVP16 + + + + + +

Strain VBY567, a derivative of L40 in which the yeast FKBI gene has been deleted, was transformed with the two humanFKBP12 fusion constructs as well as pLexA-da, a fusion containing an unrelated insert as a negative control. Each was pairedwith the activation domain control plasmid pVP16. +, Growth; -, no growth; +/-, slow growth and reduced colony size.

over, FK506, at a 13- to 40-fold excess over rapamycin,inhibited interaction ofmouse RAPT1 with FKBP12 (data notshown).

Identification of the RAPT1-Binding Domain Containing a

Critical Serine Residue. The smallest mouse Raptl clone thatinteracted with the FKBP12/rapamycin complex was 399 bp,defining the Raptl-binding domain. The Raptl-binding do-main corresponds to a region in yeast Tor located immedi-ately upstream but outside of the lipid kinase consensussequence. This region contains the serine residue that whenmutated in yeast TORI (also called DRRI) confers resistanceto rapamycin (20, 21) (Fig. 2A). We constructed the corre-sponding mutation in mouse Rapt]. The serine-to-argininemutation abolishes interaction of mouse Raptl with theFKBP12/rapamycin complex (Fig. 1), activating neitherHIS3 nor lacZ expression in the two-hybrid assay, indicatingthat the serine residue is involved in the association of theFKBP12/rapamycin complex with mouse Raptl.Human RAPTi Is Homologous to Yeast TOR. Mouse Raptl

sequences were used to probe an Epstein-Barr virus-transformed B-cell cDNA library (24) to isolate homologoushuman sequences. Overlapping human clones encompassinga 4.3-kb region were isolated. A region of 160 amino acids inhuman and mouse RAPTi encompassing the RAPTI-bindingdomain were compared with corresponding sequences inyeast Torl and Tor2 (Fig. 2A). The human RAPTi-bindingdomain, like the yeast and mouse homologs, contains theserine residue essential for rapamycin sensitivity in yeast (20,21). Within the RAPTi-binding domain, the deduced proteinsequences are highly conserved among the mammalian andyeast homologs. The mouse and human protein sequencesare 98% similar to each other and each is 55-60% similar toyeast Torl and Tor2. The unusually high degree of similarity

Table 3. Summary of cDNA library screens forrapamycin targets

Screen I Screen II

Binding domainfusion LexA-FKBP12 LexA-G6-FKBP12

Yeast transformantsscreened 1.5 x 107 1.5 x 107

Screening conditionsNo histidine, X-Gal Rapamycin Rapamycin

(100 ng/ml) (125 ng/ml) (15.6 ng/ml)His+ LacZ+ positives 107 101Rapamycin-

dependent, His+LacZ+ positives 24 20

Plasmid-linked 17 11Number of clones by

sequenceMouse Raptl 11 10Others 6 1

Screens I and II were conducted in parallel using the indicatedhuman FKBP12 binding domain fusions. Seven unique sequences,each encoding a different protein that interacts with the FKBP12/rapamycin complex, were obtained. X-Gal, 5-bromo-4-chloro-3-indolyl l3-D-galactoside.

between the yeast and mammalian sequences suggests thatthis region is important for the normal function ofRAPTi andTor.

RAPT1 RAPT1Ser-to-Arg + +

D

FIG. 1. Rapamycin-dependent interaction between humanFKBP12 fusion proteins and wild-type and mutant RAPT1 fusionproteins. Mouse Raptl clones, wild type or the serine-to-argininemutant, were used to transform two derivatives of strain VBY567containing different baits: plexA-FKBP12, the original bait used inscreen I (+; see Table 3) and plexA-da, a lexA fusion bait containingan unrelated insert as a negative control (-). Two independenttransformants containing the plexA-FKBP12 bait (+) are shown.Yeast transformants were tested on 5-bromo-4chloro-3-indolyl f-D-galactoside medium lacking histidine and containing no rapamycin(A), low rapamycin (15.6 ng/ml) (B), or high rapamycin (125 ng/ml)(C). Strains showing no growth on medium lacking histidine, whentested on medium containing histidine, showed no detectable /-ga-lactosidase activity (data not shown).

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Al Ir

I r/A i

h RAPTI Arg His Asn Ala Ala Asn Lys Ile Leu Lys Asn Met Cys Glu His 15m RAPTI Arg His Asn Ala Ala Asn Lys Ile Leu Lys Asn Met Cys Glu His 15sc TORl Arg Gln Lys Ala Ala Leu Ser Ile Ile Glu Lys Ile Arg Ile His 15sc TOR2 Arg Gln Lys Ala Ala Leu Ser Ile Ile Glu Lys Met Arg Ile His 15

h RAPTI Ser Asn Thr Leu Val Gln Gln Ala Met MetIVal Ser Glu Glu Le 30m RAPTI Ser Asn Thr Leu Val Gln Gln Ala Met Met Val Ser Glu Glu Leu 30sc TOR1 Ser Pro Val Leu Val Asn Gln Ala Glu Leu Val Ser His Glu Leu 30sc TOR2 Ser Pro Val Leu Val Asp Gln Ala Glu Leu VI .SerJ His Glu Leu 30

h RAPTI Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His Glu Gly Leu 45m RAPTI Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His Glu Gly Leu 45sc TOR1 Ile Arg Val Ala Val Leu Trp His Glu Leu Trp Tyr Glu Gly Leu 45sc TOR2 Ile Arg Met Ala Val Leu Trp His Glu Gln Trp Tyr Glu Gly Leu 45

h RAPTI Glu Glu Ala Ser Arg Leu Tyr Phe Gly Gin Arg As Val Lys Gly 60m RAPT1 Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn| Val Lys Gly 60sc TOR1 Glu Asp Ala Ser Arg Gln Phe Phe Val Glu His Asn Ile Glu Lys 60sc TOR2 Asp Asp Ala Ser Arg Gln Phe Phe Gly Glu His Ass Thr Glu Lys 60

h RAPTI Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 75m RAPTI Met Phei Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 75sc TORi Met Phel Ser Thri Leu Glu Pro Leu His Lys His Leu Gly Asn Glu 75sc TOR2 Met Phe Ala Ala Leu Glu Pro Leu Tyr Glu Met Leu Lys Arg Gly 75

h RAPTI Pro Gln Thr Leu Lys G Thr Ser Phe Asn Gln Ala Tyr GlY Arg 90m RAPTI Pro Arg Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg 90sc TORi Pro Gln Thr Leu Ser Gli Val Ser Phe Gln Lys Ser Phe Gly Arg 90sc TOR2 Pro Glu Thr Leu Arg GlI Ile Ser Phe Gln Asn Ser Phe |GlY Arga 90

h RAPTI Asp Leu Met Glu Ala Gln GiU Trp Cys Arg Lys Tyr Met Lys Ser 105m RAPTI Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser 105sc TOR1 Asp Leu| Asn Asp Ala Tyr Glu Trp Leu Asn Asn Tyr Lys LYS Ser 105sc TOR2 Asp Leu Asn Asp GAla i Trp Leu Met Asn T ys L|yLvs Ser| 105

h RAPT1 Gly Asn Val Lys Asp Leu Thr Gln Ala Trp Asp Leu Tyr Tyr His 120m RAPTI Gly Asn Val Lys Asp Leu Thr Gln Ala Trp Asp Leu |Tyr Tyr| His 120sc TORl Lys Asp Ile Asn Asn Leu Asn Gln Ala Trp Asp Ile |Tyr Tyr| Asn 120sc TOR2 Lys Asp Val Ser Asn Leu Asn Gln Ala Trp Asp Ile Tyr Tyr Asn 120

h RAPT1 Val Phe Arg Arg Ile Ser Lys Gln Leu Pro Gln Leu Thr Ser Len 135m RAPTI Val Phe Arg Arg Ile Ser Lys Gln Leu Pro Gln Leu Thr Ser Leu 135sc TORI Val Phe Arg Lys Ile| Thr Arg Gln Ile Pro Gln Leu Gln Thri Leu 135sc TOR2 Val Phe Arg Lys Ile Gly Lys Gln Leu Pro Gln Leu Gln Thr LeJ 135

h RAPTI Glu Leu Gln Tyr Val Ser Pro Lys Leu Leu Met Cys Arg Asp Leu 150m RAPTI Glu Leu Gln Tyr Val Ser Pro Lys Leu Leu Met Cys Arg Asp Leu 150sc TORi Asp Leu Gln His Val Ser Pro Gln Leu Leu Ala Thr His Asp Leu 150sc TOR2 Glu Leu Gln His Val Ser Pro Lys Leu Leu Ser Ala His Asp Leu 150

h RAPTI Glu Leu Ala Val Pro Gly Thri TyryAsp Pro 160m RAPT1 Glu Leu Ala Val Pro Gly Thri Tyr Asp Pro 160sc TORi Glu Leu Ala Val Pro Gly Thri Tyr Phe Pro 160sc TOR2 Glu Leu Ala Val Pro Gly Thri Arg Ala Ser 160

BVPS34 RNILNDQVRLINVLRECCETIKRLKDTTAKKMELLVHLLETKVRP--LVKVRPIALPLDPDVLSCDVCPETSKVFKSSLS 605pllO LKHLNRQVEAMEKLINLTDILKQEKKDETQKVQM-KFLVEQMRRPDFMDALQGFLSPLNPAHQLGNLRLEECRIMSSAKR 777RA00 FRRISKOQLPQLTSL ---------ELQYVSPKL-LMCRDLELAVPGTYDPN-QPIIRIQSIAPSL -------- QVITSKQR 2168TC01 FRKITRQIPQLQTL---------DLQHVSPQL-LATHDLBLAVPGTYFPG-KPTIRIAKFEPLF--------SVISSKQR 21057102 FRKIGKQLPQLQTL-- ELQHVSPKL-LSAHDLELAVPGTRASGGKPSIVKISKFEPVF- SVISSKQR 2109

VPS34 PLKITFKTT------LNQPYHLMFKVGDDLRQDQLVVQIISLMNELLKNENV ----DLKLTPYKILATGPQEGAIEFIPN 675pllO PLWLNWENPDIMSELLFQNNEIIKNGDDLRQDMLTLQIIRIMENIWQNQGL---- DLRMLPYGCLSIGDCVGLIEVVRN 853RA01 PRKLTLMGSN------GHEFVFLLKGHEDLRQDERVMQLFGLVNTLLANDPTSLRKNLSIQRYAVIPLSTNSGLIGWVPH 2242T7F1 PRKFSIKGSD----- GKDYKYVLKGHEDIRQDSLVMQLFGLVNTLLKNDSECFKRHLDIQQYPAIPLSPKSGLLGWVPN 2179TCR2 PRKFCIKGSD------ GKDYKYVLKGHEDIRQDSLVMQLFGLVNTLLQNDAECFRRHLDIQQYPAIPLSPKSGLLGWVPN 2183

VPS34 -DT_--------------ASILSK--YHGILGY--LKLH -------YPDENATLGVQGWVL --------------- DNFVK 715pll SHTI--------------MQIQCKGGLKGALQFNSHTLHQ------ WLKDKNKGEIYDAAI --------------DLFTR 899RAPrl CDTLHALIRDYREKKKILLNIEHiRIMLRMAPDYDHLTLMQKVEVFEHAVNNTAGDDLAKLLWLKSPSSEVWFDRRTNYTR 2322T301 SDTFHVLIREHRDAKKIPLNIEQWVMLQMAPDYENLTLLQKIEVFTYALDNTKGQDLYKILWLKSRSSETWLERRTTYTR 2259TCR2 SDTFHVLIREHREAKKIPLNIEHWVMLQMAPDYDNLTLLQKVEVFTYALNNTEGQDLYKVLWLKSRSSETWLERRT'YTR 2263

VPS34 SCAGYCVITYILGVGDRHLDNLLVTP-DGHFFHADFGYILGQDPKPFP-PLMKLPPQIIEAF------GGAESSN --- YD 784pllO SCAGYCVATFILGIGDRHNSNIMVKD-DGQLFHIDFGHFLDHKKKKFGYKRERVPFVLTQDFLIVISKGAQECTKTREFE 978RApTl SLAVMSMVGYILGLGDRHPSNLMLDRLSGKILHIDFGDCFEVAMTREKFP-EKIPFRLTRMLTNAMEVTGLD-------G 2394T731 SLAVMSMTGYILGLGDRHPSNLMLDRITGKVIHIDFGDCFEAAILREKYP-EKVPFRLTRMLTYAMEVSGIE-------G 2331T702 SLAVMSMTGYILGLGDRHPSNLMLDRITGKVIHIDFGDCFEAAISLREKFP-EKVPFRLTRMLTYAMEVSGIE-------G 2335

70PS34 KFRSYCFVAYSILRRNAGLILNLFELM ---------------KTSNIPDIRIDPNGAILRVR----------ERFNLNMS 839pllO RFQEMCYKAYLAIRQHANLFINLFSMM---------------LGSGMPE--LQSFDDIAYIR----------KTLALDKT 1031RAP00 NYRITCHTVMEVLREHKDSVMAVLEAFVYDPLLNWRLMDTNTKGNKRSRTRTDSYSAGQSVEILDGVELGEPAHKKTGTT 247470R1 SFRITCENVMRVLRDNKESLMAILEAFALDPLIHWGFDLPPQKLTEQT --------- GIPLPLINPSEL-----LRKGAI 23977012 SFRITCENVMKVLRDNKGSLMAILEAFAFDPLINWGFDLPTKKIEEET ---------GIQLPVMNANEL -----LSNGAI 2401

FIG. 2. Characterization of human and mouse RAPTI. (A) Align-ment of amino acid sequences deduced from the human and mouseRAPTI and the yeast TORI and TOR2 genes. Predicted amino acidsequences for the human and mouse RAPT1-binding domains areshown aligned with those for S. cerevisiae Torl (positions 1924-2083) and Tor2 (positions 1927-2086). Enclosed in boxes are aminoacids that are identical in all four sequences. Smallest interactingclone contains the fragment found between the two arrowheads (fromresidues 26 to 158 of the alignment). Open box above sequencesrepresents the 7.4-kb open reading frame (-2470 amino acids)encoded by either of the Tor genes. Arrow delineates boundary of theTOR1/TOR2 hybrids reported by Helliwell et al. (position 1680 ofTorl; ref. 21). The 160 residues of Torl and Tor2 shown herecorrespond to the region depicted by the solid box. Position of thedominant serine-to-arginine mutation found in the rapamycin-resistant strain drrl-I (position 1972 of Torl) is marked by an asterisk

01)

Cf)CC -

$) >, XL a)a

c - a)cL YC/v)

"0)

(J CJ

a)

c)

Co

c)

a)

o ~ ~ c c

lo CZ - 0 a)ca)m

E > Z

J Fla-~~~~~~~~~~*co.a..COa- ..:...~~~~~~~~~~~~~~~~~~~~~..... ...... .. ,_9.5 kb7.5 kb4.4 kb-

2.4 kb

1.35 kb

FIG. 3. Tissue distribution of human RAPTI mRNA. Northernblots of poly(A)+ RNA isolated from 12 human tissues were hybrid-ized with a 32P-labeled probe derived from the 480-bp fragment of theinteraction domain from human RAPTI.

The human and mouse RAPT1 sequences are more similarto each other (98%) than yeast Torl and Tor2 are to eachother (79%), suggesting that the former are homologs of thesame isozyme. However, these results do not exclude thepossibility that there are multiple RAPT1 isozymes. Outsideof the binding domain, other similarities exist between thededuced protein sequences of human RAPT1 and yeast Tor.In each homolog, both the RAPT1-binding domain and thelipid kinase consensus sequence are contained within theC-terminal half in the region found to be functionally inter-changeable between Torl and Tor2 (19-21). Conservation ofthe lipid kinase motif suggests that human RAPT1, like Torland Tor2, encodes a putative lipid kinase (Fig. 2B). Withinthe lipid kinase motif are residues conserved in the moregeneral class of protein kinases (19, 26).RAPTI Specifies a 9-kb mRNA Transcript. Expression of

RAPTI sequences was analyzed on Northern blots of mRNAisolated from various human tissues (Fig. 3). RAPTI specifiesa single transcript of -9 kb that is present in all tissuesexamined, exhibiting the highest levels in testis. The tran-script is sufficient to encode a protein equivalent to the sizeof yeast Tor, which is 284 kDa. Assuming that RAPT1 is ofsimilar size, we have homed in on a small segment of 133amino acids within a large protein that is sufficient to bind tothe FKBP12/rapamycin complex (Fig. 2A).

DISCUSSIONRapamycin is of general interest because of its potent immu-nosuppressive and antifungal activity. Rapamycin blocksgrowth factor-induced signal transduction pathways and in-hibits the G1/S transition in yeast and mammalian cells.Understanding rapamycin mechanism of action will elucidatethe link between these two aspects of cell growth. Moreover,identification of the rapamycin targets in lymphocytes, non-

in the diagram and is underlined in the sequence alignment (residue49). Lipid kinase motif derived from a comparison between Torl,Tor2, the catalytic domain of the bovine PI 3-kinase pllO (27), andthe yeast Vps34 (28) is found within the hatched box and is shown inB. (B) Lipid kinase consensus sequence. A conserved sequence motiffound in human RAPT1; yeast Torl, Tor2, and Vps34; and p110, thecatalytic subunit of bovine PI 3-kinase, is shown. First amino acid ineach line corresponds to amino acid position 699, 532, 2110, 2047,and 2050 for p110, Vps34, RAPT1, Torl, and Tor2, respectively.Amino acids in boldface are conserved in all five proteins. Conservedresidues that lie within the lipid kinase consensus described by Kunzet al. (19) are underlined.

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lymphoid cells, and yeast will facilitate the design of smallmolecule inhibitors of cell proliferation specific for each celltype.Using the two-hybrid system, we determined that the

mammalian homolog of yeast Tor, RAPT1, interacts with theFKBP12/rapamycin complex. This result substantiates pre-vious genetic analyses in yeast that implicated Tor as therapamycin target but did not rule out the possibility that Toracted downstream of the rapamycin target. The interactionbetween FKBP12/rapamycin and RAPT1 in the two-hybridsystem may involve endogenous yeast proteins. This is themost likely explanation for the interaction of FKBP12/FK506 with calcineurin A that we observed in the two-hybridsystem. In this case, yeast calcineurin B may mediate theinteraction of the two human fusion proteins, FKBP12 andcalcineurin A, since it is required for complex formation (25).The conservation of FKP12 and calcineurin between yeastand humans supports this notion. Future studies shoulddetermine whether the FKBP12/rapamycin/RAPT1 com-plex also involves other proteins. In either case, our resultsestablish that RAPT1 is a member of the FKBP12/rapamycincomplex and that its association with the complex occursonly in the presence of rapamycin.Demonstration that RAPT1, the mammalian homolog of

Tor, interacts with the FKBP12/rapamycin complex sug-gests that FKBP12, versus another member of the FKBPfamily, mediates the antiproliferative action of rapamycin inmammalian cells as it does in yeast. RAPT1 like yeast Torcontains a lipid kinase consensus sequence present in bovinePI 3-kinase. Future studies should determine whether thishomology is functionally significant. Although RAPT1 inter-acts directly with the FKBP12/rapamycin complex, a criticalquestion is whether RAPT1 is the relevant target in vivo and,if so, in which cell types.

This study represents an example of using the two-hybridsystem to detect protein-protein interaction mediated by asmall molecule. We demonstrated FK506-dependent inter-action between FKBP12 and calcineurin in the two-hybridsystem, which we then extended to the identification ofrapamycin-dependent FKBP12 interactors. A practical ap-plication of this approach would be to use the two-hybridsystem in the testing and design of small molecule modulatorsof protein-protein interaction that have potential therapeuticvalue.From the screens ofthe mouse cDNA library reported here

and a human cDNA library (V.B., unpublished results), wehave identified several other proteins that interact with theFKBP12/rapamycin complex, which we anticipate are in-volved in the antiproliferative activities of rapamycin in cellsfrom various tissues. Characterization of these targets willelucidate the precise details of how rapamycin blocks cellproliferation and will define aspects of cell division amenableto therapeutic intervention.

Note Added in Proof. After completion of this work, two reports werepublished describing proteins identical to RAPT1, designated FRAP(29) and RAFT1 (30), shown by two different biochemical ap-proaches to bind to the FKBP12/rapamycin complex. The tissuedistribution of FRAP mRNA is consistent with that of RAPT1.

We thank F. McKeon and D. Milan for providing the humancalcineurin A clone and S. Hollenberg for providing two-hybridplasmids and the mouse cDNA library.

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