The Rapamycin-binding Domain of the Protein Kinase Mammalian Target of Rapamycin Is a Destabilizing Domain

Abstract

Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. A common approach for studying complex biological processes is to perturb a protein of interest and observe subsequent changes to the biological system. The classical approach of disrupting a gene and studying the resultant phenotype is problematic for examining essential genes. As a result, several conditional techniques have been developed including the tetracycline/doxycycline and Cre recombinase/lox P systems, where control is exerted at the transcriptional level (1Furth P.A. St. Onge L. Boeger H. Gruss P. Gossen M. Kistner A. Bujard H. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9302-9306Crossref PubMed Scopus (667) Google Scholar, 2Ryding A.D.S. Sharp M.G.F. Mullins J.J. J. Endocrinol. 2001; 171: 1-14Crossref PubMed Scopus (118) Google Scholar). At the post-transcriptional level, RNA interference (RNAi) 2The abbreviations used are: RNAi, RNA interference; FKBP, 12-kDa FK506- and rapamycin-binding protein; FRB, FKBP12-rapamycin binding domain; Gal4AD, Gal4 activation domain; Gal4BD, Gal4 binding domain; iRap, C-16-(S)-3-methylindolerapamycin; mTOR, mammalian target of rapamycin; SeAP, secreted alkaline phosphatase; TMOP-rap, C-16-(R)-trimethoxyphenyl rapamycin; wtFRB, wild-type FRB; YFP, yellow fluorescent protein; yeGFP, yeast-enhanced green fluorescent protein; FACS, fluorescence-activated cell sorter; MaRap, methallyl addition at the C-20 position in rapamycin. 2The abbreviations used are: RNAi, RNA interference; FKBP, 12-kDa FK506- and rapamycin-binding protein; FRB, FKBP12-rapamycin binding domain; Gal4AD, Gal4 activation domain; Gal4BD, Gal4 binding domain; iRap, C-16-(S)-3-methylindolerapamycin; mTOR, mammalian target of rapamycin; SeAP, secreted alkaline phosphatase; TMOP-rap, C-16-(R)-trimethoxyphenyl rapamycin; wtFRB, wild-type FRB; YFP, yellow fluorescent protein; yeGFP, yeast-enhanced green fluorescent protein; FACS, fluorescence-activated cell sorter; MaRap, methallyl addition at the C-20 position in rapamycin. is becoming rapidly used as a method of gene silencing (3Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11430) Google Scholar), but RNAi lacks tunability and does not affect existing protein levels. An ideal perturbation method would directly affect the protein with a cell-permeable probe that rapidly and reversibly targets a specific protein of interest. Small molecules have been very useful as conditional perturbants; however, the lack of specificity of many of these reagents can make it difficult to unambiguously interpret results. One approach to ensure specificity is to chemically modify a small molecule with a large substituent, and this technique has been widely used to interrogate specific protein tyrosine kinases that possess a complementary cavity-forming mutation (4Shah K. Liu Y. Deirmengian C. Shokat K.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3565-3570Crossref PubMed Scopus (351) Google Scholar, 5Bishop A.C. Shah K. Liu Y. Witucki L. Kung C.Y. Shokat K.M. Curr. Biol. 1998; 8: 257-266Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). An alternative system has recently been developed using a cell-permeable small molecule to regulate the stability of specific proteins in a rapid, reversible, and dose-dependent manner (6Banaszynski L.A. Chen L.-C. Maynard-Smith L.A. Ooi A.G.L. Wandless T.J. Cell. 2006; 126: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar). A third approach for directly controlling protein function is by perturbing subcellular localization. Conditional regulation of protein localization has been achieved using the FKBP·rapamycin·FRB complex (7Ho S.N. Biggar S.R. Spencer D.M. Schreiber S.L. Crabtree G.R. Nature. 1996; 382: 822-826Crossref PubMed Scopus (218) Google Scholar, 8Luengo J.I. Yamashita D.S. Dunnington D. Beck A.K. Rozamus L.W. Yen H.-K. Bossard M.J. Levy M.A. Hand A. Newman-Tarr T. Badger A. Faucette L. Johnson R.K. D'Alessio K. Porter T. Shu A.Y.L. Heys R. Choi J. Kongsaeree P. Clardy J. Holt D.A. Chem. Biol. 1995; 2: 471-481Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 9Klemm J.D. Beals C.R. Crabtree G.R. Curr. Biol. 1997; 7: 638-644Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Inoue T. Heo W.D. Grimley J.S. Wandless T.J. Meyer T. Nat. Meth. 2005; 2: 415-418Crossref PubMed Scopus (287) Google Scholar, 11Heo W.D. Inoue T. Park W.S. Kim M.L. Park B.O. Wandless T.J. Meyer T. Science. 2006; 314: 1458-1461Crossref PubMed Scopus (525) Google Scholar, 12Suh B.-C. Inoue T. Meyer T. Hille B. Science. 2006; 314: 1454-1457Crossref PubMed Scopus (389) Google Scholar). With this method a protein of interest is fused to either FKBP, the 12-kDa FK506- and rapamycin-binding protein or FRB, the FKBP-rapamycin binding domain of the protein kinase, mTOR. The other rapamycin-binding protein is modified to include a domain that regulates subcellular localization (e.g. nuclear export or plasma membrane-associated). The addition of rapamycin induces heterodimerization of the FRB and FKBP fusions and rapid formation of a ternary complex, which causes the protein of interest to be rapidly rerouted within the cell. One disadvantage of this system is that rapamycin has biological activity; it binds to endogenous mTOR kinase, inhibition of which may confound interpretation of the experiment (13Brown E.J. Albers M.W. Shin T.B. Ichikawa K. Keith C.T. Lane W.S. Schreiber S.L. Nature. 1994; 369: 756-758Crossref PubMed Scopus (1626) Google Scholar, 14Vilella-Bach M. Nuzzi P. Fang Y. Chen J. J. Biol. Chem. 1999; 274: 4266-4272Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). To engineer biologically silent ligands to induce dimerization, a “bump-hole” strategy has been pursued in which rapamycin derivatives were designed to possess bulky substituents within the FRB/mTOR binding region. These derivatives bind poorly to mTOR, and thus are relatively nontoxic to cells, but can bind to FRB domains that contain compensatory cavityforming mutations. One such derivative contains a methallyl addition at the C-20 position in rapamycin (MaRap) and binds a triple mutant of FRB (K2095P, T2098L, W2101F) designated FRB* (15Liberles S.D. Diver S.T. Austin D.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7825-7830Crossref PubMed Scopus (160) Google Scholar). Fusing the essential gene encoding GSK-3β to FRB* and a nuclear export sequence to FKBP, the addition of MaRap should allow the formation of a ternary complex and a resultant ligand-dependent translocation of GSK-3β to the cytoplasm would be expected. However, in the absence of MaRap levels of GSK-3β expressed as a fusion to FRB* are significantly decreased with respect to the native protein, indicating that the FRB* domain confers instability (16Stankunas K. Bayle J.H. Gestwicki J.E. Lin Y.-M. Wandless T.J. Crabtree G.R. Mol. Cell. 2003; 12: 1615-1624Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). To conditionally mislocalize a protein of interest in a rapid and reversible fashion, the protein domains that comprise the ternary complex should not be inherently unstable, and the small molecule should be easy to prepare and possess desirable pharmacokinetics in vivo. We set out to develop a conditional mislocalization system that would have these characteristics. To this end, we synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, C-16-(R)-trimethoxyphenyl rapamycin (TMOP-rap) and C-16-(S)-3-methylindolerapamycin (iRap) (8Luengo J.I. Yamashita D.S. Dunnington D. Beck A.K. Rozamus L.W. Yen H.-K. Bossard M.J. Levy M.A. Hand A. Newman-Tarr T. Badger A. Faucette L. Johnson R.K. D'Alessio K. Porter T. Shu A.Y.L. Heys R. Choi J. Kongsaeree P. Clardy J. Holt D.A. Chem. Biol. 1995; 2: 471-481Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 10Inoue T. Heo W.D. Grimley J.S. Wandless T.J. Meyer T. Nat. Meth. 2005; 2: 415-418Crossref PubMed Scopus (287) Google Scholar). Screening a library of FRB mutants using a three-hybrid assay in yeast (17Licitra E.J. Liu J.O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12817-12821Crossref PubMed Scopus (267) Google Scholar) provided many FRB mutants that activated reporter gene expression in response to iRap. Further analyses in mammalian cells revealed that these FRB mutants, as well as wild-type FRB (wtFRB), significantly reduced expression levels of fusion proteins, and addition of iRap caused a dose-dependent increase in expression levels. An awareness of this ligand-dependent behavior should aid investigators in interpreting experiments based on this conditional dimerization technology. FRB Library Generation—The FRB gene used for these studies encodes residues 2015–2114 of mTOR. Diversity in the FRB sequence was generated using a combination of error-prone PCR and nucleotide analog mutagenesis. Primers for mutagenic PCR were designed to contain 50 bases of homology to the yeast expression vector pGAD-T7 (Clontech, Palo Alto, CA) including the desired restriction site and 18 bases complementary to either the 5′- or 3′-terminus of the FRB gene. For the first library, wtFRB was used as the PCR template for mutagenesis. One set of library conditions utilized 10 ng of wtFRB template, each oligonucleotide primer at 0.5 μm, 8 mm MgCl2, 0.2 mm dATP and dGTP, 1.0 mm dCTP and dTTP, 2.5 units Taq polymerase, and either 0.5 or 0.05 mm MnCl2. For the second set of conditions, the non-natural nucleotides 8-oxo-dGTP and dPTP were utilized to encourage misincorporation (18Zaccolo M. Williams D.M. Brown D.M. Gherardi E. J. Mol. Biol. 1996; 255: 589-603Crossref PubMed Scopus (269) Google Scholar). For the second FRB library, a mixed template was generated consisting of equimolar ratios of wtFRB, FRB21, and FRB22 as well as a FRB21/FRB22 double mutant. Three independent condition sets were utilized to generate diversity within the FRB coding sequence and are described in Ref. 6Banaszynski L.A. Chen L.-C. Maynard-Smith L.A. Ooi A.G.L. Wandless T.J. Cell. 2006; 126: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar. Synthesis of Rapamycin Analogs—C-16-(R)-Trimethoxyphenyl rapamycin (TMOP-rap) and C-16-(S)-3-methylindolerapamycin (iRap) were synthesized as described previously (8Luengo J.I. Yamashita D.S. Dunnington D. Beck A.K. Rozamus L.W. Yen H.-K. Bossard M.J. Levy M.A. Hand A. Newman-Tarr T. Badger A. Faucette L. Johnson R.K. D'Alessio K. Porter T. Shu A.Y.L. Heys R. Choi J. Kongsaeree P. Clardy J. Holt D.A. Chem. Biol. 1995; 2: 471-481Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 10Inoue T. Heo W.D. Grimley J.S. Wandless T.J. Meyer T. Nat. Meth. 2005; 2: 415-418Crossref PubMed Scopus (287) Google Scholar). AP21967 (C-16-(S)-7-methylindolerapamycin) was obtained from Ariad Pharmaceuticals, Inc., Cambridge, MA. Yeast Strains—YSE2 (Matα leu2-3,112 ura3-52 trp1-901 his3-Δ200 ade2-101 gal4Δ gal80Δ fpr1Δ::ADE2 TOR1-1 LYS2::GAL1 → HIS3 GAL1 → LacZ ura3::GAL1 → GAL1 → → → was by S.R. Crabtree G.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with the yeast-enhanced and the the and the a fusion and the the Yeast for and library were PubMed Scopus Google Scholar) with the A for library of the was in for 8 of to a of For the library of library with 10 of pGAD-T7 library vector was to the cells. of the was to the cells a at were in and to for at and on with Saccharomyces and of FRB cells the FRB library in were with ligand for library and iRap for library to and the of fluorescent cells were and at for were analyzed for and at the Library was with a yeast CA) and cells gene 2.5 cell were in modified with mm and and cell or cells were (7Ho S.N. Biggar S.R. Spencer D.M. Schreiber S.L. Crabtree G.R. Nature. 1996; 382: 822-826Crossref PubMed Scopus (218) Google Scholar) with to of by Gal4 binding and a which fused to of FKBP, and which an FRB wild-type or to the activation were as described previously (16Stankunas K. Bayle J.H. Gestwicki J.E. Lin Y.-M. Wandless T.J. Crabtree G.R. Mol. Cell. 2003; 12: 1615-1624Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, J.H. Grimley J.S. K. Gestwicki J.E. Wandless T.J. Crabtree G.R. Chem. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), and were analyzed with and and mutants were the expression vector as fusions to yellow fluorescent protein the cell or cells were with the using For a cells were in for To generate cells, were from the cells and cells were with the with for at were in for to allow for with were at of a and with iRap. For the stability cells were with iRap for and For the cells were with iRap and at each were from the with and in of were analyzed at the with for for the mTOR cells were at of a and for in modified were or with rapamycin iRap or AP21967 for at was to a of for at were as described previously E. Nuzzi P. Chen J. 2005; PubMed Scopus Google Scholar), and cell were by proteins were to a and with a against of Inc., were analyzed using and Screening FRB in FRB mutants to either or iRap, we used a three-hybrid transcriptional assay in S. cerevisiae. this FKBP was fused to the Gal4 domain and FRB was fused to the Gal4 activation domain The addition of rapamycin to the formation of the FKBP·rapamycin·FRB complex, the Gal4 We a yeast in which was the control of the and reporter gene expression formation of the ternary complex The rapamycin derivatives and iRap were synthesized with at the C-16 position within the FRB-binding to binding to the native target mTOR To the of the rapamycin a compensatory in FRB to be and we used error-prone PCR and nucleotide analog to prepare a library of FRB The FRB library was designed and the vector by in a in which yeast cells a library of a vector through cells to on yeast were to of for cells that expressed A library was to of using For each the cells were with to and the fluorescent cells were and further from to The library upon and was to by the These fluorescent cells were and the and of revealed two the first was and the second a mutation of the to one encoding The encoding FRB a second residues the thus the second mutant a at the To the method for an FRB mutant that bind and to FRB mutants in the pGAD-T7 vector were and were with and analyzed by for expression of the reporter gene the two FRB mutants responded to and the expression levels not of the library To an FRB mutant responsive to ligand a second library was generated in a method to the The two from the first were in the library the mixed FRB template wild-type FRB, FRB mutants from the first library and the double mutant. rapamycin derivative, iRap, was for the A second FRB library was to two of with cells were with iRap to and the of cells were and in the absence of ligand for These cells were with iRap to At this of the was fluorescent The of cells were and the and of revealed FRB For these FRB mutants were as described each mutant were with iRap, and expression levels were measured by levels of the protein from to of these levels of yeGFP, were in mammalian cells using a three-hybrid transcriptional Further of FRB in cells were by with the and either wild-type or mutant of J.H. Grimley J.S. K. Gestwicki J.E. Wandless T.J. Crabtree G.R. Chem. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). FRB the twenty from the iRap library as well as of the mutants from the first the of these FRB cells were for in with rapamycin or iRap of were measured for FRB and a is in FRB mutants response to iRap, and the for the mutants from to iRap induces expression with an of indicating that wtFRB and the FRB mutants from the library to iRap in this transcriptional mutants with iRap a of by the in a the of FRB for a conditional mislocalization system that in a rapid is that proteins are in the absence of ligand. was not the for the FRB mutant (16Stankunas K. Bayle J.H. Gestwicki J.E. Lin Y.-M. Wandless T.J. Crabtree G.R. Mol. Cell. 2003; 12: 1615-1624Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). To the stability of the FRB mutants from the library FRB mutants and as well as wtFRB were fused to the of YFP, and the were expressed in mammalian cells. These FRB mutants were displayed the for iRap in the cells were with and or with iRap to cells were with the wtFRB and as fluorescence levels were measured in the presence and absence of iRap the absence of iRap, fluorescence levels for the FRB were that the FRB proteins were when expressed in mammalian cells. as for FRB* with MaRap, the FRB proteins to be when with iRap. A 10-fold increase in fluorescence was when cells wtFRB fused to were with iRap stability was when expression levels for cells with the of the FRB mutants in fluorescence upon treatment with iRap. of determine the to fluorescence as well as the to this fluorescence level, a of cells the was cells were with of iRap, and fluorescence was as a function of We that fluorescence at the for the cell with μm, and 10 iRap, the cell with 10 iRap decreased fluorescence levels at cell achieved fluorescence at and levels by indicating that fluorescence was by but not in the absence of further A assay was to determine the of iRap to the fusion cells with were with iRap and fluorescence levels The cells achieved fluorescence with iRap and the at To that fluorescence levels to protein expression cells with the and with iRap for were and with a against The protein expression levels were with fluorescence levels not mTOR experiments out utilized FRB, which is a relatively small domain within the mTOR kinase. To determine the rapamycin derivatives with the to mTOR as to FRB, a of was The mTOR protein at the ability of rapamycin and its to inhibit endogenous mTOR can be using a for this cells were with rapamycin iRap or the derivative conditions that mTOR were by and with a for A is in in which the for of as iRap mTOR. compounds were found to inhibit endogenous mTOR, with of for for iRap, and for AP21967 We set out to a ternary complex FKBP, an FRB mutant and a rapamycin derivative that be used to conditionally mislocalize proteins to rapidly and reversibly probe The FRB library screened with the rapamycin derivative iRap revealed twenty mutants that expressed levels a mammalian three-hybrid system using the reporter gene SeAP, these FRB mutants a of expression levels. mutants were wtFRB but most mutants were The iRap was by but we that of rapamycin be for the behavior of of the mutants from the library by quantitative mass spectrometry, we that the iRap rapamycin. Rapamycin was not in the of that the rapamycin analogs the ability to bind to wtFRB and mTOR but of rapamycin may the potency of the this it was difficult to determine of mutants, activation of is to the rapamycin The fusion protein of wtFRB to was in and cells, and the 10-fold increase in expression levels in the presence of iRap was for wtFRB and the FRB mutant proteins were by as the fluorescence levels in the presence of iRap were of a although the FRB proteins ligand-dependent the of in the presence of ligand was relative to The behavior of FRB* was to the cavity-forming but it that the wild-type FRB protein was To a to control protein function based on rapid subcellular the proteins should be for protein levels to the of mislocalization. a mislocalization system upon domains that ligand-dependent stability may as it would be difficult to of an phenotype is to mislocalization changes in protein expression levels. We found by fluorescence cells with a fusion and with 10 iRap of the that the with of iRap and expression levels at when cells with a ligand at low it is to that protein expression levels are examining the of iRap, and AP21967 in mTOR iRap and AP21967 were found to inhibit mTOR with in the low studies to rapamycin levels in these revealed that rapamycin was not in AP21967 the C-16 of iRap was to that of this that the ability of iRap to inhibit mTOR or fusion proteins T. Heo W.D. Grimley J.S. Wandless T.J. Meyer T. Nat. Meth. 2005; 2: 415-418Crossref PubMed Scopus (287) Google Scholar) is to of contaminating rapamycin. for a ternary complex that be used to rapidly mislocalize proteins of we have that the FRB domain of the mTOR is inherently FRB is fused to the fluorescent protein and in the presence and absence of the expression levels of the fusion protein increase from to 10-fold for of the FRB proteins the ligand-dependent stability of FRB fusion proteins as well as the purity of rapamycin derivatives should be considered as potentially confounding variables when using systems. We the Crabtree and for yeast and Grimley for the of iRap, for the of TMOP-rap, and for with