US20040072207A1

US20040072207A1 - Nucleic acids encoding a mammalian raptor polypeptide and uses therefor

Info

Publication number: US20040072207A1
Application number: US10/437,421
Authority: US
Inventors: David Sabatini; Do-hyung Kim; Dos Sarbassov
Original assignee: Whitehead Institute for Biomedical Research
Current assignee: Whitehead Institute for Biomedical Research
Priority date: 2002-05-14
Filing date: 2003-05-13
Publication date: 2004-04-15
Also published as: US20080260721A1; WO2004029276A9; AU2003295314A1; US8809500B2; AU2003295314A8; US8486643B2; US20140004532A1; WO2004029276A3; US20110143370A1; WO2004029276A2

Abstract

The present invention relates to isolated raptor nucleic acid molecules of mammalian origin (e.g., human) and complements, portions and variants thereof. Another aspect of the invention are isolated raptor polypeptides of mammalian origin and portions thereof, and antibodies or antigen binding fragments thereof that specifically bind a raptor polypeptide. The present invention also relates to constructs and host cells comprising the nucleic acid molecules described herein. In addition, the present invention relates to uses of the nucleic acid and polypeptide molecules provided herein.

Description

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No. 60/378,153, filed May 14, 2002. [0001]
The entire teachings of the above application(s) are incorporated herein by reference.[0002]

GOVERNMENT SUPPORT

[0003] The invention was supported, in whole or in part, by a grant R01 A147389 from the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Increasing evidence indicates that in eukaryotes cell growth (mass accumulation) is finely regulated in response to environmental and developmental conditions and can be deranged in human diseases such as cancer and diabetes (reviewed by Dixon and Fordham-Skelton, Curr. Opin. Plant Biol. 1:1 (1998); Johnston and Gallant, Bioessays 24: 54-64 (2002); Katso et al., Annu. Rev. Cell Dev. Biol. 17:615-75 (2001); Kozma and Thomas, Bioessays 24:65-71 (2002); Schmelzle and Hall, Cell 103:253-62 (2000)). The rate of mass accumulation is controlled not simply by the availability of nutrients, but by signaling pathways that coordinate the activity of the cell growth machinery with nutritional, hormonal and mitogenic signals. Studies into the mechanism of action of rapamycin, an immunosuppressive and anti-cancer drug, led to the discovery of an evolutionarily conserved regulator of cell growth, the TOR (Target of Rapamycin) pathway (Brown et al., Nature 369:756-758 (1994); Chiu et al., Proc. Natl. Acad. Sci. USA 91:12574-12578 (1994); Kunz et al., Cell 73:585-596 (1993); Oldham et al., Genes Dev 14:2689-94 (2000); Sabatini et al., Cell 78:35-43 (1994); Sabers et al., J. Biol. Chem. 270:815-822.(1995); Zhang et al., Genes Dev 14:2712-24 (2000)). The complex of rapamycin with its receptor, FKBP12, binds directly to TOR and perturbs its function in a poorly understood fashion (Brown et al., Nature 377:441-446 (1995); Burnett et al., PNAS 95:1432-1437 (1998); Peterson et al., J Biol Chem 275:7416-23 (2000); Zheng et al., Cell 82:121-130 (1995)). TOR is a member of the PIK-related family of proteins (Keith and Schreiber, Science 270:50-5 (1995)) that share homology with the catalytic domain of phosphatidylinositol 3-kinase (PI-3K), but appear to function as serine/threonine rather than lipid kinases. Studies in several organisms have shown that the TOR pathway regulates a variety of processes contributing to cell growth, including initiation of mRNA translation, ribosome synthesis, expression of metabolism-related genes, autophagy and cytoskeletal reorganization (recently reviewed by Schmelzle and Hall, Cell 103:253-62 (2000) and by Gingras et al., Genes Dev 15:807-26 (2001)). By interfering with the function of mammalian TOR, rapamycin inhibits progression through the G1 phase of the cell cycle in various cell types. Because of these anti-proliferative effects, rapamycin is a clinically valuable drug that is currently used to block immune rejection of transplanted organs (Saunders et al., Kidney Int. 59:3-16 (2001)) and in trials for the treatment of cancer (Dudkin et al., Clin. Cancer Res 7:1758-64 (2001); Hidalgo and Rowinsky, Oncogene 19, 6680-6 (2000)) and for the prevention of restenosis after angioplasty (Sousa et al., Circulation 104:2007-11 (2001)).

Mammalian TOR, mTOR (also known as RAFT1 or FRAP), phosphorylates at least two regulators of protein synthesis: S6K1 (formerly called p70 ribosomal S6 kinase) and an inhibitor of translation initiation, the eIF-4E binding protein 1 (4E-BP1) (Brunn et al., Science 277, 99-101 (1997); Burnett et al., PNAS 95:1432-1437 (1998); Hara et al., J. Biol. Chem. 272, 26457-63 (1997); Isotani et al., J Biol Chem 274:34493-8 (1999)). In mammalian cells, amino-acid deprivation leads to the dephosphorylation of both S6K1 and 4E-BP1 and to decreased rates of protein synthesis, effects that are rapidly reversed by the re-addition of amino acids (Fox et al., American Journal of Physiology Cell Physiology 274:43-1 (1998); Hara et al., J Biol Chem 273:14484-94 (1998)). Among amino acids, changes in leucine levels alone are sufficient to regulate the phosphorylation state and activity of both downstream components of the mTOR pathway (Hara et al., J Biol Chem 273:14484-94 (1998); Lynch et al., J. Cell Biochem. 77:234-51 (2000)). In addition to amino acid levels, mitochondrial function (Xu et al., Diabetes 50:353-60 (2001)), glycolysis (Dennis et al., Science 294:1102-5 (2001)), and cell stress (Parrott and Templeton, J. Biol. Chem. 274:24731-6.(1999)) regulate S6K1, as do growth factors, such as insulin (Lawrence and Brunn, Prog. Mol. Subcell. Biol. 26:1-31 (2001)).

Despite extensive efforts, how nutrients regulate the mTOR signaling pathway remains poorly understood. In particular, stimuli that activate (e.g. amino acids) or inhibit (e.g. mitochondrial uncouplers) downstream effectors of mTOR, such as S6K1 and 4E-BP1, fail to change the in vitro kinase activity of mTOR (Dennis et al., Science 294:1102-5 (2001); Hara et al., J. Biol. Chem. 272:26457-63 (1997)).

Thus, a greater understanding of mTOR/RAFT1/FRAP, a central component of a signaling pathway that modulates cell growth in response to nutrients and hormones and which is the target of the immunosuppressive drug rapamycin, would be useful in the diagnosis and treatment of cell growth disorders such as cancer.

SUMMARY OF THE INVENTION

As shown herein, in vivo mTOR exists in a stoichiometric complex with raptor, a novel, evolutionarily conserved protein that plays at least two roles in the mTOR pathway. Raptor is required for mTOR protein expression, nutrient-stimulated signaling to the downstream effector S6K1, and maintenance of cell size. The association of raptor with mTOR also negatively regulates the mTOR kinase activity. Conditions that repress the pathway, such as nutrient deprivation and mitochondrial uncoupling, stabilize the mTOR-raptor association and inhibit the kinase activity. Overexpression of wild-type raptor mimics nutrient deprivation, causing the formation of a stable mTOR-raptor complex with decreased mTOR kinase activity. Thus, raptor is a missing component of the TOR pathway that through its association with mTOR adjusts the rate cell of growth to nutrient levels.

Accordingly, the present invention relates to an isolated nucleic acid molecule comprising SEQ ID NO: 1 or the complement of SEQ ID NO: 1. In one embodiment, the isolated nucleic acid molecule that encodes an amino acid sequence comprises SEQ ID NO: 2. In another embodiment, the isolated nucleic acid molecule comprises a sequence that hybridizes under highly stringent conditions to SEQ ID NO: 1 or a complement of SEQ ID NO: 1. In a particular embodiment, the isolated nucleic acid molecule that comprises a sequence that hybridizes under highly stringent conditions to a complement of SEQ ID NO: 1, encodes a mammalian raptor protein.

The present invention also relates to an isolated polypeptide encoded by a nucleic acid comprising SEQ ID NO: 1. In one embodiment, the isolated polypeptide has an amino acid sequence comprising SEQ ID NO: 2.

The present invention also relates to an expression construct comprising SEQ ID NO:1. In one embodiment, SEQ ID NO: 1 in the expression construct is operably linked to a regulatory sequence.

Another aspect of the invention is a host cell comprising an isolated nucleic acid described herein. In one embodiment, the isolated nucleic acid is operably linked to a regulatory sequence.

Mammalian raptor polypeptide can be produced in a method comprising culturing a host cell comprising an isolated nucleic acid described herein under conditions in which the raptor polypeptide is produced. The method can further comprise isolating the raptor polypeptide from the cell. The present invention also relates to an isolated raptor polypeptide produced by the method.

The present invention also relates to an antibody (e.g., polyclonal antibody, monoclonal antibody) or antigen binding fragment thereof that specifically binds to a mammalian raptor polypeptide, wherein the mammalian raptor polypeptide is encoded by an isolated nucleic acid that encodes SEQ ID NO: 2. In one embodiment, the antibody recognizes an epitope from about amino acid 985 to about amino acid 1001 of SEQ ID NO: 2.

The present invention also relates to a method of identifying a nucleic acid that encodes a mammalian raptor polypeptide in a sample comprising contacting the sample with a nucleotide sequence comprising SEQ ID NO: 1 under conditions in which hybridization occurs between SEQ ID NO: 1 and the nucleic acid in the sample using high stringency conditions. The nucleic acid which hybridizes to SEQ ID NO: 1 under high stringency conditions is identified, thereby identifying a nucleic acid that encodes a mammalian raptor polypeptide in a sample.

The present invention also relates to a method of identifying a mammalian raptor polypeptide in a sample comprising contacting the sample with an antibody or antigen binding fragment thereof that specifically binds to a mammalian raptor polypeptide wherein the mammalian raptor polypeptide is encoded by an isolated nucleic acid that encodes SEQ ID NO: 2. The polypeptide which specifically binds to the antibody is identified, thereby identifying a mammalian raptor polypeptide in a sample.

The present invention also relates to a method of identifying an agent that alters interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising contacting a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 with mTOR protein under conditions in which the raptor protein interacts with the mTOR protein, with an agent to be assessed. The extent to which raptor interacts with mTOR in the presence of the agent to be assessed is determined, wherein if the extent to which raptor interacts with mTOR is altered in the presence of the agent compared to the extent to which raptor interacts with mTOR in the absence of the agent, then the agent alters interaction of a mammalian raptor protein with mTOR protein.

The present invention also relates to a method of identifying an agent that alters interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising contacting a host cell which comprises isolated nucleic acid that encodes a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 wherein the raptor protein, when expressed, interacts with mTOR protein in the cell, with an agent to be assessed. The growth rate and/or size of the host cell can then be assessed, wherein an altered growth rate and/or size of the host cell compared to growth rate and/or size of a control cell indicates that the agent alters interaction of a mammalian raptor protein with mTOR protein.

The present invention also relates to a method of identifying an agent that inhibits interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising contacting a host cell which comprises isolated nucleic acid that encodes a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 wherein the raptor protein, when expressed, interacts with mTOR protein in the cell, with an agent to be assessed. The growth rate and/or size of the host cell is then assessed, wherein a decrease in growth rate and/or size of the host cell compared to growth rate and/or size of a control cell indicates that the agent inhibits interaction of a mammalian raptor protein with mTOR protein. In one embodiment, the growth rate or size of the cell can be assessed by measuring phosphorylation of a regulator of protein synthesis selected from the group consisting of:

S6 kinase

1, 4E-BP1 and combinations thereof. In another embodiment, the growth rate or size is assessed by measuring binding of the mammalian raptor protein with mTOR protein.

The present invention also relates to a method of identifying an agent that enhances interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising contacting a host cell which comprises isolated nucleic acid that encodes a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 wherein the raptor protein, when expressed, interacts with mTOR protein in the cell, with an agent to be assessed. The growth rate and/or size of the host cell is then assessed, wherein an increase in growth rate and/or size of the host cell compared to growth rate and/or size of a control cell indicates that the agent enhances interaction of a mammalian raptor protein with mTOR protein.

The present invention provides a method of altering the growth and/or size of a cell comprising introducing into the cell an agent that alters interaction of raptor protein with mTOR protein. In one embodiment, the present invention provides a method of enhancing growth rate and/or size of a cell comprising introducing into the cell an agent that inhibits interaction of mammalian raptor protein with mammalian TOR protein. The agent can be, for example, exogenous nucleic acid that inhibits activity of a mammalian raptor protein. In one embodiment, the exogenous nucleic acid can result in overexpression of mammalian raptor protein in the cell. In one embodiment, the exogenous nucleic acid is mRNA that specifically targets and destroys the mammalian raptor protein. In a particular embodiment, the mRNA that specifically targets and destroys the mammalian raptor protein comprises pairs of oligoribonucleotides which correspond to nucleotides from about nucleotide 1531 to about nucleotide 1551 of SEQ ID NO: 1.

In another embodiment, the present invention provides a method of inhibiting growth rate and/or size of a cell comprising introducing into the cell an agent that enhances interaction of mammalian raptor protein with mammalian TOR protein. The agent can be, for example, exogenous nucleic acid that enhances activity of a mammalian raptor protein.

The present invention also relates to a method of altering kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that alters interaction of a mammalian raptor protein with the mTOR. In one embodiment, the invention relates to a method of enhancing kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that inhibits interaction of a mammalian raptor protein with the mTOR. In another embodiment, the invention relates to a method of inhibiting kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that enhances interaction of a mammalian raptor protein with the mTOR.

The present invention also relates to a method of treating cancer in an individual comprising administering to the individual an agent that enhances an interaction of raptor protein with mTOR protein.

Another aspect of the present invention is a method of identifying proteins that associate with mTOR within a cell comprising contacting a lysate of the cell with a reversible crosslinker (e.g., dithiobis(succinmidylpropionate) (DSP)) thereby forming a crosslink between proteins that associate with mTOR within the cell. Proteins that are associated by the crosslink are separated from the lysate, thereby identifying proteins that associate with mTOR within a cell. In one embodiment, the proteins that are associated by the crosslink are separated from the lysate using an antibody that specifically binds one of the proteins (e.g., mTOR). The method can further comprise reducing the crosslink between the proteins using a reducing agent (e.g., dithiothreitol) and isolating the proteins.

The present invention also relates to a method of identifying proteins that associate with mTOR within a cell, comprising preparing a lysate of the cell with a buffer comprising a detergent other than Triton (e.g., CHAPS), thereby preserving an association between the proteins. The proteins that are associated are then separated from the lysate, thereby identifying proteins that associate with mTOR within a cell. The method can further comprise isolating the proteins that are associated from one another. In one embodiment, the buffer comprises about 0.05% to about 2% CHAPS. In a particular embodiment, the buffer comprises about 0.3% CHAPS and can further comprise 120 mM NaCl. In one embodiment a crosslinker is not used.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [0027]
FIGS. [0028] 1A-1C are the nucleotide sequence (SEQ ID NO: 1) of human raptor.
FIG. 2 is the amino acid sequence (SEQ ID NO: 2) of human raptor. [0029]
FIGS. [0030] 3A-3C are gels which identify a 150 kDa mTOR-associated protein.
FIG. 3A is an autoradiograph of SDS-PAGE analysis of mTOR immunoprecipitates prepared from metabolically labeled HEK293T cells lysed in the absence (−) or presence (+) of the chemical cross-linker DSP, or from lysates treated with [0031] DSP 30 minutes after lysis (30′+). A 150 kDa protein is present only in immunoprecipitates prepared with an anti-mTOR antibody and not with antibodies recognizing the EGF receptor (EGFR), the 110 kDa catalytic subunit of PI-3-kinase (PI3K p110), the ribosomal S6 kinase (S6K1), actin, or lamin. Bands representing EGFR and PIK3 are visible on the autoradiograph, while S6K1, actin and lamin have run off the gel.
FIG. 3B is a silver stained SDS-PAGE analysis of mTOR immunoprecipitates isolated from cells lysed in the absence (−) or presence (+) of DSP. Quantitation of the bands corresponding to mTOR and p150 reveals a ratio of 1.0 mTOR to 0.84 p150. [0032]
FIG. 3C is a gel showing near stoichiometric amounts of mTOR and p150 in mTOR immunoprecipitates prepared from cells metabolically labeled to equilibrium. Quantitation of the bands correspond to mTOR and p150 reveals, after normalization to the methionine content of each protein, a ratio of 0.92 mTOR to 1.0 p150. [0033]
FIGS. [0034] 4A-4C show that raptor is an evolutionarily conserved protein and is widely expressed in human tissues in a similar pattern to mTOR.
FIG. 4A is a schematic representation of structural features of Raptor showing the conserved domain organization of raptor homologues from five eukaryotic species. Boxes labeled 1, 2, and 3 are sequence blocks that make up the Raptor N-terminal Conserved (RNC) domain and are 67-79% similar in the species shown. [0035] Block 2 of the S. cerevisiae raptor has a small insertion that makes it slightly larger than the same block in the other species. The seventh WD-40 repeat of C. elegans raptor is indicated with an empty green box because it lacks the prototypical Trp/Asp pattern. The accession numbers for the raptor homologues are: D. melanogaster (AAF46122), S. pombe (P87141), S. cerevisiae (P38873), C. elegans (T19183), A. thaliana (NP_—87497).
FIG. 4B is an amino acid sequence alignment of the RNC domain from the [0036] H. sapiens (SEQ ID NO: 3), D. melanogaster (SEQ ID NO: 4), S. pombe (SEQ ID NO: 5), S. cerevisiae (SEQ ID NO: 6), C. eleganns (SEQ ID NO: 7), and A. thanliana (SEQ ID NO: 8) species in FIG. 4A, showing identical (*), conserved (double dot) and partially conserved (single dot) residues. A ClustalX color scheme is used to depict characteristics of amino acids (Thompson et al., Nucleic Acids Res., 25:4876-4882, (1997)).
FIG. 4C is a northern blot analysis of a multiple human tissue blot (Clontech) for raptor, mTOR and β-actin showing similar patterns of expression for raptor and mTOR. [0037]
FIGS. [0038] 5A-5E show specific in vivo interactions between endogenous raptor and mTOR and recombinant versions of both proteins.
FIG. 5A is a western blot analysis showing levels of raptor (bottom panel) and mTOR (top panel) in immunoprecipitates prepared with two different mTOR antibodies or 5 control antibodies. mTOR Ab1 and Ab2 are anti-peptide antibodies produced against amino acids 1-18 and 221-237, respectively, of human mTOR. [0039]
FIG. 5B is a western blot showing that in the absence of the cross-linker, cell lysis conditions affect the mTOR-raptor association. Western blot showing levels of raptor (bottom panel) and mTOR (top panel) in mTOR immunoprecipitates from cells lysed in buffers containing the indicated concentrations of Triton X-100 or CHAPS. Increasing concentrations (100, 200, 300 mM) of NaCl in the lysis buffer dissociate raptor from mTOR. [0040]
FIG. 5C is a western blot showing that Raptor and mTOR associate in cell lines derived from several human tissues. mTOR immunoprecipitates prepared from lysates (1 mg total protein) of the indicated cell types were analyzed as in FIG. 5B. [0041]
FIG. 5D is a western blot showing that endogenous mTOR interacts with recombinant raptor but not γ-tubulin. Western blot showing levels of mTOR (top panel) and myc-raptor and myc-tubulin (bottom panel) in anti-myc immunoprecipitates from HEK293T cells transfected with 100 ng of either a myc-raptor- or a myc-γ-tubulin-encoding plasmid. [0042]
FIG. 5E is a western blot showing that recombinant mTOR and raptor interact in transfected HEK293T cells. Western blot showing levels of HA-raptor (top panel) and myc-mTOR and myc-γ-tubulin (middle panel) in anti-myc immunoprecipitates prepared from HEK293T cells transfected with 1 ug of a plasmid encoding HA-raptor and 100 ng of either a myc-raptor- or a myc-γ-tubulin-encoding plasmid. HA-raptor expression levels in the cell lysates used for immunoprecipitations (bottom panel). [0043]
FIGS. [0044] 6A-6B show identification of protein domains involved in the mTOR-raptor interaction.
FIG. 6A is a gel showing that Raptor interacts with the N-terminal portion of mTOR containing the HEAT repeats. Myc-tagged full-length mTOR, its indicated fragments or γ-tubulin were co-expressed in HEK293T cells with HA-raptor, and anti-myc immunoprecipitates analyzed by SDS-PAGE and anti-HA immunoblotting (top panel). Western blot showing amounts of mTOR, its fragments or γ-tubulin in the immunoprecipitates (middle panel). HA-raptor expression levels in the cell lysates used for the immunoprecipitations (bottom panel). [0045]
FIG. 6B is a schematic and a western blot showing the requirement of the complete raptor protein for the interaction with mTOR and identification of raptor mutants incapable of binding mTOR. Raptor fragments and location of raptor mutations used in this study are indicated in the schematic. Western blot analysis showing levels of mTOR (top panel) in anti-HA immunoprecipitates prepared from HEK293T cells transfected with 1 μg of plasmids encoding HA-tagged raptor, its fragments or the mutants shown in the schematic. Anti-HA western blot analysis showing levels of full-length raptor or fragments in the immunoprecipitates (bottom panel). [0046]
FIGS. [0047] 7A-7E show that Raptor participates in nutrient signaling to S6K1, maintenance of cell size and in cell growth.
FIG. 7A is a western blot showing that siRNA-induced reductions in raptor levels inhibit leucine-stimulated signaling to S6K1. HEK293T cells transfected with siRNAs targeting lamin, mTOR, or raptor, were incubated in leucine-free RPMI for 50 minutes. Cells were left unstimulated (first lane in each group of four) or stimulated for 10 minutes with increasing amounts of leucine (5.2, 16, and 52 μg/ml). Cell lysates were prepared and 20 μg of each analyzed by western blotting to determine levels of indicated proteins or protein phosphorylation states. P-S6K1 and P-PKB/Akt indicate S6K1 phosphorylated on T389 and PKB/Akt on S473, respectively. [0048]
FIG. 7B is a bar graph showing reductions in raptor or mTOR inhibit leucine-stimulated S6K1 phosphorylation and lower protein levels of both mTOR and raptor. The graph shows mean±S.D. derived from three independent experiments performed as in FIG. 5A and quantitated by densitometry. Table shows means±S.D. (n=4) of protein levels of mTOR, raptor, S6K1, PkB/Akt, and ATM determined by densitometry from immunoblots in FIG. 5A. [0049]
FIG. 7C is a western blot and graphs showing that in actively growing HEK293T cells, reductions in the levels of raptor or mTOR reduce the phosphorylation state of S6K1 and cell size. Cells were transfected with siRNAs and the cell lysates analyzed by western blotting for mTOR, raptor, phospo-S6K1, and S6K1. Cell diameters and volumes were determined three days after transfection using a particle size counter (Coulter Multisizer II). Graphs show size distributions of cells transfected with the indicated siRNAs or treated with 20 nM rapamycin for 48 hours (red lines) overlaid on size distribution of non-transfected cells (black line). The mean±S.D. (μm) cell diameters are: non-transfected cells 15.92±0.11 (n=5); lamin siRNA cells 15.89±0.10 (n=5); mTOR siRNA 15.43±0.09 (n=6); raptor siRNA 15.45±0.07 (n=6); rapamycin treated cells 14.61±0.10 (n=5). The mean±S.D. (pL) for the cell volumes are: non-transfected cells 2111±44 (n=5); lamin siRNA cells 2099±39 (n=5); mTOR siRNA 1922±33 (n=6); raptor siRNA 1930±26 (n=6); rapamycin treated cells 1632±33 (n=5). Mean±S.D. values were obtained from the indicated numbers of independent trials in which at least 10,000 cells were analyzed per trial. The reductions in size caused by the raptor and mTOR siRNA and by rapamycin are significant to p<0.001. [0050]
FIG. 7D is a graph showing transfection of HEK293T cells with siRNA targeting raptor or mTOR reduces the capacity of cells to attain a normal mean size after emerging from a confluence-induced shrinking in cell size. Cells transfected with the indicated siRNA were grown to confluence, induced into active growth by dilution into fresh media and cell volumes determined at 1, 2, and 3 days after dilution. For comparison, non-transfected cells were treated with 20 nM rapamycin at 1 day after dilution. Shown are mean±S.D. from three independent experiments. The reductions in size caused by the raptor and mTOR siRNA are significant to p<0.01 and 0.05 at the 2 and 3 day time points, respectively. [0051]
FIG. 7E is a graph showing that transfection of HEK293T cells with siRNA targeting raptor or mTOR or treatment with rapamycin inhibits leucine-induced increases in cell size. Cells were transfected with the indicated siRNAs and the experiment performed as described herein. The growth rates were estimated as 1.85, 1.06, 1.03, and 1.02% volume per hour for the cells transfected with the lamin, mTOR, and raptor siRNAs, and for cells treated with rapamycin, respectively. Shown are mean ±S.D. from samples for each time point. The reductions in size caused by the raptor and mTOR siRNAs and by rapamycin at 10 hours are significant to p<0.05. [0052]
FIGS. [0053] 8A-8E are gels showing that nutrients, mitochondrial function, and glycolysis regulate both the mTOR-raptor interaction and mTOR kinase activity.
FIG. 8A is a gel showing deprivation of amino acids, leucine, or glucose increases the stability of raptor-mTOR complex and decreases mTOR kinase activity, effects reversed by stimulation with leucine or glucose. HEK293T cells were incubated for 50 minutes in RPMI lacking amino acids (−amino acids), leucine (−leucine), or glucose (−glucose) or left untreated (control). Duplicate plates of cells deprived of leucine or glucose were stimulated for 10 minutes with 52 μg/ml leucine (−leu+leu) or 11 mM glucose (−gluc+gluc), respectively. Cells were lysed, anti-mTOR immunoprecipitates prepared, and mTOR kinase activity determined with in vitro kinase assays containing ATP-[γ-[0054] ³²P] and a GST-S6K1 fusion protein (Burnett et al., PNAS 95:1432-1437 (1998)). Kinase reactions were resolved by SDS-PAGE, proteins transferred to PVDF, ³²p-incorporation into GST-S6K1 detected with autoradiography (third panel from top) and levels of mTOR (top panel) and raptor (middle panel) determined by immunoblotting. Western blot analyses of cell lysates used to prepare mTOR immunoprecipitates showing effects of nutrient conditions on the phosphorylation state and gel mobility of S6K1 in vivo (bottom two panels).
FIG. 8B is a gel showing that the kinase activity of immunoprecipitates of endogenous mTOR depends on the presence of mTOR and is sensitive to 20 μM LY294002 (LY) but not to 40 nM Protein Kinase A Inhibitor (PKI), 20 μM H-8 (H8), and 20 μM PD98059 (PD). The kinase activity of recombinant, myc-tagged mTOR (wt) is absent in the D2357E mutant (kd) and is sensitive to 20 μM LY294002 (LY). [0055]
FIG. 8C is a gel showing that mitochondrial function, glycolysis and oxidative stress regulate the mTOR-raptor interaction and the mTOR kinase activity. Cells were treated for 10 minutes with 500 mM sucrose, 1 mM H[0056] ₂O₂, 1 μm valinomycin A, or 100 mM 2-deoxyglucose (2-DG) or left untreated (control). mTOR kinase activity, the amounts of mTOR and raptor in mTOR immunoprecipitates, and the in vivo phosphorylation states of S6K1 were analyzed as in FIG. 8A.
FIG. 8D is a gel showing that overexpression of wild-type raptor increases the amount of the stable mTOR-raptor complex, leading to decreases in the in vitro kinase activity of mTOR and in the in vivo phosphorylation state of S6K1. HEK293T cells were transfected with 5 μg of an empty vector (prk5), a mammalian expression vector encoding wild-type raptor (raptor wt) or a mutant incapable of interacting with mTOR (raptor mut 1). mTOR activity and levels of the mTOR-raptor interaction were analyzed as in FIG. 8A. Western analyses of cell lysates used to prepare mTOR immunoprecipitates shows the effects of overexpression of wild type raptor on the phosphorylation state and gel mobility of S6K1 and amounts of expressed wild-type and mutant raptor (bottom panel). [0057]
FIG. 8E is a gel showing that overexpression of wild-type raptor increases the amount of 4E-BP1 bound to eIF-4E. Experiments were performed as in FIG. 8D except the cells were also transfected with 50 ng of a plasmid encoding myc-4E-BP1. 7-methyl-GTP-affinity chromatography and eIF-4E western blotting were performed as described in Burnett et al., [0058] PNAS, 95:1432-1437 (1998).
FIGS. [0059] 9A-9C are gels that provide evidence that the mTOR-raptor complex exists in two binding states.
FIG. 9A is a gel showing that raptor amounts in mTOR immunoprecipitates prepared from cells grown in leucine-rich or -poor conditions are similar when cells are lysed in the presence of cross-linker DSP. [0060]
FIG. 9B is a gel showing that [0061] raptor mutant 5 forms an unstable complex with mTOR. Experiments were performed under nutrient-rich conditions as in FIG. 9A except that cells were transfected with 10 ng of the indicated raptor plasmid 24 hours before mTOR immunoprecipitations. Western blots show amounts of mTOR and indicated HA-raptors recovered in mTOR immunoprecipitates (top panels) prepared from cells lysed in the absence or presence of the cross-linker DSP and expression levels of the raptor mutants in the cell lysates (bottom panels).
FIG. 9C is a gel showing that the interactions of [0062] raptor mutants 4 and 5 with mTOR are not regulated by nutrients. HEK293T cells transfected with 10 ng of the indicated raptor plasmids were incubated for 50 minutes in leucine-free (−) or rich (+) media. The amounts of mTOR and HA-raptor in the mTOR immunoprecipitates were analyzed as in FIG. 9B (short exp). A longer exposure of the western blot was necessary to see that leucine levels do not regulate the residual interaction between raptor mutant 5 and mTOR (long exp).
FIGS. [0063] 10A-10C show that rapamycin destabilizes the mTOR-raptor interaction under either leucine-rich or poor conditions.
FIG. 10A is a gel showing that rapamycin added before or after cell lysis disrupts the mTOR-raptor interaction. HEK293 cells were treated for 10 min with 10 nM rapamycin, 10 nM FK506, or appropriate amounts of ethanol vehicle before cell lysis or with 10 nM rapamycin for 30 minutes after lysis. mTOR immunoprecipitates were obtained and the amounts of mTOR (top panel) and co-precipitating Raptor determined (bottom panel). The inclusion of the DSP cross-linker in the lysis buffer maintains the mTOR-raptor interaction even with rapamycin treatment. [0064]
FIG. 10B is a gel showing that rapamycin destabilizes the mTOR-raptor interaction in leucine-poor or rich conditions. HEK293 cells were treated with increasing concentrations of rapamycin (0, 2, 5, 10, 20, or 30 nM) for 20 min before (leftmost panels) or after (middle panels) incubation of cells for 30 minutes in leucine-free RPMI or after incubation for 30 minutes in leucine-rich RPMI (rightmost panels). The amount of raptor (bottom panels) coprecipitated with mTOR was quantitated, normalized to the amount obtained in the absence of rapamycin (first lane in each group) and plotted against the rapamycin concentration. The EC50 of rapamycin for dissociation of raptor from mTOR decreases from about 5 nM in the absence of leucine (irrespective of order of rapamycin addition or leucine deprivation) to about 1.5 nM in the presence of leucine (graph). [0065]
FIG. 10C is a model to explain effects of nutrients on the mTOR-raptor complex and the mTOR activity. It is propose that raptor makes a constitutive interaction with mTOR that provides a positive input to mTOR function (+) and a nutrient-sensitive interaction that inhibits the mTOR kinase activity (−). In the model, high nutrient conditions lead to the formation of the ‘unstable complex’ from the ‘stable complex’ by displacing the nutrient-sensitive interaction. [0066]
FIGS. [0067] 11A-11B show a sequence alignment of full-length sequences of raptor homologues H. sapiens (SEQ ID NO: 9), D. melanogaster (SEQ ID NO: 10), S. pombe (SEQ ID NO: 11), S. cerevisiae (SEQ ID NO: 12), C. eleganns (SEQ ID NO: 13), and A. thanliana (SEQ ID NO: 14). Sequences were aligned with ClustalX v1.81 (Thompson et al., Nucleic Acids Res. 25:4876-82 (1997)) using the Gonnet series weight matrix. Both pairwise gap opening and gap extension penalties were set at 10.00 and 0.10, respectively. Multiple alignment gap opening and gap extension penalties were set at 10.00 and 0.20. Black boxes frame regions of sequence similarity. Three sequence blocks were found to be common among all six sequences, as described in FIGS. 4A-4C. Three HEAT and seven WD40-like repeats were aligned between most of the sequences. However, alignment of several of the repeats did require minor manual adjustments.
FIG. 12 is a gel showing that transfections into the same cells of the siRNAs targeting both mTOR and raptor do not have additive effects on the phosphorylation state of S6K1. Experiment was performed and analyzed as in FIG. 7C except that each plate was transfected with equal amounts of the two indicated siRNAs. For the negative control twice the total amount of lamin siRNA was used (2× lamin). [0068]
FIG. 13 is a gel showing that stimulation with insulin (200 nM for 25 minutes) of HEK293T cells cultured in serum-free media for 24 hours does not affect the mTOR-raptor association but does increase the phosphorylation level of S6K1. The amounts of mTOR and raptor in mTOR immunoprecipitates, and the in vivo phosphorylation state of S6K1 were analyzed as in FIG. 8A. [0069]
FIG. 14 is a gel showing that cell lysis conditions that destabilize the mTOR-raptor interaction increase mTOR kinase activity. mTOR kinase activity was determined in mTOR immunoprecipitates prepared from cells lysed in unmodified Buffer B (see methods), or Buffer B with 200 mM NaCl (B w/NaCl), or with 0.5% Triton X-100 (B w/TX-100). Although the amounts of mTOR are the same in all the immunoprecipitates (top panel), there is an inverse correlation between the levels of raptor (middle panel) and mTOR kinase activity (bottom panel).[0070]

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery that in vivo mTOR exists as a complex with one or more regulatory subunits that are lost during isolation of mTOR for in vitro assays. As shown herein, several lines of evidence support the notion that mTOR associates with other proteins: mTOR contains HEAT repeats, which are known protein-protein interaction domains; it migrates at a disproportionately large apparent molecular weight (1.5-2.0 mDa) in gel filtration chromatography; and the expression in transfected cells of mTOR fragments lacking the catalytic site has dominant negative effects on the pathway. However, conventional biochemical attempts to purify mTOR-interacting proteins have been fruitless, since, as shown herein mTOR-containing complexes are unstable under standard purification conditions. [0071]
As described herein, a purification scheme that uses a reversible chemical cross-linker to stabilize putative mTOR-containing complexes was devised. Using this strategy, raptor (Regulatory Associated Protein of mTOR), a protein that is required for nutrient signaling to S6K1 and for control of cell growth, has been discovered. The mTOR-raptor interaction also regulates the kinase activity of mTOR and is sensitive to conditions, such as nutrient availability, that signal through the pathway. Thus, provided herein is a mechanism for how nutrients regulate mTOR activity in vivo. Although rapamycin has been generally regarded as mimicking the effects of nutrient deprivation, it was found that the two conditions have opposite effects on the mTOR-raptor interaction. [0072]
As shown herein, in vivo mTOR exists in a stoichiometric complex with raptor, a novel, evolutionarily conserved protein that plays at least two roles in the mTOR pathway. Raptor is required for mTOR protein expression, nutrient-stimulated signaling to the downstream effector S6K1, and maintenance of cell size. The association of raptor with mTOR also negatively regulates the mTOR kinase activity. Conditions that repress the pathway, such as nutrient deprivation and mitochondrial uncoupling, stabilize the mTOR-raptor association and inhibit the kinase activity. Overexpression of wild-type raptor mimics nutrient deprivation, causing the formation of a stable mTOR-raptor complex with decreased mTOR kinase activity. Thus, raptor is a missing component of the TOR pathway that through its association with mTOR adjusts the rate cell of growth to nutrient levels. [0073]
Accordingly, the present invention relates to isolated raptor nucleic acid molecules of mammalian origin (e.g., human, murine (rat, mouse), bovine, feline, canine) and complements, portions and variants thereof. The present invention also relates to isolated raptor polypeptides of mammalian origin and portions thereof, and antibodies or antigen binding fragments thereof that specifically bind a raptor polypeptide. The present invention also relates to constructs and host cells comprising the nucleic acid molecules described herein. In addition, the present invention relates to uses of the nucleic acid and polypeptide molecules provided herein. [0074]
In one embodiment, the present invention relates to an isolated nucleic acid sequence comprising SEQ ID NO: 1. In another embodiment, the isolated nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 2. The Genbank Accession number for the human raptor cDNA is AY090663 [0075]
As used herein “nucleic acid molecule” includes DNA (e.g., cDNA, genomic DNA, a gene), RNA (e.g., mRNA) and analogs thereof. The nucleic acid molecule can be single stranded or double stranded and can be the coding strand (sense strand) or the noncoding strand (antisense strand). The nucleic acid can include all or a portion of the coding strand and can further comprise additional non-coding sequences such as introns and non-coding 5′ and 3′ sequences (e.g., regulatory sequences). [0076]
An “isolated” nucleic acid molecule indicates that the nucleic acid molecule is in a form that is distinct from the form in which it occurs in nature. Isolated nucleic acid molecules of the present invention are separated from other nucleic acid molecules which are present in its natural state (e.g., free of sequences which normally flank the nucleic acid in the genome of the organism from which it is derived). In one embodiment, the isolated nucleic acid molecule is part of a composition (e.g., a crude extract). In another embodiment, the isolated nucleic acid molecule is substantially free from the cellular material in which it occurs, and in yet another embodiment, the isolated nucleic acid molecule is purified to homogeneity. Various methods, such as gel electrophoresis or chromatography can be used to identify nucleic acid molecules that are substantially free from cellular materials or purified to homogeneity. [0077]
A nucleic acid molecule of the present invention can be isolated using standard recombinant or chemical methods and the sequences provided herein. For example, using all or a portion of SEQ ID NO: 1 as a hybridization probe, a raptor sequence can be isolated using standard hybridization and cloning methods (Sambrook et al., eds., [0078] Molecular Cloning: A Laboratory Manual, 2^nded., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Speing Harbor, N.Y., 1989). A nucleic acid of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate primers according to standard polymerase chain reaction (PCR) methodology. The amplified nucleic acid can then be cloned into an appropriate vector and characterized using DNA sequence analysis. Raptor nucleic acids can also be prepared using, for example, an automated DNA synthesizer.
In another embodiment, the invention relates to an isolated nucleic acid molecule which is the complement of SEQ ID NO:1 or a portion thereof. A complement of SEQ ID NO: 1 is a sequence which is sufficiently complementary so that it hybridizes to SEQ ID NO: 1, thereby forming a stable duplex. In a particular embodiment, the complement hybridizes to SEQ ID NO: 1 and encodes a raptor polypeptide. [0079]
The nucleic acid molecule of the invention can comprise a portion of a nucleic acid sequence encoding raptor. In one embodiment, the portion is a fragment that can be used as a probe or primer. In another embodiment, the portion encodes a biologically active portion of a raptor protein. The portion of a nucleic acid sequence encoding raptor can include all or a portion of the raptor coding sequence and can further include non-coding sequences such as introns and 5′ and 3′ sequences (e.g., regulatory sequences). The nucleotide sequence determined from the cloning of the human raptor gene allows for the generation of probes and primers designed for use in identifying and/or cloning raptor homologues in other cell types, e.g., from other tissues, as well as raptor homologues from other mammals. The portion (e.g., probe/primer) can comprise a substantially purified raptor oligonucleotide. The portion is generally of a length and composition that hybridizes to all or a characteristic portion of a nucleic acid sequence under stringent conditions. The portion typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 10, and more particularly about 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 1000 contiguous nucleotides of the sense or anti-sense sequence of SEQ ID NO:1 or of a naturally occurring mutant of SEQ ID NO:1. [0080]
Probes based on the human raptor nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or identical proteins, or splice variants or polymorphisms of raptor. A label group (e.g., a radioisotope, a fluorescent compound, an enzyme) can be attached to the probe. Such probes can be used as a part of a diagnostic test kit to assess expression (e.g., aberrant expression) of a raptor protein in a cell or tissue sample by measuring a level of a raptor-encoding nucleic acid in a sample from an individual (e.g., detecting raptor mRNA levels or determining whether a genomic raptor gene has been mutated or deleted). For example, a nucleic acid fragment of the raptor nucleic acid sequence that can be used as a probe or primer includes the raptor N-terminal conserved (RNC) domain and/or one or more of the WD40 repeats. [0081]
A nucleic acid fragment encoding a “biologically active portion of raptor” can be prepared by isolating a portion of SEQ ID NO:1 which encodes a polypeptide having a raptor biological activity, expressing the encoded portion of raptor protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of raptor. A biologically active portion of raptor includes portion which, for example, interact with mTOR, participate in nutrient signaling, participate in maintenance of cell size and negatively regulate the mTOR kinase activity. [0082]
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO:1 due to degeneracy of the genetic code and thus encode the same raptor protein as that encoded by the nucleotide sequence shown in SEQ ID NO:1. For example, the present invention relates to nucleic acid sequence polymorphisms that lead to changes in the amino acid sequences of raptor which exist within a population (e.g., the human population). Such genetic polymorphism in the raptor gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a raptor polypeptide (e.g., a mammalian raptor polypeptide). Such nucleotide variations and resulting amino acid polymorphisms in raptor that are the result of natural allelic variation and that do not alter the functional activity of raptor are within the scope of the invention. [0083]
Moreover, nucleic acid molecules encoding raptor proteins from other species (raptor homologues), which have a nucleotide sequence which differs from that of a human raptor, are within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the raptor cDNA of the invention can be isolated based on their identity to the human raptor nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. In one embodiment, the nucleic acid molecule of the present invention comprises a nucleotide sequence that is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 or a complement thereof. [0084]
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000 or 1300 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, and in a particular embodiment the coding sequence, of SEQ ID NO:1 or the complement thereof. In one embodiment, the nucleic acid molecule hybridizes to the coding sequence of SEQ ID NO: 1. In a particular embodiment, the nucleic acid molecule hybridizes to SEQ ID NO: 1 and encodes a raptor polypeptide. [0085]
As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. In one embodiment, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:1 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to a nucleic acid molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). [0086]
In addition to naturally-occurring allelic variants of the raptor sequence that may exist in the population, it is known in the art that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO:1, thereby leading to changes in the amino acid sequence of the encoded raptor protein, without altering the functional (biological) ability of the raptor protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made. Alteration of a “non-essential” amino acid residue in the wild-type sequence of raptor (e.g., the sequence of SEQ ID NO:2) will not affect the biological activity of raptor. Conversely, an “essential” amino acid residue is required for biological activity of raptor. Therefore, alteration of an essential amino acid in the wild-type sequence of raptor will affect the biological activity of raptor. Amino acid residues that are conserved among the raptor proteins of various species will likely be essential amino acids. For example, as described herein, several mutations in the RNC or WD40 domains of raptor, generated by changing evolutionarily conserved residues (FIG. 6B), eliminated the interaction with mTOR, whereas other RNC domain mutants, as well as a mutation in the region between the HEAT and WD40 repeats, did not affect it. [0087]
In one embodiment, raptor proteins of the present invention, contain at least one RNC domain. Additionally, a raptor protein also contains at least one WD40 repeat. As shown herein, such conserved domains contain essential or conserved amino acids. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved among raptor of various species) are likley not essential for activity and thus can be altered without altering the biological activity of raptor. [0088]
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding raptor proteins that contain changes in amino acid residues that are not essential for activity. Such raptor proteins differ in amino acid sequence from SEQ ID NO:2 and retain raptor biological activity (e.g., interaction with mTOR, participation in nutrient signaling, participation in maintenance of cell size and negative regulation of the mTOR kinase activity). In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 45%, 50%, 60%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2. [0089]
An isolated nucleic acid molecule encoding a raptor protein having a sequence which differs from that of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of raptor nucleic acid molecule (SEQ ID NO:1) such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. A predicted nonessential amino acid residue in raptor is preferably replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a raptor coding sequence, and the resultant mutants can be screened for raptor biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using methods described herein. [0090]
In a preferred embodiment, a mutant Raptor protein can be assayed for the ability to interact with mTOR, participate in nutrient signaling, participate in maintaining cell size and negatively regulate the mTOR kinase activity [0091]
The present invention also encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a raptor polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA raptor molecule or complementary to an mRNA raptor sequence. The antisense nucleic acid can be complementary to an entire raptor coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding raptor. The noncoding regions (5′ and 3′ untranslated regions) are the 5′ and 3′ sequences which flank the coding region and are not translated into amino acids. The antisense nucleic acid molecule can be complementary to the entire coding region of raptor mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of raptor mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using procedures known in the art (e.g., using chemical synthesis and enzymatic ligation reactions). [0092]
The invention also relates to isolated raptor protein or polypeptides, and portions (e.g., biologically active portions) thereof. An “isolated” or “purified” (e.g., partially or substantially) polypeptide or biologically active portion thereof is in a form that is distinct from the form in which it occurs in nature. In one embodiment, the polypeptide is part of a composition (crude extract). In another embodiment, the polypeptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the raptor protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of raptor protein in which the protein is separated from cellular components of the cells from which it is isolated, recombinantly produced or chemically synthesized. Such preparations of raptor protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or non-raptor chemicals. Various methods, such as gel electrophoresis or chromatography can be used to identify polypeptides that are substantially free of cellular material. In one embodiment, the present invention relates to an isolated polypeptide encoded by a nucleic acid comprising SEQ ID NO:1. In another embodiment, the present invention relates to an isolated polypeptide having an amino acid sequence comprising SEQ ID NO: 2. [0093]
The present invention also relates to portions of a raptor polypeptide. In one embodiment, the portions are biologically active portions of a raptor protein and include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the raptor protein (e.g., the amino acid sequence shown in SEQ ID NO:2). Biologically active portions include a portion of the full length raptor proteins, and exhibit at least one activity of a raptor protein (e.g., interaction with mTOR, participation in nutrient signaling, participation in maintenance of cell size and negative regulation of the mTOR kinase activity). Typically, biologically active portions comprise one or more domains or regions with at least one activity of the raptor protein. A biologically active portion of a raptor protein can be a polypeptide which is, for example, at least about 10, 25, 50, 100 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length. Biologically active polypeptides include one or more identified raptor domains, e.g., RNC domain. Other biologically active portions can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native raptor protein. [0094]
Other raptor polypeptides of the present invention are substantially identical to SEQ ID NO:2, retain the functional activity of the protein of SEQ ID NO:2, yet differ in amino acid sequence due to natural allelic variation or mutagenesis. Raptor is involved in nutrient signaling and cell growth. Accordingly, a useful raptor polypeptide includes an amino acid sequence at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the raptor polypeptide of SEQ ID NO:2. In other instances, the raptor polypeptide has an [0095] amino acid sequence 55%, 65%, 75%, 85%, 95%, or 98% identical to the raptor RNC domain. In one embodiment, the raptor polypeptide retains a functional activity of the Raptor protein of SEQ ID NO:2.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes, wherein gaps are introduced in the sequences being compared. The amino acid residues at corresponding amino acid positions or nucleotides at corresponding nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % identity=# of identical positions/total # of positions×100). [0096]
As described herein, the determination of percent homology between two sequences can be accomplished using a mathematical algorithm. Examples of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1993) [0097] Proc. Nat'l Acad. Sci. USA 90: 5873-5877 and the algorithm incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).
Native raptor polypeptides can be isolated from cells or tissue sources using the purification schemes described herein. In one embodiment, the present invention relates to a method of identifying proteins that associate with mTOR within a cell comprising contacting a lysate of the cell with a reversible crosslinker (e.g., dithiobis(succinmidylpropionate) (DSP)) thereby forming a crosslink between proteins that associate within the cell. Proteins that are associated by the crosslink from the lysate are then separated, thereby identifying proteins that associate within a cell. The method can further comprising reducing the crosslink (e.g., using a reducing agent such as dithiothreitol ) between the proteins and isolating the proteins. In addition, the method can further comprise separating the proteins that are associated by the crosslink from the lysate using an antibody that specifically binds one of the proteins. [0098]
The present invention also relates to a method of identifying proteins that associate with mTOR within a cell, comprising preparing a lysate of the cell with a buffer comprising a detergent other than Triton, thereby preserving an association between the proteins. The proteins that are associated are separated from the lysate, thereby identifying proteins that associate within a cell under conditions in which a crosslinker is not used. The method can further comprising isolating the proteins that are associated from one another. In one embodiment, the buffer comprises CHAPS at a concentration from about 0.05% to about 2% CHAPS. In a particular embodiment, the buffer comprises about 0.3% CHAPS. In addition, the buffer can further comprise 120 mM NaCl. In another embodiment, the method of identifying proteins that associate with mTOR within a cell is performed without the use of a crosslinker. [0099]
The present invention also provides a method of producing raptor polypeptides using recombinant DNA techniques. Alternative to recombinant expression, a raptor protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques. [0100]
The invention also provides raptor chimeric or fusion proteins. As used herein, a raptor “chimeric protein” or “fusion protein” comprises a raptor polypeptide fused in-frame to an additional component (a non-raptor polypeptide). Within a raptor fusion protein, the raptor polypeptide can correspond to all or a portion of a raptor protein, preferably at least one biologically active portion of a raptor protein. The additional component can be fused to the N-terminus or C-terminus of the raptor polypeptide. An example of a fusion protein is a GST-raptor fusion protein in which the raptor sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant raptor. Another example of a fusion protein is a raptor-immunoglobulin fusion protein in which all or part of raptor is fused to sequences derived from a member of the immunoglobulin protein family. The raptor-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-raptor antibodies in a subject, to purify raptor ligands and in screening assays to identify molecules which inhibit the interaction of raptor with a raptor ligand (e.g., mTOR). [0101]
A raptor chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques (e.g., using blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion, and enzymatic ligation). In another embodiment, conventional techniques such as an automated DNA synthesizer can be used. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A raptor-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the raptor protein. [0102]
The present invention also pertains to variants of raptor proteins or polypeptides which function as either raptor agonists (mimetics) or as raptor antagonists. Variants of the raptor protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the raptor protein). [0103]
Variants of the raptor polypeptide which function as either raptor agonists or as raptor antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants of the raptor polypeptide for raptor polypeptide agonist or antagonist activity. There are a variety of methods which can be used to produce libraries of potential raptor variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes provides, in one mixture, of all of the sequences encoding the desired set of potential raptor sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) [0104] Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198: 056; Ike et al. (1983) Nucleic Acid Res. 11:477). Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property (e.g., a biased library).
The present invention also relates to an antibody or antigen binding fragment thereof that specifically binds to a mammalian raptor polypeptide. In one embodiment, the antibody or antigen binding fragment thereof specifically binds to mammalian raptor polypeptide encoded by an isolated nucleic acid that encodes SEQ ID NO: 2. In another embodiment, the antibody or antigen binding fragment thereof specifically binds to mammalian raptor polypeptide comprising SEQ ID NO: 2. An isolated raptor protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind raptor using standard techniques for polyclonal and monoclonal antibody preparation. The full-length raptor protein or antigenic peptide fragments of the raptor protein can be used as immunogens. For example, an antigenic peptide of raptor can comprise at least about 10, 12, 15, 20, 30, 50 or 100 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompass an epitope of raptor such that an antibody raised against the peptide forms a specific immune complex with raptor. Particular epitopes encompassed by the antigenic peptide are regions of raptor that are located on the surface of the protein, e.g., hydrophilic regions. [0105]
Generally, a suitable subject, (e.g., rabbit, goat, mouse, rat, hamster or other mammal) is immunized with a raptor immunogen to prepare antibodies or antigen binding fragments thereof that specifically bind raptor. The raptor immunogen can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic raptor preparation induces a polyclonal anti-raptor antibody response. [0106]
A molecule which specifically binds to raptor is a molecule which binds raptor, but does not substantially bind other molecules in a sample, e.g., a biological sample, which contains raptor. As used herein “antibody” includes full length antibodies or immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Immunologically active portions of immunoglobulin molecules include, for example, F(ab) and F(ab′)[0107] ₂fragments which can be generated by treating the antibody with an enzyme such as pepsin. The term “antibody” also includes polyclonal and monoclonal antibodies that bind raptor.
Polyclonal anti-raptor antibodies can be prepared as described above by immunizing a suitable subject with a raptor immunogen. The antibody molecules directed against raptor can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques (e.g., protein A chromatography). In addition, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) [0108] Nature 256:495-497. The technology for producing various antibodies monoclonal antibody hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.). A monoclonal anti-raptor antibody can also be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with raptor to thereby isolate immunoglobulin library members that bind raptor. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP.TM. Phage Display Kit, Catalog No.240612).
The term “antibody” also includes chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. [0109]
An anti-raptor antibody (e.g., monoclonal antibody) can be used to isolate raptor by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-raptor antibody can facilitate the purification of natural raptor from cells, recombinantly produced raptor expressed in host cells and chemically synthesized raptor. Moreover, an anti-raptor antibody can be used to detect raptor protein in a sample (e.g., in a cellular lysate or cell supernatant) and also to evaluate the quantity and expression pattern of the raptor protein. Anti-raptor antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure (e.g., to determine the efficacy of a given treatment regimen). A detectable substance or tag can be coupled to the antibody to facilitate detection. Examples of detectable substances include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. [0110]
The present invention also provides expression constructs (expression vectors) containing a nucleic acid encoding a raptor polypeptide or a portion thereof. Examples of vectors include plasmids and viral vector (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses). [0111]
The expression constructs of the invention comprise a raptor nucleic acid of the invention operably linked to one or more regulatory sequences. In one embodiment, the expression construct comprises SEQ ID NO: 1. The regulatory sequence is selected based on the vector and host cell used for expression of raptor. As used herein “operably linked” indicates that the raptor nucleic acid is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). As used herein, a “regulatory sequence” includes promoters, enhancers and other expression control elements such as polyadenylation signals which direct constitutive expression or tissue-specific expression of a nucleic acid. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). The vector used in the present invention depends on several factors such as the choice of the host cell to be transformed, the level of expression of protein desired, etc. When introduced into a host cell the vectors of the invention can be used to produce raptor proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., raptor proteins, mutant forms of raptor, fusion proteins). [0112]
The vectors of the invention can be designed for expression of raptor in prokaryotic or eukaryotic cells, e.g., bacterial cells such as [0113] E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. The vectors described herein can also comprise a nucleic acid molecule of the invention cloned into the vector in an antisense orientation.
Another aspect of the invention pertains to host cells into which an expression vector of the invention has been introduced (recombinant cells). In one embodiment, a host cell of the present invention comprises a nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 2. The term “host cell” refers to the particular subject cell and to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. [0114]
A host cell can be a prokaryotic or eukaryotic cell. For example, raptor protein can be expressed in bacterial cells (e.g., [0115] E. coli), insect cells, yeast cells or mammalian cells (e.g., Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells using a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell. For example, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation can be used. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals. [0116]
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. Optionally, a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the nucleic acid encoding raptor to identify and select cells that include the nucleic acid. Examples of selectable markers include G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding raptor or can be introduced on a separate vector. [0117]
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a (i.e., express) raptor polypeptide. Accordingly, the invention further provides methods for producing a raptor polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell comprising nucleic acid encoding a raptor polypeptide or portion thereof under conditions in which (e.g., in a suitable medium) raptor polypeptide is produced. In another embodiment, the method further comprises isolating raptor polypeptide from the medium or the host cell. The present invention also relates to the isolated raptor polypeptide. [0118]
The raptor nucleic acid molecules, raptor proteins, and anti-raptor antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. [0119]
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal, transdermal (topical), transmucosal, and rectal administration (e.g., suppositories). The pharmaceutical compositions of the present invention can also include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents; antioxidants; chelating agents; buffers and agents for the adjustment of tonicity. The can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [0120]
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol and polyol (e.g., glycerol, propylene glycol). In addition, a coating (e.g., lecithin) or a surfactant can be used. Antibacterial and antifungal agents, (e.g., thimerosal) can also be included. Moreover, sugars, polyalcohols and sodium chloride can be included in the pharmaceutical composition. An agent which delays absorption, for example, aluminum monostearate and gelatin can also be used. [0121]
Oral compositions can include an inert diluent or an edible carrier and can be in the form of capsules (e.g., gelatin), pills or tablets. The tablets, pills or capsules, can contain a binder, an excipient, a lubricant, a sweetening agent or a flavoring agent. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. [0122]
In one embodiment, the active compounds can be administered as a controlled release formulation, including implants and microencapsulated delivery systems (e.g., biodegradable, biocompatible polymers can be used). Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. [0123]
The dosage of the pharamceutical compositions of the invention depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. [0124]
The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. [0125]
The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in a variety of methods. [0126]
The isolated nucleic acid molecules of the invention can be used to express raptor protein (e.g., via a recombinant expression vector in a host cell), to detect raptor mRNA (e.g., in a biological sample) and to modulate raptor activity. In addition, the raptor proteins can be used to screen drugs or compounds which modulate the raptor activity or expression as well as to treat disorders characterized by a decreased or excessive production of raptor protein or production of raptor protein forms which have decreased or aberrant activity compared to raptor wild type protein. In addition, the anti-raptor antibodies of the invention can be used to detect and isolate raptor proteins and modulate raptor activity. This invention further pertains to novel agents identified by the above-described screening assays and their use for treatments as described herein. [0127]
Another aspect of the present invention relates to diagnostic assays for determining raptor polypeptide and/or nucleic acid expression as well as raptor activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant raptor expression or activity. The invention also provides for determining whether an individual is at risk of developing a disorder associated with raptor protein, nucleic acid expression or activity. For example, mutations in a raptor gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to prophylactically treat an individual prior to the onset of a disorder characterized by or associated with raptor protein, nucleic acid expression or activity (e.g., cancer). [0128]
The present invention also pertains to a method for detecting the presence or absence of raptor in a sample (e.g., a biological sample) comprising contacting a sample with a compound or an agent capable of detecting raptor protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes raptor protein such that the presence of raptor is detected in the sample. The method can further comprise obtaining the sample. In one embodiment, a labeled nucleic acid sequence (probe) capable of hybridizing to raptor mRNA or genomic DNA is used to detect raptor nucleic acid (e.g., mRNA or genomic DNA). The nucleic acid sequence can be, for example, a full-length raptor nucleic acid, such as the nucleic acid of SEQ ID NO: 1 or a portion thereof, such as an oligonucleotide of at least about 10, 20, 30, 50, 100, 350, 500, 1000 or 2000 nucleotides in length and sufficient to specifically hybridize under stringent conditions to raptor nucleic acid. Other suitable probes for use in the diagnostic assays of the invention are described herein. [0129]
In another embodiment, an antibody, preferably an antibody with a detectable label, capable of binding to raptor protein or a characteristic portion thereof is used. Thus, the present invention also provides a method of identifying a mammalian raptor polypeptide in a sample comprising contacting the sample with an antibody or antigen binding fragment thereof that specifically binds to a mammalian raptor polypeptide wherein the mammalian raptor polypeptide is encoded by an isolated nucleic acid that encodes SEQ ID NO: 2. The polypeptide which specifically binds to the antibody is identified, thereby identifying a mammalian raptor polypeptide in a sample. [0130]
Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)[0131] ₂) can be used. Examples of detectable labels include a fluorescently labeled secondary antibody, and biotin such that it can be detected with fluorescently labeled streptavidin.
A “sample” includes biological samples such as tissues, cells and biological fluids of a subject which contain raptor protein molecules, mRNA molecules or genomic DNA molecules from the test subject. The detection method of the invention can be used to detect raptor mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of raptor mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of raptor protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of raptor genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of raptor protein include introducing into a subject a labeled anti-raptor antibody, wherein the antibody is labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. [0132]
In another embodiment, the methods further involve obtaining a control sample from a control subject, contacting the control sample with a compound or agent capable of detecting raptor protein, mRNA, or genomic DNA, such that the presence of raptor protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of raptor protein, mRNA or genomic DNA in the control sample with the presence of raptor protein, mRNA or genomic DNA in the test sample. [0133]
The invention also encompasses kits for detecting the presence of raptor in a sample. Such kits can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of Raptor (e.g., a proliferative cell disorder such as cancer). For example, the kit can comprise a labeled compound or agent capable of detecting raptor protein or mRNA in a sample and means for determining the amount of raptor in the sample (e.g., an anti-raptor antibody or an oligonucleotide probe which binds to DNA encoding Raptor such as SEQ ID NO: 1). Kits may also include instruction for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of raptor if the amount of raptor protein or mRNA is above or below a normal level. [0134]
For antibody-based kits, the kit may comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to raptor protein; and, optionally, (2) a second, different antibody which binds to raptor protein or the first antibody and is conjugated to a detectable agent. For oligonucleotide-based kits, the kit may comprise, for example: (1) a oligonucleotide, e.g., a detectably labelled oligonucleotide, which hybridizes to a raptor nucleic acid sequence or (2) a pair of primers useful for amplifying a raptor nucleic acid molecule. [0135]
The kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit may also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit may also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of raptor. [0136]
The invention provides a method (also referred to herein as a “screening assay”) for identifying agents that alter raptor expression and/or activity. For example, such agents (modulators) include candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules such as small organic molecules or other drugs) which bind to a raptor polypeptide and/or inhibit or enhance (partially, completely) raptor expression or raptor activity. In one embodiment, the ability of an agent to alter raptor expression and/or activity is accomplished by determining the ability of the agent to alter the activity of (e.g., interaction of) raptor with a raptor target molecule. As used herein, a “target molecule” is a molecule with which a raptor protein binds to or interacts with in nature. In one embodiment, a raptor target molecule is mTOR. Thus, the present invention relates to a method of identifying an agent that alters interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising contacting a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 with mTOR protein under conditions in which the raptor protein interacts with the mTOR protein, with an agent to be assessed. The extent to which raptor interacts with mTOR in the presence of the agent to be assessed is determined, wherein if the extent to which raptor interacts with mTOR is altered in the presence of the agent compared to the extent to which raptor interacts with mTOR in the absence of the agent, then the agent alters interaction of a mammalian raptor protein with mTOR protein. [0137]
Determining the ability of the raptor protein to bind to or interact with a raptor target molecule can be accomplished by methods which detect binding directly or indirectly. In one embodiment, determining the ability of the raptor protein to bind to or interact with a raptor target molecule can be accomplished by directly detecting the binding of raptor to the target molecule using, for example, one or more antibodies to detect raptor and/or its target molecule, or gel electrophoresis. In another embodiment, determining the ability of the raptor protein to bind to or interact with a raptor target molecule can be accomplished by determining the activity of raptor and/or the target molecule. For example, the activity of raptor or a raptor target molecule such as mTOR, can be determined by detecting interaction of raptor and mTOR, the ability of mTOR to participate in nutrient signaling, the ability of mTOR to participate in maintenance of cell size and the ability of raptor to negatively regulate the mTOR kinase activity. [0138]
In one embodiment, the method comprises contacting a raptor protein or biologically active portion thereof with an agent and determining the ability of the agent to bind to the raptor protein or biologically active portion thereof. Binding of the test compound to the raptor protein can be determined either directly or indirectly. In one embodiment, the assay includes contacting the raptor protein or biologically active portion thereof with a raptor target molecule which binds raptor (e.g., mTOR) to form an assay mixture; contacting the assay mixture with an agent; and determining the ability of the agent to interact with a raptor protein. In this embodiment, the ability of the agent to interact with a raptor protein comprises comparing the extent to which the agent binds to raptor or a biologically active portion thereof, to the extent to which mTOR binds to raptor or a biologically active portion thereof. If raptor preferentially binds the agent as compared to mTOR, then the agent alters raptor expression and/or activity. [0139]
In the screening methods of the present invention, the raptor or its target molecule can be immobilized to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of an agent to raptor, or interaction of raptor with a target molecule in the presence and absence of an agent to be assessed, can be accomplished using, for example, microtitre plates, test tubes, and micro-centrifuge tubes. Examples of methods for immobilizing proteins on matrices include the use of glutathione-S-transferase/raptor fusion proteins or glutathione-S-transferase/target fusion proteins adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates and the use biotin and streptavidin conjugation. [0140]
In another embodiment, modulators of raptor expression are identified in a method in which a cell is contacted with an agent and the expression of raptor mRNA or protein in the cell is determined. The level of expression of raptor mRNA or protein in the presence of the agent is compared to the level of expression of raptor mRNA or protein in the absence of the agent. The agent can then be identified as a modulator of raptor expression based on this comparison. For example, when expression of raptor mRNA or protein is greater in the presence of the agent than in its absence, the candidate compound is identified as a stimulator of raptor mRNA or protein expression. Alternatively, when expression of raptor mRNA or protein is less in the presence of the agent than in its absence, the candidate compound is identified as an inhibitor of raptor mRNA or protein expression. The level of raptor mRNA or protein expression in the cells can be determined by methods described herein for detecting raptor mRNA or protein. [0141]
In another embodiment, the present invention relates to a method of identifying an agent that alters interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising contacting a cell (e.g., a host cell) which comprises nucleic acid that encodes a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 wherein the raptor protein, when expressed, interacts with mTOR protein in the cell, with an agent to be assessed. The growth rate and/or size of the cell is then determined, wherein an altered growth rate and/or size of the cell compared to growth rate and/or size of a control cell indicates that the agent alters interaction of a mammalian raptor protein with mTOR protein. In one embodiment, the present invention relates to a method of identifying an agent that inhibits an interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising contacting a cell which comprises nucleic acid that encodes a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 wherein the raptor protein, when expressed, interacts with mTOR protein in the cell, with an agent to be assessed. The growth rate and/or size of the cell is assessed, wherein a decrease in growth rate or size of the cell compared to growth rate and/or size of a control cell indicates that the agent inhibits interaction of a mammalian raptor protein with mTOR protein. [0142]
In another embodiment, the invention relates to a method of identifying an agent that enhances an interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising contacting a cell which comprises nucleic acid that encodes a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 wherein the raptor protein, when expressed, interacts with mTOR protein in the cell, with an agent to be assessed. The growth rate and/or size of the cell is assessed, wherein an increase in growth rate and/or size of the cell compared to growth rate and/or size of a control cell indicates that the agent enhances interaction of a mammalian raptor protein with mTOR protein. [0143]
In the methods of the present invention, the growth rate and/or size can be assessed by measuring phosphorylation of a regulator of protein synthesis (e.g., [0144] S6 kinase 1, 4E-BP1 and combinations thereof). The growth rate and/or size can also be assessed by measuring binding of the mammalian raptor protein with mTOR protein.
As shown herein, raptor forms a nutrient sensitive complex with mTOR and is necessary for the activity of the mTOR pathway. Specifically, it has been shown that two interactions exist between raptor and mTOR: a ‘constitutive’ interaction that is required for in vivo mTOR function and a ‘nutrient sensitive’ interaction that forms in the absence of nutrients and negatively regulates mTOR kinase activity. As also shown herein, growing cells transfected with interfering RNA (siRNA) targeting raptor were reduced in size. [0145]
Accordingly, the present invention provides a method of altering the growth and/or size of a cell comprising introducing into the cell an agent that alters interaction of raptor protein with mTOR protein. [0146]
In one embodiment, the present invention provides a method of enhancing growth rate and/or size of a cell comprising introducing into the cell an agent that inhibits interaction of mammalian raptor protein with mammalian TOR protein. The agent can be, for example, exogenous nucleic acid that inhibits activity of a mammalian raptor protein. In one embodiment, the exogenous nucleic acid results in overexpression of mammalian raptor protein in the cell. The exogenous nucleic acid can be, for example, mRNA that specifically targets and destroys the mammalian raptor protein (e.g., siRNA), such as mRNA comprising pairs of oligoribonucleotides which correspond to nucleotides from about nucleotide 1531 to about nucleotide 1551 of SEQ IDNO: 1. [0147]
In another embodiment, the present invention provides a method of inhibiting growth rate and/or size of a cell comprising introducing into the cell an agent that enhances interaction of mammalian raptor protein with mammalian TOR protein. The agent can be, for example, exogenous nucleic acid that enhances activity of a mammalian raptor protein. [0148]
The present invention also provides a method of altering in vivo activity of mTOR protein in a cell comprising introducing into the cell an agent that alters an interaction of a mammalian raptor protein with the mTOR. In one embodiment, the invention relates to a method of enhancing in vivo activity of mTOR protein in a cell comprising introducing into the cell an agent that inhibits interaction of a mammalian raptor protein with the mTOR. In another embodiment, the invention relates to a method of inhibiting in vivo activity of mTOR protein in a cell comprising introducing into the cell an agent that enhances constitutive interaction of a mammalian raptor protein with mTOR. [0149]
The present invention also relates to a method of altering kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that alters interaction of a mammalian raptor protein with the mTOR. In one embodiment, the invention relates to a method of enhancing kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that inhibits interaction of a mammalian raptor protein with the mTOR. In another embodiment, the invention relates to a method of inhibiting kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that enhances interaction of a mammalian raptor protein with the mTOR. [0150]
The present invention also provides for prophylactic and therapeutic methods of treating a subject at risk of or susceptible to a disorder or having a disorder associated with aberrant raptor and/or mTOR expression or activity. In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant raptor expression or activity, by administering to the subject an agent which alters raptor expression or at least one raptor activity. Subjects at risk for a disease which is caused or contributed to by aberrant raptor expression or activity can be identified by, for example, any of a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the raptor aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of raptor aberrancy, for example, a raptor agonist or raptor antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. [0151]
Another aspect of the invention pertains to methods of modulating raptor expression or activity for therapeutic purposes. The method of the invention involves contacting a cell with an agent that alters one or more of the activities of raptor protein activity associated with the cell. An agent that alters raptor protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a raptor protein, a peptide, a raptor peptidomimetic, or other small molecule (e.g., small organic molecule). In one embodiment, the agent stimulates one or more of the biological activities of raptor protein. Examples of such stimulatory agents include active raptor protein and a nucleic acid molecule encoding raptor that has been introduced into the cell. In another embodiment, the agent inhibits one or more of the biological activities of raptor protein. Examples of such inhibitory agents include antisense raptor nucleic acid molecules and anti-raptor antibodies. These methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a raptor protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) raptor expression or activity. In another embodiment, the method involves administering a raptor protein or nucleic acid molecule as therapy to compensate for reduced or aberrant raptor expression or activity. [0152]
Stimulation of raptor activity is desirable in situations in which raptor is abnormally downregulated and/or in which increased raptor activity is likely to have a beneficial effect. Conversely, inhibition of raptor activity is desirable in situations in which raptor is abnormally upregulated, e.g., in cancer, and/or in which decreased raptor activity is likely to have a beneficial effect. [0153]
In a particular embodiment, the present invention relates to a method of treating cancer in an individual comprising administering to the individual an agent that enhances interaction of raptor protein with mTOR protein. [0154]

EXEMPLIFICATION

Materials and Methods

Materials [0155]
Reagents were obtained from the following sources: DSP and Protein G-Sepharose from Pierce; methionine-L-[[0156] ³⁵S] and ATP-[γ-³²P] from NEN; mTOR, EGFR, S6K1, actin, lamin, and PI3-K antibodies as well as HRP-labeled anti-mouse, anti-goat and anti-rabbit secondary antibodies from Santa Cruz Biotechnology; Phospho-T389 S6K1 and Phospho-S473 PKB/Akt antibodies from Cell Signaling; HA monoclonal antibody from Covance; myc monoclonal antibody from Oncogene Research Products; myc rabbit polyclonal antibody from Upstate Biotechnology; eIF-4E antibody from Transduction Laboratories; DMEM, leucine, glucose, RPMI, and RPMI without amino acids, leucine, or glucose, from Life Technologies; and rapamycin, FK506, valinomycin, antimycin A, and 2-deoxyglucose from Calbiochem. Rabbit polyclonal anti-peptide antibodies recognizing mTOR (amino acids 221-237) and human raptor (amino acids 985-1001) were produced using the regular antibody service from Zymed.
Crosslinking Assay and Immunoprecipitations [0157]
3 million HEK293T cells growing in 6 cm dishes in DMEM with 10% dialyzed fetal calf serum were metabolically labeled by the addition of 0.4 mCi of [[0158] ³⁵S] methionine for 2 hours. Cells were rinsed once with PBS and lysed in 300 μl of ice-cold Buffer A (40 mM Hepes pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na₃Vo₄, 1% Triton X-100, and one tablet EDTA-free protease inhibitors (Roche) per 10 ml) with or without 2.5 mg/ml DSP and incubated for 30 min on ice. Crosslinking reactions were quenched by adding 75 μl 1M Tris-HCl pH 7.4 followed by an additional 30 min incubation. After clearing the lysates by centrifugation at 10,000×g for 10 min, 30 μl of a 50% slurry of protein G-Sepharose and 4 μg of the anti-mTOR antibody or control antibodies were added to the supernatant. After a 3 hour incubation at 4° C., immunoprecipitates were washed once each with Tris-buffered saline containing 0.05% Tween 20, Wash Buffer 1 (50 mM Hepes pH 7.5, 40 mM NaCl, and 2 mM EDTA) with 1% Triton X-100, Wash Buffer 1 with 500 mM LiCl and 0.5% Triton X-100, Wash Buffer 1 with 500 mM LiCl, and Wash Buffer 2 (50 mM Hepes, pH 7.5, and 150 mM NaCl). 5× sample buffer (0.242 M Tris, 10% SDS, 25% glycerol, 0.5M dithiothreitol and bromophenol blue) was added to washed immunoprecipitates and incubated for 1 h at 37° C. to reduce the DSP crosslinking. Samples were resolved by 3-8% SDS-PAGE, proteins transferred to PVDF and the blot exposed to film. Cross-linking experiment using unlabeled cells were analyzed by silver staining of gels.
Unless otherwise stated, for mTOR immunoprecipitates prepared in the absence of the cross-linker, cells were lysed in ice-cold Buffer B (Buffer A in which Triton X-100 is replaced by 0.3% CHAPS) and immunoprecipitates were washed four times in Buffer B and twice in [0159] Wash Buffer 2.
Protein Sequencing of Raptor [0160]
mTOR immunoprecipitates prepared from 200 million HEK293T cells were prepared as above, resolved by SDS-PAGE, and proteins visualized by Coomassie blue staining. The band corresponding to raptor was excised and trypsinized as described (Erdjument-Bromage et al., [0161] Protein Sci 3:2435-46 (1994)). A hundred percent of the generated peptides were subjected to a micro-clean-up procedure using 2 μL bed-volume of Poros 50 R2 (PerSeptive) reversed-phase beads packed in an Eppendorf gel-loading tip. Mass spectrometry (MALDI-ReTOF) was then carried out on two peptide pools (16 and 30% MeCN) recovered from the RP-microtip column using a Bruker REFLEX III instrument with delayed extraction. For mass fingerprinting, top major experimental masses (m/z) combined from both MALDI-ReTOF experiments were used to search a non-redundant human protein database (NR; ˜66,605 entries; NCBI; Bethesda, Md.), using the PeptideSearch (M. Mann, University of Southern Denmark) algorithm. A molecular weight range twice the predicted weight was covered with a mass accuracy restriction better than 40 ppm, and maximum one missed cleavage site was allowed per peptide. Alternatively, mass spectrometric-based sequencing (ESI-MS/MS) of selected peptides from partially fractionated pools was carried out using a PE-SCIEX API300 triple quadrupole instrument, fitted with a continuous flow nano-electrospray source (JaFIS). All peptide masses in pools were obtained by DE-MALDI-reTOF MS (BRUKER Reflex III). Peptide sequences were obtained by nano-electrospray tandem MS (JaFIS® source with SCIEX API300 triple quadrupole).
Cloning of the Raptor cDNA, DNA Manipulations and Mutagenesis [0162]
The cDNA for the KIAA1303 protein was obtained from the Kazusa DNA Research Institute. It encodes amino acids 200-1335 of raptor downstream of 126 bases of unknown origin that are not present in the EST databases and likely represent unspliced intronic sequence. To identify the full-length raptor sequence, EST databases with bases 127-500 of KIAA1303 were searched and cDNAs that extended the sequence in the 5′ direction were identified. The 5′ sequences of these cDNAs were used to search the EST databases for cDNAs that further extended the 5′ end of the raptor mRNA and this process was repeated until no additional cDNAs were found. The sequences obtained in this fashion allowed us to design PCR primers and amplify the 5′ end of raptor using first strand cDNA prepared from human BJAB cell RNA as template. PCR products were subcloned into the pCRII vector using a T/A cloning kit (Invitrogen) and sequenced. To create the 7051 base pair cDNA for the full length raptor mRNA, the PCR fragments and KIAA1303 were assembled in pBluescript SK-II(+) (Stratagene) using restriction sites found in overlapping regions. For expression in mammalian cells, the entire raptor open reading frame was subcloned into the myc- and HA-prk5 vectors (Sabatini et al., [0163] Science 284:1161-4 (1999)).
The raptor and mTOR fragments indicated in FIGS. [0164] 6A-6B were expressed from cDNAs subcloned into the HA- and/or myc-prk5 vectors. The raptor open reading frame in pBluescript SK-II(+) was mutagenized using the QuikChange mutagenesis kit (Stratagene) and subcloned into the Sal 1 and Not 1 sites of HA-prK5. Mutants used in this study are: raptor mut1 (₁₉₄YDC₁₉₆-AAA), mut2 (₂₆₁DLF₂₆₃-AAA), mut3 (₃₁₃NWIF₃₁₆-AAAA), mut4 (₃₉₁SQ₃₉₂-PA), mut 5 (₄₇₃FPY₄₇₅-AAA), mut 7 (₇₃₈SLQN₇₄₁-PAAA), and mut 9 (₁₁₉₁RVYDRR₁₁₉₆-DAAADD).
Sequence Analysis and Alignments [0165]
Unaligned sequences of raptor homologues were submitted to the MEME server (Multiple Em for Motif Elicitation v3.0) (Bailey and Elkan, [0166] Proc. Int. Conf. Intell. Syst. Mol. Biol. 2:28-36 (1994)) at http://meme.sdsc.edu/meme/website/ to identify blocks of similar sequence between the proteins. Three blocks (1, 2, and 3) of similar sequence were identified in the N-terminal half of all the homologues and collectively named the RNC (Raptor N-terminal Conserved) domain. The sequences were then examined for the presence of common protein motifs using a Pfam v6.6 fragment search (Sonnhammer et al., Proteins, 28:405-420 (1997)) at http://pfam.wustl.edu/ and three HEAT-like and seven WD40-like repeats were found in each protein. The sequences of the raptor homologues were also aligned with ClustalX (v1.81) (Thompson et al., Nucleic Acids Res. 25:4876-82 (1997)) using the Gonnet series weight matrix. Pairwise gap opening and gap extension penalties were set at 10.00 and 0.10, respectively. Multiple alignment gap opening and gap extension penalties were set at 10.00 and 0.20, respectively. The ClustalX alignment recapitulated the results of the MEME search, highlighting the three blocks of conserved identity and similarity corresponding to the RNC domain. In addition, the HEAT-like and WD40-like repeats were also aligned. Secondary structure prediction (Rost and Sander, J. Mol. Biol. 232:584-99 (1993)) was performed at http://cubic.bioc.columbia.edu.
Plasmid and siRNA Transfections [0167]
3 million HEK293T cells in 6-cm dishes were transfected with plasmid constructs and amounts indicated in the Figure legends using the Lipofectamine 2000 transfection reagent (Life Technology). 24 hours after DNA addition, cells were rinsed once with PBS and lysed in 300 μl of ice-cold Buffer B. Immune complexes were prepared from cleared supernatants using 3 μg polyclonal anti-myc or monoclonal anti-HA antibodies and 20 μl of a 50% slurry protein G-Sepharose. After a 3 hour incubation, immuneprecipitates were washed six times with Buffer B and twice with [0168] Wash Buffer 2. Bound proteins were eluted in 1× sample buffer, and mTOR or HA- or myc-tagged proteins were detected by immunoblotting as described (Burnett et al., PNAS 95:1432-143 (1998)).
For the siRNA experiments, 21-nucleotide complementary RNAs with symmetrical 2-nucleotide overhangs were obtained from Dharmacon (Boulder, Colo.) and designed to target the following regions of the open reading frames: bases 1531-1551 or 3374-3394 of raptor, bases 2241-2261 of human mTOR (FRAP), and bases 608-630 of human lamin. Oligonucleotides were annealed into duplexes as described (Elbashir et al., [0169] Embo J. 20:6877-88 (2001)) and transfected at 40 riM with Effectene (Qiagen) into HEK293T cells cultured in Optimem media (Life Technology) without serum. Unless otherwise described, 48 hours after adding siRNAs, the medium was replaced with DMEM containing 10% serum, and cells were cultured for a further 24 hours before use in experiments. mRNA levels of raptor and mTOR in siRNA transfected cells were determined by quantitative RT-PCR using a SYBR green assay as described (Alfonso et al., J. Neurosci. Res. 67:225-34 (2002)).
Cell Size Determinations [0170]
For the regrowth after confluence experiment, HEK293T cells were transfected with siRNAs as described above and grown to confluence in a 6-cm culture dish for 24 hours. Cells were then harvested, diluted 1:10 or 1:20 and replated in fresh media. At 1, 2, and 3 days after replating, the cells were harvested by trypsinization in 2 ml media, diluted 1:20 with counting solution (Isoflow Sheath Fluid, Coulter Corp.) and cell diameters and volumes determined using a partilce size counter (Coulter Multisizer II). Cells between 11 and 21 μm in diameter were used for analysis. For the leucine-induced growth experiment, the siRNA transfected cells were diluted into fresh media one day after transfection and, at 24 hours after dilution the cells were deproved of leucine for a further 24 hours. The cells were then provided with leucine (52 μg/ml) and cell diameters, volumes, and numbers determined as above at 6 and 10 hours after the leucine supplementation. [0171]
In Vitro Kinase Assay for mTOR Activity [0172]
Kinase assays were performed in a volume of 10 μl at 30° C. for 20 min and contained ¼ the washed mTOR immunoprecipitates from 10 million HEK293T cells growing on a 10-cm dish, 200 ng of a GST-S6K1 fusion protein (amino acids 332-502), 1 μCi of [γ-[0173] ³²P]ATP, 25 mM Hepes-KOH, pH 7.4, 50 mM KCl, 20% glycerol, 10 mM MgCl₂, 4 mM MnCl₂, 1 mM DTT, and 50 μM unlabeled ATP. When inhibitors were used they were added to the mTOR immunoprecipitates 10 minutes prior to the start of the kinase assay. Reactions were stopped by the addition of 5 μl of 2× sample buffer. After boiling the samples for 3 min, proteins were resolved by 8% SDS-PAGE, transferred to PVDF (poly(vinylidene difluoride)), and phosphorylated proteins visualized with autoradiography. The same blots were then used to determine the levels of mTOR and raptor by western analysis. Kinase assays using recombinant mTOR were performed as described in Burnett et al., PNAS, 95:1432-1437 (1998), except that they were washed as above with Buffer B.

Results

Identification of Raptor as an mTOR-Associated Protein [0174]
HEK 293T cells were metabolically labeled with [[0175] ³⁵S]-methionine and lysed in the presence or absence of the reversible cross-linker dithiobis(succinimidylpropionate) (DSP). mTOR immunoprecipitates prepared from these lysates were then treated with dithiothreitol to reduce the DSP cross-links and analyzed by SDS-PAGE and autoradiography. A protein with an apparent molecular weight of 150 kDa co-precipitated with mTOR only in immunoprecipitates recovered from lysates prepared in the presence of DSP (FIG. 3A). The intensity of this band decreased significantly when DSP was added 30 minutes after cell lysis, indicating that the protein interacts weakly or transiently with mTOR or that the lysis procedure disrupts the association (FIG. 3A). Immunoprecipitates prepared using antibodies against five other proteins did not co-precipitate the 150-kDa protein, indicating that it specifically interacts with mTOR. For reasons that will become apparent, the 150 kDa protein was designated raptor (Regulatory Associated Protein of mTOR). Raptor was also detected in silver (FIG. 3B) or Coomasie blue stained gels of mTOR immunoprecipitates prepared from DSP-treated lysates of unlabelled cells. Quantitation of the amount of raptor in these gels indicated that it is present in near stoichiometric amounts with mTOR. Furthermore, once sequence information allowed normalization of radioactive band intensities to the methionine content of raptor and mTOR, near stoichiometric amounts of the two proteins were found in complexes isolated from cells metabolically labeled to equilibrium (FIG. 3C).
Raptor is an Evolutionarily Conserved Protein with a Novel N-Terminal Domain and Seven WD40 Repeats [0176]
To characterize raptor sequence information was obtained by mass spectrometry and three peptides (SVSSYGNIR (SEQ ID NO: 15), LDDQIFLNR (SEQ ID NO: 16), and IPEEHDLESQIR (SEQ ID NO: 17)), all of which are contained within KIAA1303 (NCBI # 7242961), a partial ORF for a human protein of unknown function, were identified. Using the BLAST homology search algorithm, it was found that KIAA1303 likely represents an N-terminally truncated fragment of the human homologue of fission yeast Mip1p. The function of Mip1 is not clear, but, interestingly, it has been implicated in cell growth and nitrogen sensing (Shinozaki-Yabana et al., [0177] Mol. Cell Biol. 20:1234-42 (2000)), processes regulated by TOR proteins in a variety of organisms. By searching EST databases, overlapping cDNAs that extended the 5′ end of the KIAA1303 cDNA were identified and sequence information that allowed the design of PCR primers and assembly of a full-length cDNA for human raptor were provided. The 7051 nucleotide mRNA contains an open reading frame encoding a protein of 1335 amino acids (predicted molecular weight of 149 kDa) that is flanked by 5′ and 3′ untranslated regions of 0.9 and 2.1 kb, respectively. Human genome sequence information for the raptor locus permitted localization of the gene to human chromosome 17q25.3.
Raptor shows a high degree of conservation amongst all eukaryotes with completed genome projects, including [0178] D. melanogaster, S. pombe, S. cerevisiae, C. elegans, and A. thaliana (FIG. 4A and FIGS. 11A-11B). The raptor homologues in budding (Winzeler et al., Science 285:901-6. (1999)) and fission (Shinozaki-Yabana et al., Mol. Cell Biol. 20:1234-42 (2000)) yeast are encoded by essential genes. All raptor homologues have a novel N-terminal domain that we have named RNC (for Raptor N-terminal Conserved) domain, and consists of three highly conserved sequence blocks that share at least 67% similarity amongst raptor homologues (FIG. 4B). The RNC domain is unrelated to other sequences in the public databases and is predicted to have a high propensity to form a-helixes. Following the RNC domain, all raptor homologues have three HEAT repeats (Andrade and Bork, Nat. Genet. 11:115-6 (1995)), with highly conserved aspartate and arginine residues at motif positions 19 and 25, respectively. The HEAT repeats are followed by seven WD40 repeats in the C-terminal third of the protein (FIG. 4A). Both HEAT and WD-40 repeats are common protein-protein interaction motifs found in many eukaryotic regulatory proteins (Kobe et al., Structure Fold Des. 7:R91-7. (1999); Smith et al., Trends Biochem. Sci. 24:181-5 (1999)). Northern blot analysis shows that raptor is expressed in all human tissues in a pattern similar to that of mTOR, with the greatest levels of both mRNAs found in skeletal muscle, brain, kidney, and placenta (FIG. 4C). This indicates that both proteins are coordinately expressed in different tissues.
Specific mTOR-Raptor Interaction in Vivo [0179]
To characterize the mTOR-raptor interaction, a polyclonal antibody that specifically recognizes amino acids 985 to 1001 of human raptor was generated. This antibody detected raptor in immunoprecipitates prepared from DSP-treated cell lysates using two different anti-mTOR antibodies but not in immunoprecipitates obtained with five control antibodies incapable of recognizing mTOR (FIG. 5A). To enable a study of the physiological regulation of the mTOR-raptor interaction lysis conditions that might preserve the complex in the absence of the chemical cross-linker were investigated. This was achieved by avoiding in the lysis buffer Triton X-100, a detergent which eliminated the interaction at all concentrations tested (FIG. 5B). Lysis of cells in buffers containing the detergent CHAPS instead of Triton X-100 preserved the interaction with the greatest amount of co-precipitating raptor obtained at 0.3% CHAPS. Interestingly, the majority of in vitro studies on mTOR function have been performed on mTOR isolated from cells lysed with Triton X-100 or the related detergent NP-40, conditions that were found to completely disassociate raptor from mTOR. All further experiments were performed using a lysis buffer (Buffer B, see methods) containing 0.3% CHAPS and 120 mM NaCl, conditions that allowed the recovery of a complex containing a ratio of about 1.0 mTOR to about 0.7 raptor. In addition to HEK293T cells, the mTOR-raptor complex was detected in all other human cell lines tested, including the uterine cancer HeLa, B-cell lymphoma BJAB, neuroblastoma SK-N-MC, and lung cancer A549 derived cell lines (FIG. 5C), indicating that the association likely occurs in most cell types. In transfected HEK293T cells the interaction of epitope-tagged raptor with endogenous mTOR (FIG. 5D) as well as between epitope-tagged versions of both proteins (FIG. 5E) were detected. [0180]
The mTOR-Raptor Interaction Requires the mTOR HEAT Repeats and Multiple Sites on Raptor [0181]
To identify which region(s) of mTOR interacts with raptor, several myc-tagged mTOR fragments together with the full-length HA-tagged raptor protein were expressed in HEK293T cells. The N-terminal region of mTOR ([0182] amino acids 1 to 1482) containing all the HEAT motifs interacted with raptor almost as strongly as the full length protein, whereas the C-terminal region (amino acids 1348-2549) exhibited a weak but greater than background interaction (FIG. 6A). Further shortening of the N-terminal region of mTOR by 297 residues severely weakened the interaction and fragments of the N-terminal region of mTOR (amino acids 21-551 or 652-1185) were incapable of interacting with raptor (FIG. 6A). These results indicate that the overall structure of the N-terminal portion of mTOR is required for the association with raptor and that the C-terminal half of mTOR contains a weaker interaction site.
Whether individual domains of recombinant HA-tagged raptor expressed in HEK293T cells could interact with the endogenous mTOR was also tested (FIG. 6B). None of the raptor fragments bound to mTOR, indicating that the overall structure of raptor is required for the interaction. Several mutations in the RNC or WD40 domains of raptor, generated by changing evolutionarily conserved residues (FIG. 6B), eliminated the interaction with mTOR, whereas another RNC domain mutation (mut 4), as well as a mutation in the region between the HEAT and WD40 repeats (mut 7), did not affect it. These observations indicate that the mTOR-raptor interaction involves multiple sites in raptor and a large region of mTOR and, therefore, indicate that both proteins make extensive contacts with each other. Alternatively, the mutations in raptor could destabilize its entire structure and, thus, disturb the mTOR-raptor association without directly being part of the mTOR-interaction site. Interestingly, ATR, a protein which like mTOR is a member of the PIK-related family of kinases, makes extensive contacts with ATRIP (ATR-Interacting Protein), a recently discovered interacting partner of ATR (Cortez et al., [0183] Science 294:1713-6 (2001)).
Raptor Participates in Nutrient Signaling and Maintenance of Cell Size [0184]
A major role of the mTOR pathway is to coordinate the synthesis of ribosomal proteins with the levels of available amino acids. This is accomplished by controlling the translational regulator S6K1, a kinase whose phosphorylation state and in vivo activity are regulated by amino acid levels (Fox et al., [0185] American Journal of physiology Cell Physiology 2 74:43-1(1998); Hara et al., J Biol. Chem. 273:14484-94. (1998)) and is phosphorylated in vitro by mTOR (Burnett et al., PNAS 95:1432-1437. (1998); Isotani et al., J. Biol. Chem. 274:34493-8. (1999)). To investigate the role of raptor in mTOR-mediated signaling, small interfering RNA (siRNA) (Elbashir et al., Nature 411:494-8 (2001)) were used to decrease endogenous levels of raptor in HEK293T cells and the phosphorylation state of S6K1 in response to stimulation with increasing concentrations of leucine was measured. Consistent with a critical role for raptor in nutrient signaling to S6K1, decreased levels of raptor attenuated leucine-stimulated phosphorylation of S6K1 to a similar extent as decreased levels of mTOR achieved with an siRNA specific to mTOR (FIGS. 7A and 7B). After stimulation with the highest concentration of leucine the cells with reduced amounts of raptor or mTOR had only about 30% of the level of S6K1 phosphorylation as the cells transfected with the control siRNA (FIG. 7B). Reductions in the levels of raptor or mTOR did not significantly affect the amounts of S6K1 and ATM, or the phosphorylation state or amount of PKB/Akt, a downstream effector of PI 3-Kinase (FIGS. 7A and 7B). Interestingly, mTOR and raptor expression appear to be coordinately regulated because reduced levels of either protein induced by its specific siRNA also decreased the level of the other without affecting the amount of its mRNA. The inhibitory effect on S6K1 signaling of the raptor-targeted siRNA is not simply due to its decrease of mTOR levels. In control experiments using the mTOR-targeted siRNA, it was found that to observe any effect on S6K1 phosphorylation mTOR had to be reduced to less than 25% its normal level, a far higher reduction than caused by the raptor-targeted siRNA (FIG. 7B). The mutual dependence of expression observed between mTOR and raptor is another similarity the partners share with the ATR-ATRIP interacting pair (Cortez et al., Science 294:1713-6 (2001)).
In Drosophila, the TOR pathway is a major regulator of cell growth and, thus, cell size (Oldham et al., [0186] Genes Dev. 14:2689-94 (2000); Zhang et al., Genes Dev. 14: 2712-24. (2000)). A large part of this regulation is exerted through S6K1, and, in Drosophila and mice, loss of function mutations in S6K1 lead to smaller animals with smaller cells (Montagne et al., Science 285:2126-9 (1999); Shima et al., Embo J. 17:6649-59 (1998)). A role for mTOR in regulating cell growth can also be demonstrated in tissue culture, as inhibition of the pathway with rapamycin treatment reduces the size of many mammalian cell types, including HEK293Ts (FIG. 7C). Consistent with a role for raptor in growth control, it was found that actively growing cells transfected with siRNAs targeting raptor or mTOR underwent comparable reductions in size and that these correlated with a decrease in the phosphorylation state of S6K1 (FIG. 7C). The siRNA-mediated reductions in cell size are highly significant (p<0.001) but less than the reduction caused by treatment of cells with rapamycin for 48 hours. This is expected because rapamycin completely inhibits S6K1 phosphorylation in all of the cells, while the raptor and mTOR siRNAs partially inhibit S6K1 only in transfected cells.
Raptor Participates in Cell Growth [0187]
HEK293T cells grown to confluence in a tissue culture dish become smaller than actively growing cells and, after dilution and plating into fresh media, regain their normal mean size over a period of three days. The effects of reducing raptor and mTOR levels on the capacity of cells to increase in mean size after emerging from confluence were examined (FIG. 7D). Cells were transfected with siTNAs targeting lamin, mTOR or raptor and plated at high density so that they reached confluence within 24 hours. The confluent cells were harvested, diluted into fresh media, replated and their mean size measured for three days (FIG. 7D). As the cells transfected with the lamin siRNA emerged from confluence they gradually increased in mean size over a period of three days, an effect that was inhibited in the cells transfected with the mTOR or raptor siRNAs and dramatically reduced by rapamycin. [0188]
The effects of reducing raptor and mTOR levels on the capacity of cells, in the absence of proliferation, to grow after a reduction in size caused by prolonged nutrient tdeprivation were also examined. Cells transfected with siRNAs targeting lamin, mTOR or raptor and, 24 hours after transfection, cells were incubated in leucine-free media for an additional 24 hours. During this period of leucine deprivation the cells ceased to divide and became extremely small so that, irrespective of transfected siRNA, the mean cell volume in all the samples was about 67% of the mean volume of cells in leucine-containing media (FIG. 7E). Leucine was then added to the media and cell size measured at 6 and 10 hours after leucine addition. The growth of cells transfected with the mTOR or raptor siRNAs or treated with rapamycin at the same time of leucine addition was impaired as these cells increased in size significantly less than those transfected with the lamin siRNA. During this period of growth the cells did not divide and resumption of cell proliferation did not begin until 15-20 hours after leucine addition. [0189]
As described above, reductions in raptor and mTOR protein levels have similar effects on nutrient-stimulated phosphorylation of S6K1, cell size, and cell growth. In addition, it was found that mTOR and raptor exist in a near stoichiometric complex (FIGS. [0190] 3A-3C). An efficient explanation for these findings is that raptor and mTOR are part of the same signaling system that regulates S6K1 phosphorylation. Formally, however, it is possible that mTOR and raptor are in independent pathways that both converge on nutrient-stimulated S6K1 phosphorylation. If this was the case, it would be expected that reducing both raptor and mTOR levels in the same cells would have additive inhibitory effects on the nutrient-stimulated S6K1 phosphorylation because two independent pathways would not be affected. It was found that in cells co-transfected with siRNAs targeting both raptor and mTOR S6K1 phosphorylation is reduced to a similar extent in cells transfected with either the mTOR or raptor siRNA (FIG. 12).
This finding supports mTOR and raptor being part of the same nutrient-regulated growth pathway. [0191]
Nutrients and Mitochondrial Function Regulate the Stability of the mTOR-Raptor Complex and the mTOR Kinase Activity [0192]
To determine if the mTOR-raptor interaction is regulated by conditions that are known to affect the activity of downstream effectors of mTOR, the effects of different nutrient conditions on the stability of the mTOR-raptor complex were tested. The amount of raptor recovered bound to mTOR was markedly increased when HEK293T cells were incubated in amino acid deprived medium, an effect that was mimicked by the removal of just leucine from the culture medium (FIG. 8A). More importantly, a ten-minute stimulation with leucine, which activates the phosphorylation of S6K1 (FIGS. [0193] 7A-7B and lower panels of FIG. 8A), reversed the effect of leucine deprivation and restored the interaction to the level observed in cells grown in nutrient-rich medium. Furthermore, the in vitro kinase activity of mTOR towards S6K1 inversely correlated with the amount of raptor recovered with mTOR. Greater activity was observed in raptor-deprived complexes obtained after nutrient stimulation. Conversely, reduced activity was observed in raptor-enriched complexes obtained after nutrient starvation (FIG. 8A). It was also found that glucose deprivation and re-addition affected the mTOR-raptor interaction and mTOR activity in the same way as did changes in leucine levels (FIG. 8A).
Although mild buffers were used to rpepare the mTOR immunoprecipitates (in order to preserve the raptor-mTOR association), control kinase assays confirm that the observed activity is that of mTOR and not of a contaminating kinase that also might be capable of phosphorylating S6K1 (FIG. 8B). The kinase activity in the mTOR immunoprecipitates depends on the presence of mTOR and is sensitive to LY294002, a known inhibitor of mTOR (Brunn et al., [0194] Science, 277:99-101 (1997)), but not to high concentrations of PKI (an inhibitor of PKA), H-8 (an inhibitor of PKA, PKG, and PKC) and PD98059 (an inhibitor of MEK) (FIG. 8B top panel). Moreover, kinase assays performed on recombinant wild-type and kinase-dead mTOR isolated under mild buffer conditions show that the phosphorylation of S6K1 depends on a wild-type mTOR kinase domain and is sensitive to LY294002 (FIG. 8B, bottom panel). Thus, these findings show that, when mTOR is isolated under conditions designed to preserve its interaction with raptor, the activity of the isolated kinase does change in concert with the in vivo effects of stimuli that regulate the pathway. Moreover, two different nutrients, leucine and glucose, similarly affect the mTOR-raptor interaction, indicating that its regulation is a common event downstream of diverse nutrient signals.
The identity of the intracellular messengers that signal nutrient availability to mTOR is unknown, but several lines of evidence suggest that the mitochondrial metabolism of nutrients is necessary to activate the pathway (Dennis et al., [0195] Science 294:1102-5. (2001); Xu et al., Diabetes 50:353-60. (2001)). It was found that valinomycin, a mitochondrial uncoupler (Bernard and Cockrell, Biochim. Biophys. Acta, 548:173-186 (1979)); antimycin A, an electron transport inhibitor (Wolvetang, E. J., et al., FEBS Letters, 339:40-44 (1994)); and 2-deoxyglucose, a glycolytic inhibitor, stabilized the mTOR-raptor interaction and inhibited mTOR kinase activity in a similar fashion as nutrient deprivation (FIG. 8C). Other cell stressing conditions known to inhibit S6K1 in vivo, such as the oxidative stress caused by H₂O₂treatment, which affects mitochondrial function (Majumder et al., Cell Growth Differ. 12:465-70. (2001)), also stabilized the interaction and inhibited the mTOR kinase (FIG. 8C). On the other hand, a sucrose-induced osmotic shock decreased the phosphorylation state of S6K1 but had only a modest effect on complex stability and kinase activity (FIG. 8C). In addition to nutrients and cell stress, growth factors such as insulin are also known to regulate downstream components of the mTOR pathway, like S6K1 (Lawrence and Brunn, Prog. Mol. Subcell. Biol., 26:1-31 (2001). However, it was found that while treatment of serum-starved cells with insulin increased the phosphorylation state of S6K1, it did not affect the raptor-mTOR interaction (FIG. 13), indicating that its regulation is independent of growth factor signaling.
Increasing the Amount of Raptor Bound to mTOR Leads to an Inhibition of the mTOR Kinase Activity [0196]
The inverse correlation between the stability of the mTOR-raptor interaction and the kinase activity of mTOR indicates that a strong association between raptor and mTOR leads to an inhibition the mTOR catalytic activity. To substantiate this correlation, the activity of mTOR isolated under cell lysis conditions that differentially affect the mTOR-raptor association was determined (mutant 1, FIG. 14). A small increase in salt concentration in the lysis buffer slightly reduced the amount of raptor bound to mTOR and this correlated with a corresponding increase in mTOR activity. Strikingly, the addition of Triton X-100 to the lysis buffer completely eliminated the interaction and also strongly activated in vitro mTOR activity. [0197]
To exclude the possibility that the different isolation conditions directly affect mTOR activity, whether an increase in the intracellular concentration of raptor could drive, even in cells growing in nutrient-rich conditions, the formation of stable mTOR-raptor complexes and inhibit mTOR kinase activity was determined. Overexpression of wild-type raptor, but not of a mutant that cannot interact with mTOR ([0198] mutant 1, FIGS. 6A-6B), resulted in an increased amount of raptor bound to mTOR and a decrease in its in vitro kinase activity (FIG. 8D). In addition, the overexpression of wild type, but not mutant raptor, decreased the in vivo phosphorylation state of S6K1 and increased the amount of 4E-BP1 bound to eIF-4E (FIGS. 8D and 8E), providing in vivo correlates of the inhibitory effects of raptor on the in vitro mTOR kinase activity. These results strongly indicate that a function of a tightly bound raptor leads to a decrease mTOR kinase activity and that raptor overexpression can circumvent the normal nutrient-regulated mechanism(s) that control the strength of the interaction.
Evidence that the mTOR-Raptor Complex Exists in Two Binding States [0199]
Despite the negative role of raptor in regulating the nTOR kinase activity, the experiments using the siRNA indicate that in vivo raptor also has a positive function in the mTOR pathway as decreases in raptor levels reduce S6K1 phosphorylation, cell size, rate of cell growth, and mTOR expression (FIGS. [0200] 7A-7E). Moreover, when a cross-linker was present during the lysis of cells raptor was isolated in a stiochiometric complex with mTOR (FIGS. 3A-3C). Thus, it is unlikely that the decrease amount of raptor recovered with mTOR isolated from cells grown in nutrient-rich conditions (FIG. 8A) reflects a dissociation of the mTOR-raptor complexes in vivo. This was proven to be the case, as the amounts of raptor recovered with mTOR isolated from leucine deprived or stimulated cells were similar when cells were lysed in the presence of the chemical cross-linker (FIG. 9A).
These findings show that in vivo raptor and mTOR physically interact under all nutrient conditions and indicate that mTOR-raptor complexes exist in at least two nutrient-determined states with differential stability: an unstable complex that does not survive in vitro isolation and a stable complex that does. As nutrient-rich conditions decreased the amount of raptor recovered with mTOR, it is likely that nutrients lead to the formation of the unstable complex. Evidence for these two binding states was sought by asking if any of the raptor mutants identified, formed complexes with mTOR that were permanently in the unstable or stable state. A mutant that forms an unstable complex with mTOR was searched for by screening the mutants for those that are recovered with mTOR only when cells are lysed in the presence of a cross-linker (FIG. 9B). Only one mutant (mutant 5) had this characteristic, indicating that in [0201] vivo mutant 5 does asscociate with mTOR but that the interaction does not survive the in vitro isolation conditions. As expected, the small residual interaction of mutant 5 with mTOR was no longer sensitive to levels of leucine in the media (FIG. 9C). Next, a mutant that forms a stable complex with mTOR irrespective of nutrient conditions was searched for. Of the two raptor mutants (mutants 4 and 7) that associate with mTOR in the absence of a cross-linker (FIG. 9B), the interaction of mutant 4 was not sensitive to leucine levels while that of mutant 7 was still regulated (FIG. 9C). These findings indicate that mutants 5 and 4 form complexes similar to those found under nutrient-rich and -poor conditions, respectively. Complexes containing mutant 5 are in the unstable state characteristic of nutrient-rich conditions while those containing mutant 4 are in the stable state characteristic of nutrient-poor conditions.
Rapamycin Severely Weakens the mTOR-Raptor Interaction [0202]
In vivo, rapamycin, like nutrient deprivation, inhibits the activation of downstream effectors of mTOR, but exactly how the drug perturbs mTOR function is unknown. It was found that treatment of HEK293T cells with rapamycin or the addition of the drug to cell lysates significantly destabilized the mTOR-raptor complex (FIG. 10A). The effect was specific to rapamycin, as FK506, an immunosuppressant that also binds FKBP12 but does not target mTOR, had no effect on the interaction, nor did ethanol, the vehicle used for both drugs. However, when lysates were prepared in the presence of the cross-linker almost normal levels of raptor were recovered with mTOR (FIG. 10A). Therefore, as with nutrient stimulation and [0203] raptor mutant 5, rapamycin destabilizes the raptor-mTOR complex but does not abolish it in vivo.
The effect of increasing concentrations of rapamycin on the mTOR-raptor interaction in cells deprived of or stimulated with leucine was investigated in order to understand the inhibitory mechansim of rapamycin. Interestingly, irrespective of nutrient conditions, rapamycin dominantly destabilized the raptor-mTOR complex (FIG. 10B). The order of rapamycin addition and leucine deprivation did not matter as the drug had similar effects when it was added before or after leucine withdrawal. In addition, leucine-rich conditions enhanced the destabilizing effects of rapamycin, decreasing its EC50 for mTOR-raptor dissociation by about 3-fold when compared to leucine-poor conditions. A simple explanation for this latter result is that leucine-poor conditions lead to a decrease in the affinity between mTOR and FKBP12-rapamycin, likely by inducing a conformational change in the FKBP12-rapamycin binding site in mTOR. Alternatively, leucine deprivation likely increases the affinity between raptor and mTOR so that the complex is less susceptible to the destabilizing effects of FKBP12-rapamycin. The finding that the unstable and stable complexes found in nutrient-rich and poor conditions, respectively, are differentially sensitive to rapamycin provides further evidence that the raptor-mTOR complex can exist in two-binding states. [0204]

Discussion

Raptor Forms a Nutrient Sensitive Complex (NSC) with mTOR [0205]
Described herein is raptor, a 149-kDa protein that participates in the mTOR pathway and associates in a near stoichiometric ratio with mTOR to form a nutrient-sensitive complex (NSC). It was found that the strength of the association between raptor and mTOR modulates the kinase activity of mTOR and is controlled by cellular conditions known to regulate S6K1, such as nutrient availability, mitochondrial function and cell stress. The mTOR-raptor complex may have escaped prior detection because it is unstable under commonly used cell lysis solutions and is particularly sensitive to the detergent Triton X-100. Its identification was first made possible by stabilization of the association through the use of a reversible chemical cross-linker. [0206]
Raptor and mTOR associate under all cellular conditions but the stability of the complex changes with the activity of the pathway. Under nutrient-poor conditions the mTOR-raptor association is strong and high levels of the complexes are recovered even when cell lysates are prepared without the cross-linker. On the other hand, in nutrient-rich conditions, the association is weak and most of the complex readily falls apart in the absence of the cross-linker. These findings are consistent with a model in which mTOR and raptor are held together in a constitutive, easily disrupted association, which, under nutrient-poor conditions, is strengthened by an additional interaction(s) that also represses the kinase activity of mTOR. Thus, it is likely that at least two interactions exist between raptor and mTOR: a ‘constitutive’ interaction that is required for in vivo mTOR function and a ‘nutrient sensitive’ interaction that forms in the absence of nutrients and negatively regulates mTOR kinase activity (FIG. 10C). In support of this model, it was found that two raptor mutants associate with mTOR in ways that indicate that they have selective defects in the nutrient-sensitive interaction described above. The behavior of [0207] raptor mutant 5 is consistent with it having lost the nutrient-sensitive interaction and it appears to associate with mTOR only through the constitutive interaction. The behavior of rator mutant 4 is consistent with it retaining both interactions but with the nutrient-sensitive interaction no longer being regulated by nutrients.
The molecular mechanisms by which raptor regulates mTOR function are unknown. The nutrient sensitive mTOR-raptor interaction could decrease the catalytic activity of mTOR by inducing a conformational change in the mTOR kinase domain (mechanism shown in FIG. 10C) or by sterically preventing substrates from accessing the mTOR active site. On the other hand, the constitutive interaction is clearly not required for in vitro kinase activity, since, as shown herein, raptor-depleted mTOR strongly phosphorylates S6K1. Previous studies have shown that to function in vivo mTOR requires more than its kinase activity because truncation and point mutants of mTOR that retain wild-type kinase activity cannot signal to S6K1 within mammalian cells (Brown et al., [0208] Nature 377:441-446 (1995); Sabatini et al., Science 284:1161-4. (1999)). The deletion and mutagenesis studies performed under nutrient-rich conditions and described herein, show that raptor makes extensive contacts with mTOR regions that are far from its kinase domain. Thus, it is likely that in vivo the constitutive mTOR-raptor interaction is required for a function other than mTOR kinase activity. A simple possibility, supported by the finding that a reduction in raptor levels also reduces mTOR levels, is that raptor is required for the proper folding and/or stability of mTOR. Raptor could also serve as an adaptor that brings substrates to the mTOR kinase domain and/or may be a determinant of the proper subcellular localization of mTOR.
Rapamycin Destabilizes the NSC Irrespective of Nutrient Conditions [0209]
Although rapamycin and nutrient deprivation similarly inhibit the activity of downstream components of the mTOR pathway, such as S6K and 4E-BP1, it was found that they have opposite effects on the mTOR-raptor interaction. Rapamycin destabilizes the interaction regardless of nutrient availability, and its potency for dissociation is increased under nutrient-rich conditions. [0210]
Several mechanisms can be proposed to account for the effects of FKBP12-rapamycin on the stability of the complex and to explain how the drug inhibits the pathway. Two appealing models will be considered here. In the first, FKBP12-rapamycin dislodges raptor from its nutrient-sensitive binding site on mTOR because it binds to mTOR at or near that site. By replacing raptor at this site, FKBP12-rapamycin would mimic raptor's inhibitory effect on the mTOR kinase that is manifested under nutrient poor conditions. A prediction of this model is that in vitro FKBP12-rapamycin should inhibit mTOR kinase activity, a result seen in many studies (Brown et al., [0211] Nature 377:441-446 (1995); Brunn et al., Nature 377:441-446 (1997); Brunn et al., Embo J. 15:5256-67 (1996); Burnett et al., PNAS 95:1432-1437. (1998); Isotani et al., J. Biol. Chem. 274:34493-8. (1999)). In the second model, FKBP12-rapamycin does not affect the nutrient-sensitive interaction but interferes with the constitutive mTOR-raptor interaction. Because this is a positive interaction required for the in vivo function of mTOR, its interference by FKBP12-rapamycin inhibits the pathway. Of course, it is also possible that rapamycin exerts its negative effects on the pathway independently of its perturbation of the mTOR-raptor complex, perhaps, as has been recently suggested (Fang et al., Science 294:1942-5 (2001)), by preventing the action of a small signaling molecule on mTOR.
Diverse Signals Converge on the Regulation of the NSC [0212]
How might changes in levels of diverse nutrients regulate the strength of the NSC association? Certainly, mTOR and/or raptor could be phosphorylated or modified by upstream ‘nutrient sensors’ that regulate their interaction. However, an alternate hypothesis is one in which the mTOR-raptor complex is itself the nutrient sensor. In this scenario, one or more intracellular molecules increase in concentration in nutrient-rich conditions and bind to raptor and/or mTOR, destabilizing the NSC and relieving raptor inhibition of the mTOR kinase (FIG. 10C). If a single molecular species sufficed to destabilize the interaction, its concentration would have to reflect the availability of both leucine and glucose, as well as the state of mitochondrial metabolism. Alternatively, destabilization could require several molecular species that are derived from distinct nutrients and act on multiple independent sites on the NSC. Because raptor and mTOR are large proteins with high potentials for small molecule-induced allosteric and conformational changes that may affect protein-protein interactions, the NSC appears well equipped for sensing multiple growth signals. [0213]
In this regard it is interesting that the mTOR-raptor interaction is mediated in part through the N-terminal portion of mTOR, which contains at least 16 HEAT repeats (Dennis et al., [0214] Curr. Opin. Genet. Dev. 9:49-54 (1999)). HEAT repeat-containing domains appear to posses substantial conformational flexibility and, for example, in β-importin undergo a twisting conformational change in superhelical structure that enables them to bind to distinct proteins for nuclear import (Lee et al., J. Mol. Biol. 302:251-64 9(2000)). The importance of the HEAT motifs in the mTOR-raptor interaction might indicate that conformational changes in that region of mTOR play a role in regulating the nutrient-sensitive strength of the interaction. However, the results described herein also show that in addition to the HEAT repeats, sites in the C-terminal half of mTOR also contribute to the interaction.
It is worth noting that the interaction is exquisitely sensitive to the detergent Triton X-100. This indicates that a hydrophobic molecule, such as a lipid, plays a role in maintaining the interaction, or that Triton X-100 mimics a molecule that normally weakens it in vivo. Consistent with this possibility, mTOR is found in association with membrane fractions (Sabatini et al., [0215] Science 284:1161-4 (1999)), and phosphatidic acid (PA), a component of lipid membranes as well as a signaling molecule, has been shown to interact with mTOR and stimulate the pathway through unknown mechanisms (Fang et al., Science 294:1942-5 (2001)).

Conclusions

Using a chemical cross-linker and appropriate cell lysis conditions, it has been demonstrated that in vivo mTOR interacts with raptor, a protein that has a positive role required for the in vivo activity of the mTOR pathway and for cell growth. Under nutrient deprivation conditions, however, raptor also serves as a negative regulator of mTOR kinase activity. FKBP12-rapamycin may mimic the binding of raptor to a nutrient-sensitive site on mTOR, locking mTOR in the nutrient-poor state, or it may disrupt a constitutive interaction necessary for mTOR function. It is likely that the regulation of the mTOR-raptor interaction is a critical mechanism by which eukaryotic cells coordinate the rate of cell growth with different environmental conditions. Small molecules can be designed that perturb the mTOR-raptor association in subtler ways than rapamycin, allowing a finer pharmacological control of the TOR pathway than is currently possible. [0216]
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. [0217]
1 28 1 6431 DNA homo sapien 1 gagagcaggg tcatcgtgag gcctgaagtc tcttacgctt ttggcagctc ccctcgcagc 60 ccctctggaa acgtacagcc tcaggagcag ccagtggctt gggacctggg gtggtgtgtg 120 tctgcggagc ttcttgggct gccccatttc ctagcggccc ccacctcccc acttcccgct 180 cagagttaga gataaggatc tcagactttt gcctgagtaa gggtctccgc actctttatc 240 catttggttt tcgatttccc gtttttgttt cttatttcac caattctggt acacgctagt 300 ttttaaggct ggaggttctc gagcgcttgc tgccaaggac tcccccaccc cctcccccac 360 tgatggagtc cgaaatgctg caatcgcctc ttctgggcct gggggaggaa gatgaggctg 420 atcttacaga ctggaaccta cctttggctt ttatgaaaaa gaggcactgt gagaaaattg 480 aaggctccaa atccttagct cagagctgga ggatgaagga tcggatgaag acagtcagtg 540 ttgccttagt tttgtgcctg aatgttggtg tggaccctcc cgatgtggtg aagaccacgc 600 cctgtgcacg cttggaatgc tggatcgatc ctctgtcgat gggtcctcag aaagctctgg 660 aaaccatcgg tgcaaattta cagaagcagt acgagaactg gcagccaagg gcccggtaca 720 agcagagcct tgacccaact gtggatgaag tcaagaagct ctgcacgtcc ttacgtcgca 780 acgccaagga ggagcgagtc ctctttcact acaatggcca cggggtgccc cggcccacag 840 tcaacgggga ggtctgggtc ttcaacaaga actacacgca gtacatccct ctgtccatat 900 atgacctgca gacgtggatg ggcagcccgt cgatcttcgt ctacgactgc tccaatgctg 960 gcttgatcgt caagtccttc aagcagttcg cactacagcg ggagcaggag ctggaggtag 1020 ctgcaatcaa cccaaatcac cctcttgctc agatgccttt gcctccgtcg atgaaaaact 1080 gcatccagct ggcagcctgc gaggccaccg agctgctgcc catgatcccc gacctcccgg 1140 ctgacctatt cacctcctgc ctcaccaccc ccatcaagat cgccctgcgc tggttttgca 1200 tgcagaaatg tgtcagtctg gtgcctggcg tcacactgga tttgatagaa aagatccctg 1260 gccgcctgaa cgacaggagg acgcccctgg gtgaactgaa ctggatcttc acagccatca 1320 cagacaccat cgcgtggaac gtgctccccc gggatctctt ccaaaagctc ttcagacagg 1380 acttgctggt ggctagtctg tttcgaaatt ttttattggc ggaaaggatt atgaggtcgt 1440 ataactgcac tcccgtcagc agcccgcgtc tgccgcccac gtacatgcac gccatgtggc 1500 aagcctggga cctggctgtt gacatctgtc tgtctcagct gccgacgatc atcgaggaag 1560 gcactgcgtt tcggcacagc ccgttcttcg ccgagcagct gaccgcattc caggtgtggc 1620 tcaccatggg cgtggagaac cgaaacccac ccgaacagct gcccatcgtc ctgcaggtgc 1680 tgttaagcca agtgcaccgg ctgagagcat tggacttgct tggaagattt ttggacctgg 1740 gtccctgggc agtgagcctg gccttgtctg tcggcatctt cccctacgtg ctgaagctgc 1800 tccagagctc ggcccgagag ctgcggccac ttctcgtttt catctgggcc aagatcctcg 1860 cagtggacag ctcgtgccag gcggacctcg tgaaggacaa cggccacaag tacttcctgt 1920 cggtcctggc ggacccctac atgccagctg aacaccggac catgacggct ttcattctcg 1980 ccgtgatcgt caacagctat cacacggggc aggaagcctg ccttcaggga aacctcattg 2040 ccatctgcct ggagcagctc aacgacccgc accccttgct gcgccagtgg gtggccatct 2100 gcctcggcag gatctggcag aacttcgact cggcgaggtg gtgcggcgtg agggacagcg 2160 ctcatgagaa gctctacagc ctcctctccg accccattcc cgaggtccgc tgcgcagcgg 2220 tcttcgccct tggcacgttc gtgggcaact ctgcagagag gacggaccac tccaccacca 2280 tcgaccacaa cgtggccatg atgctggccc agctggtcag cgacgggagc cccatggtcc 2340 ggaaggagct ggtggtggct ctgagtcatc ttgtggttca gtatgaaagc aatttctgca 2400 ccgtggccct gcagttcata gaagaggaaa agaactacgc cttgccttct ccagcaacca 2460 cagagggagg gagtttgacc ccagtgcgag acagcccgtg cacccccaga cttcgttctg 2520 tgagctccta tggaaacatc cgtgctgtcg ccacagccag gagcctcaac aaatctttgc 2580 agaacctgag tttgacagag gaatctggtg gcgcggtggc gttctccccc ggaaacctca 2640 gcaccagcag cagcgccagc agcaccctgg gcagccccga gaatgaggag catatcctgt 2700 ccttcgagac catcgacaag atgcgccgcg ccagctccta ctcctccctc aactccctca 2760 tcggagtttc ctttaacagt gtttacactc agatttggag agtcctgctg cacctggctg 2820 ctgaccccta tccagaggtc tcggacgtgg ccatgaaagt actcaacagc atcgcctaca 2880 aggccaccgt gaacgcccgg ccgcagcgcg tcctggacac ctcctccctc acgcagtcgg 2940 cccccgccag ccccaccaac aagggcgtgc acatccacca ggcggggggc tcccctccgg 3000 cgtccagcac cagcagctcc agcctgacca acgatgtggc caagcagccg gtcagccgag 3060 acttgccttc tggccggccg ggcaccacag gccccgctgg ggcgcagtac acccctcact 3120 cccaccagtt cccccggaca cggaagatgt tcgacaaggg cccagagcag actgcggacg 3180 acgcggacga tgctgctgga cacaaaagtt tcatctccgc cacggtgcag acggggttct 3240 gcgactggag cgcccgctat tttgcccagc ccgtcatgaa gatcccagaa gagcacgacc 3300 tggagagtca gatccgcaag gagcgggagt ggcggttcct gcgaaacagc cgtgtcagga 3360 ggcaggccca gcaagtcatt cagaagggca ttacgagatt ggacgaccaa atatttctga 3420 acaggaaccc cggcgtcccc tctgtggtga aattccaccc cttcacgccg tgcatcgctg 3480 tagccgacaa ggacagcatc tgcttttggg actgggagaa aggggagaag ctggattatt 3540 tccacaatgg gaaccctcgg tacacgaggg tcactgccat ggagtatctg aatggccagg 3600 actgctcgct tctgctgacg gccacagacg atggtgccat cagggtctgg aagaattttg 3660 ctgatttgga aaagaaccca gagatggtga ccgcgtggca ggggctctcg gacatgctgc 3720 caacgacgcg aggagctggg atggtggtgg actgggagca ggagaccggc ctcctcatga 3780 gctcaggaga cgtgcggatc gtccggatct gggacacaga ccgtgagatg aaggtgcagg 3840 acatccctac gggcgcagac agctgtgtga cgagtctgtc ctgtgattcc caccgctcac 3900 tcatcgtggc tggcctcggt gacggctcca tccgcgtcta cgacagaagg atggcactca 3960 gcgaatgccg cgtcatgacg taccgggagc acacagcctg ggtggtgaag gcctccctgc 4020 agaagcgtcc cgacggccac atcgtgagtg tgagcgtcaa tggagatgtg cgcatctttg 4080 atccccggat gcctgagtcg gtaaatgtgc ttcagatcgt gaaggggctg acggccctgg 4140 acatccaccc ccaggcggac ctgatcgcat gtggctccgt caatcagttc accgccatct 4200 acaacagcag cggagagctc atcaacaaca tcaagtacta cgacggcttc atgggccagc 4260 gggtcggcgc catcagctgc ctggccttcc acccgcactg gcctcacctg gccgtgggaa 4320 gcaacgacta ctacatctcc gtgtactcgg tggagaagcg tgtcagatag cggcgtgacc 4380 cgggcccacc aggccacggc cgcctgctgt acatagtgaa gctgtcactc gccggggcac 4440 ggggcgtcgg ctgctgcggc cccgcagtgt gaacgttggc tgctgcctta gctgctgatg 4500 acggcaggag ggccctgcta ctcgcttttg tctgtcttcg ctgtcgtgtc tggaatgtca 4560 gggaagggga gggctcgggt tgacggtggc ttcccactga gcaccagcat ccaggtgcac 4620 ccccgcggcc acggcgcctc tgtccctctc ctgttctgtg tttctctgag acgctgaaag 4680 gggaaacacc tcactttatt tccatgtaat cagagcatta gctgcagaaa aacccccaga 4740 cagagccctg gcggagaggc aggcgctggg gctcctacgg gtccctgggg cagctgtccc 4800 catcaggcca agagcgagcg agaggcgctg ccccagccag gcccaccacc tctcacagtc 4860 agtgcacgca agcagggaca tttcctagcc agctggggga cactggaaat tcgggaaacc 4920 aagagagagg aagaaggaga cgcccctcca actggcgggt gtgaaggaag ccgcccaggg 4980 gtccgggctg tccttggccg ctggcagcat cactgagcag gaagcgcaca gcccaccctc 5040 cccgcacctc caggtctctg gactccagtt ttggcccctc tcacacagag ctgtcagcag 5100 gggccgctgt ggcggtgcac aggggaggca ggtccttggc gaggtagccc ctgccttaat 5160 ccacggggct cctttccctc cgaagggctg ctcttcccca caggcgcggg gacagcagcc 5220 cgacctgtgg tctccatgcc tgtgccctca cacaggtgta gcacacgcat gtgcagatgg 5280 caccacggcc ggcacctggg ggcacacaca tgcaggcggc gtggtctccc tgctctgtcc 5340 ccacacgttc ctcacataca ggcaagaggc actgccgggt cccggacggc tccgggtgac 5400 accagccccg tctccagcct tgagccgccc atgctgatgc gacctcggct gacagctggg 5460 cctgtggtgc agacaggagc tgtgtggaca gtcccgccca ggaggggccg cagggcgtgt 5520 atgagcagtt ttgcaaacag aacacaacca caatgatggt attttgaaaa gtgttctttc 5580 cgtgttcgtc gggaatcagg attattgaga ggtgaaggag ccaggtggct tcattctggc 5640 ggtgagaggc ccatgaccac gggagtgaga gctggtgtgg cgaggcccgg ctctcctgcg 5700 gtgtggctgg tggcctgccg tggccaagag catcttctgg gtggatggaa ccctgcctgg 5760 tcacatttgg ccagagacac acctggccct cagggggctg agctggagac tgagctgggg 5820 ctggccggga cgtgacaagg caggacagag gcggcccctc cgctgctcct ttttggaatg 5880 cgagctccca ccagaagaag gttccggcac gaatcccatc cccacgtctg ggccgagaaa 5940 gcagcccggg tccggaaggt gtagagagtc ccggcctcac tcagctcaca gggcgtgcca 6000 ggcggcaaca ccagaatctt ccagaagccc agctccaccc gcacacgcag cttcccatcc 6060 agtccttcaa ctcaattctt acccaacacg cgtttccgtt tgttttgaga caaaatcacc 6120 acctgtcaaa aggcaggtgg ctccagaggg gtcaagaccc ccccccccgc ccccgctcca 6180 ccctggagcc cacccccatg ggcactgcgt gccgcctgca cgtgggctgt cttcacaggt 6240 ctgatgtgaa aattcaatca cgacgttaac cggctcgaga gagcgccggc ctagaggctc 6300 attatctatt tattttacca aacgcgaatt gagacggact ttgacaaaac acgaaatggt 6360 aatgtgaagc taagagcaga gagtgaccaa cagtaaacaa cacgcgcaga aaaaaaaaaa 6420 aaaaaaaaaa a 6431 2 1335 PRT homo sapien 2 Met Glu Ser Glu Met Leu Gln Ser Pro Leu Leu Gly Leu Gly Glu Glu 1 5 10 15 Asp Glu Ala Asp Leu Thr Asp Trp Asn Leu Pro Leu Ala Phe Met Lys 20 25 30 Lys Arg His Cys Glu Lys Ile Glu Gly Ser Lys Ser Leu Ala Gln Ser 35 40 45 Trp Arg Met Lys Asp Arg Met Lys Thr Val Ser Val Ala Leu Val Leu 50 55 60 Cys Leu Asn Val Gly Val Asp Pro Pro Asp Val Val Lys Thr Thr Pro 65 70 75 80 Cys Ala Arg Leu Glu Cys Trp Ile Asp Pro Leu Ser Met Gly Pro Gln 85 90 95 Lys Ala Leu Glu Thr Ile Gly Ala Asn Leu Gln Lys Gln Tyr Glu Asn 100 105 110 Trp Gln Pro Arg Ala Arg Tyr Lys Gln Ser Leu Asp Pro Thr Val Asp 115 120 125 Glu Val Lys Lys Leu Cys Thr Ser Leu Arg Arg Asn Ala Lys Glu Glu 130 135 140 Arg Val Leu Phe His Tyr Asn Gly His Gly Val Pro Arg Pro Thr Val 145 150 155 160 Asn Gly Glu Val Trp Val Phe Asn Lys Asn Tyr Thr Gln Tyr Ile Pro 165 170 175 Leu Ser Ile Tyr Asp Leu Gln Thr Trp Met Gly Ser Pro Ser Ile Phe 180 185 190 Val Tyr Asp Cys Ser Asn Ala Gly Leu Ile Val Lys Ser Phe Lys Gln 195 200 205 Phe Ala Leu Gln Arg Glu Gln Glu Leu Glu Val Ala Ala Ile Asn Pro 210 215 220 Asn His Pro Leu Ala Gln Met Pro Leu Pro Pro Ser Met Lys Asn Cys 225 230 235 240 Ile Gln Leu Ala Ala Cys Glu Ala Thr Glu Leu Leu Pro Met Ile Pro 245 250 255 Asp Leu Pro Ala Asp Leu Phe Thr Ser Cys Leu Thr Thr Pro Ile Lys 260 265 270 Ile Ala Leu Arg Trp Phe Cys Met Gln Lys Cys Val Ser Leu Val Pro 275 280 285 Gly Val Thr Leu Asp Leu Ile Glu Lys Ile Pro Gly Arg Leu Asn Asp 290 295 300 Arg Arg Thr Pro Leu Gly Glu Leu Asn Trp Ile Phe Thr Ala Ile Thr 305 310 315 320 Asp Thr Ile Ala Trp Asn Val Leu Pro Arg Asp Leu Phe Gln Lys Leu 325 330 335 Phe Arg Gln Asp Leu Leu Val Ala Ser Leu Phe Arg Asn Phe Leu Leu 340 345 350 Ala Glu Arg Ile Met Arg Ser Tyr Asn Cys Thr Pro Val Ser Ser Pro 355 360 365 Arg Leu Pro Pro Thr Tyr Met His Ala Met Trp Gln Ala Trp Asp Leu 370 375 380 Ala Val Asp Ile Cys Leu Ser Gln Leu Pro Thr Ile Ile Glu Glu Gly 385 390 395 400 Thr Ala Phe Arg His Ser Pro Phe Phe Ala Glu Gln Leu Thr Ala Phe 405 410 415 Gln Val Trp Leu Thr Met Gly Val Glu Asn Arg Asn Pro Pro Glu Gln 420 425 430 Leu Pro Ile Val Leu Gln Val Leu Leu Ser Gln Val His Arg Leu Arg 435 440 445 Ala Leu Asp Leu Leu Gly Arg Phe Leu Asp Leu Gly Pro Trp Ala Val 450 455 460 Ser Leu Ala Leu Ser Val Gly Ile Phe Pro Tyr Val Leu Lys Leu Leu 465 470 475 480 Gln Ser Ser Ala Arg Glu Leu Arg Pro Leu Leu Val Phe Ile Trp Ala 485 490 495 Lys Ile Leu Ala Val Asp Ser Ser Cys Gln Ala Asp Leu Val Lys Asp 500 505 510 Asn Gly His Lys Tyr Phe Leu Ser Val Leu Ala Asp Pro Tyr Met Pro 515 520 525 Ala Glu His Arg Thr Met Thr Ala Phe Ile Leu Ala Val Ile Val Asn 530 535 540 Ser Tyr His Thr Gly Gln Glu Ala Cys Leu Gln Gly Asn Leu Ile Ala 545 550 555 560 Ile Cys Leu Glu Gln Leu Asn Asp Pro His Pro Leu Leu Arg Gln Trp 565 570 575 Val Ala Ile Cys Leu Gly Arg Ile Trp Gln Asn Phe Asp Ser Ala Arg 580 585 590 Trp Cys Gly Val Arg Asp Ser Ala His Glu Lys Leu Tyr Ser Leu Leu 595 600 605 Ser Asp Pro Ile Pro Glu Val Arg Cys Ala Ala Val Phe Ala Leu Gly 610 615 620 Thr Phe Val Gly Asn Ser Ala Glu Arg Thr Asp His Ser Thr Thr Ile 625 630 635 640 Asp His Asn Val Ala Met Met Leu Ala Gln Leu Val Ser Asp Gly Ser 645 650 655 Pro Met Val Arg Lys Glu Leu Val Val Ala Leu Ser His Leu Val Val 660 665 670 Gln Tyr Glu Ser Asn Phe Cys Thr Val Ala Leu Gln Phe Ile Glu Glu 675 680 685 Glu Lys Asn Tyr Ala Leu Pro Ser Pro Ala Thr Thr Glu Gly Gly Ser 690 695 700 Leu Thr Pro Val Arg Asp Ser Pro Cys Thr Pro Arg Leu Arg Ser Val 705 710 715 720 Ser Ser Tyr Gly Asn Ile Arg Ala Val Ala Thr Ala Arg Ser Leu Asn 725 730 735 Lys Ser Leu Gln Asn Leu Ser Leu Thr Glu Glu Ser Gly Gly Ala Val 740 745 750 Ala Phe Ser Pro Gly Asn Leu Ser Thr Ser Ser Ser Ala Ser Ser Thr 755 760 765 Leu Gly Ser Pro Glu Asn Glu Glu His Ile Leu Ser Phe Glu Thr Ile 770 775 780 Asp Lys Met Arg Arg Ala Ser Ser Tyr Ser Ser Leu Asn Ser Leu Ile 785 790 795 800 Gly Val Ser Phe Asn Ser Val Tyr Thr Gln Ile Trp Arg Val Leu Leu 805 810 815 His Leu Ala Ala Asp Pro Tyr Pro Glu Val Ser Asp Val Ala Met Lys 820 825 830 Val Leu Asn Ser Ile Ala Tyr Lys Ala Thr Val Asn Ala Arg Pro Gln 835 840 845 Arg Val Leu Asp Thr Ser Ser Leu Thr Gln Ser Ala Pro Ala Ser Pro 850 855 860 Thr Asn Lys Gly Val His Ile His Gln Ala Gly Gly Ser Pro Pro Ala 865 870 875 880 Ser Ser Thr Ser Ser Ser Ser Leu Thr Asn Asp Val Ala Lys Gln Pro 885 890 895 Val Ser Arg Asp Leu Pro Ser Gly Arg Pro Gly Thr Thr Gly Pro Ala 900 905 910 Gly Ala Gln Tyr Thr Pro His Ser His Gln Phe Pro Arg Thr Arg Lys 915 920 925 Met Phe Asp Lys Gly Pro Glu Gln Thr Ala Asp Asp Ala Asp Asp Ala 930 935 940 Ala Gly His Lys Ser Phe Ile Ser Ala Thr Val Gln Thr Gly Phe Cys 945 950 955 960 Asp Trp Ser Ala Arg Tyr Phe Ala Gln Pro Val Met Lys Ile Pro Glu 965 970 975 Glu His Asp Leu Glu Ser Gln Ile Arg Lys Glu Arg Glu Trp Arg Phe 980 985 990 Leu Arg Asn Ser Arg Val Arg Arg Gln Ala Gln Gln Val Ile Gln Lys 995 1000 1005 Gly Ile Thr Arg Leu Asp Asp Gln Ile Phe Leu Asn Arg Asn Pro Gly 1010 1015 1020 Val Pro Ser Val Val Lys Phe His Pro Phe Thr Pro Cys Ile Ala Val 1025 1030 1035 1040 Ala Asp Lys Asp Ser Ile Cys Phe Trp Asp Trp Glu Lys Gly Glu Lys 1045 1050 1055 Leu Asp Tyr Phe His Asn Gly Asn Pro Arg Tyr Thr Arg Val Thr Ala 1060 1065 1070 Met Glu Tyr Leu Asn Gly Gln Asp Cys Ser Leu Leu Leu Thr Ala Thr 1075 1080 1085 Asp Asp Gly Ala Ile Arg Val Trp Lys Asn Phe Ala Asp Leu Glu Lys 1090 1095 1100 Asn Pro Glu Met Val Thr Ala Trp Gln Gly Leu Ser Asp Met Leu Pro 1105 1110 1115 1120 Thr Thr Arg Gly Ala Gly Met Val Val Asp Trp Glu Gln Glu Thr Gly 1125 1130 1135 Leu Leu Met Ser Ser Gly Asp Val Arg Ile Val Arg Ile Trp Asp Thr 1140 1145 1150 Asp Arg Glu Met Lys Val Gln Asp Ile Pro Thr Gly Ala Asp Ser Cys 1155 1160 1165 Val Thr Ser Leu Ser Cys Asp Ser His Arg Ser Leu Ile Val Ala Gly 1170 1175 1180 Leu Gly Asp Gly Ser Ile Arg Val Tyr Asp Arg Arg Met Ala Leu Ser 1185 1190 1195 1200 Glu Cys Arg Val Met Thr Tyr Arg Glu His Thr Ala Trp Val Val Lys 1205 1210 1215 Ala Ser Leu Gln Lys Arg Pro Asp Gly His Ile Val Ser Val Ser Val 1220 1225 1230 Asn Gly Asp Val Arg Ile Phe Asp Pro Arg Met Pro Glu Ser Val Asn 1235 1240 1245 Val Leu Gln Ile Val Lys Gly Leu Thr Ala Leu Asp Ile His Pro Gln 1250 1255 1260 Ala Asp Leu Ile Ala Cys Gly Ser Val Asn Gln Phe Thr Ala Ile Tyr 1265 1270 1275 1280 Asn Ser Ser Gly Glu Leu Ile Asn Asn Ile Lys Tyr Tyr Asp Gly Phe 1285 1290 1295 Met Gly Gln Arg Val Gly Ala Ile Ser Cys Leu Ala Phe His Pro His 1300 1305 1310 Trp Pro His Leu Ala Val Gly Ser Asn Asp Tyr Tyr Ile Ser Val Tyr 1315 1320 1325 Ser Val Glu Lys Arg Val Arg 1330 1335 3 158 PRT homo sapiens 3 Trp Arg Met Lys Asp Arg Met Lys Thr Val Ser Val Ala Leu Val Leu 1 5 10 15 Cys Leu Asn Val Gly Val Asp Pro Pro Asp Val Val Lys Thr Thr Pro 20 25 30 Cys Ala Arg Leu Glu Cys Trp Ile Asp Pro Leu Ser Met Gly Pro Gln 35 40 45 Lys Ala Leu Glu Thr Ile Gly Ala Asn Leu Gln Lys Gln Tyr Glu Asn 50 55 60 Trp Gln Pro Arg Ala Arg Tyr Lys Gln Ser Leu Asp Pro Thr Val Asp 65 70 75 80 Glu Val Lys Lys Leu Cys Thr Ser Leu Arg Arg Asn Ala Lys Glu Glu 85 90 95 Arg Val Leu Phe His Tyr Asn Gly His Gly Val Pro Arg Pro Thr Val 100 105 110 Asn Gly Glu Val Trp Val Phe Asn Lys Asn Tyr Thr Gln Tyr Ile Pro 115 120 125 Leu Ser Ile Tyr Asp Leu Gln Thr Trp Met Gly Ser Pro Ser Ile Phe 130 135 140 Val Tyr Asp Cys Ser Asn Ala Gly Leu Ile Val Lys Ser Phe 145 150 155 4 125 PRT homo sapiens 4 Cys Ile Gln Leu Ala Ala Cys Glu Ala Thr Glu Leu Leu Pro Met Ile 1 5 10 15 Pro Asp Leu Pro Ala Asp Leu Phe Thr Ser Cys Leu Thr Thr Pro Ile 20 25 30 Ile Pro Gly Arg Leu Asn Asp Arg Arg Thr Pro Leu Gly Glu Leu Asn 35 40 45 Trp Ile Phe Thr Ala Ile Thr Asp Thr Ile Ala Trp Asn Val Leu Pro 50 55 60 Arg Asp Leu Phe Gln Lys Leu Phe Arg Gln Asp Leu Leu Val Ala Ser 65 70 75 80 Leu Phe Arg Asn Phe Leu Leu Ala Glu Arg Ile Met Arg Ser Tyr Asn 85 90 95 Cys Thr Pro Val Ser Ser Pro Arg Leu Pro Pro Thr Tyr Met His Ala 100 105 110 Met Trp Gln Ala Trp Asp Leu Ala Val Asp Ile Cys Leu 115 120 125 5 105 PRT homo sapiens 5 Phe Phe Ala Glu Gln Leu Thr Ala Phe Gln Val Trp Leu Thr Met Gly 1 5 10 15 Val Glu Asn Arg Asn Pro Pro Glu Gln Leu Pro Ile Val Leu Gln Val 20 25 30 Leu Leu Ser Gln Val His Arg Leu Arg Ala Leu Asp Leu Leu Gly Arg 35 40 45 Phe Leu Asp Leu Gly Pro Trp Ala Val Ser Leu Ala Leu Ser Val Gly 50 55 60 Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Ser Ala Arg Glu Leu 65 70 75 80 Arg Pro Leu Leu Val Phe Ile Trp Ala Lys Ile Leu Ala Val Asp Ser 85 90 95 Ser Cys Gln Ala Asp Leu Val Lys Asp 100 105 6 158 PRT D. melanogaster 6 Trp Arg Ile Arg Glu Arg Met Lys Thr Ala Ser Val Ala Leu Val Leu 1 5 10 15 Cys Leu Asn Ile Gly Val Asp Pro Pro Asp Val Val Lys Ile Gln Pro 20 25 30 Cys Ser Arg Leu Glu Cys Trp Ile Asp Pro Ser Ser Val Ser Pro Pro 35 40 45 Lys Ala Met Glu Leu Ile Gly Ser Asn Leu Gln Met Gln Tyr Glu Arg 50 55 60 Trp Gln Pro Arg Ala Arg Tyr Lys Lys Cys Asn Asp Pro Thr Val Asp 65 70 75 80 Asp Val Lys Lys Leu Cys Thr Ser Leu Arg Arg Asn Ala Lys Gly Glu 85 90 95 Arg Ile Leu Phe His Tyr Asn Gly His Gly Val Pro Lys Pro Thr Ala 100 105 110 Asn Gly Glu Ile Trp Val Phe Asn Lys Thr Phe Thr Gln Tyr Ile Pro 115 120 125 Leu Ser Ile Phe Glu Leu Ile Thr Trp Met Ser Ala Pro Ser Ile Tyr 130 135 140 Val Tyr Asp Cys Ser Asn Ala Gly Ile Ile Ile Asn Ser Phe 145 150 155 7 125 PRT D. melanogaster 7 Cys Ile His Leu Ala Ala Cys Ala Ala Asn Glu Ile Leu Pro Met Asn 1 5 10 15 Ala Gln Leu Pro Ala Asp Leu Phe Thr Ser Cys Leu Thr Thr Pro Ile 20 25 30 Ile Pro Gly Lys Val Asn Asp Arg Arg Thr Met Met Gly Glu Leu Asn 35 40 45 Trp Ile Phe Thr Ala Ile Thr Asp Thr Ile Ala Trp Asn Thr Leu Pro 50 55 60 Arg Glu Leu Phe Gln Arg Leu Phe Arg Gln Asp Leu Leu Val Ala Ser 65 70 75 80 Leu Phe Arg Asn Phe Leu Leu Ala Glu Arg Ile Leu Arg Ser His Asp 85 90 95 Cys Thr Pro Val Ser Leu Pro Ala Leu Pro Pro Cys Tyr Arg His Pro 100 105 110 Met Trp Lys Ala Trp Asp Leu Val Val Asp Leu Ala Leu 115 120 125 8 105 PRT D. Melanogaster 8 Phe Phe Glu His Gln Leu Thr Ala Phe Gln Val Trp Leu Asp Ser Glu 1 5 10 15 Ser Glu Ser Arg Thr Pro Pro Glu Gln Leu Pro Ile Val Leu Gln Val 20 25 30 Leu Leu Ser Gln Val His Arg Leu Arg Ala Leu Glu Leu Leu Ala Arg 35 40 45 Phe Leu Asp Leu Gly Pro Trp Ala Val Asn Leu Ala Leu Gly Val Gly 50 55 60 Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Ser Thr Arg Glu Leu 65 70 75 80 Arg Pro Val Leu Val Phe Ile Trp Ala Lys Ile Leu Ala Val Asp Pro 85 90 95 Ser Cys Gln Val Asp Leu Val Lys Glu 100 105 9 158 PRT S. Pombe 9 Trp Arg Met Arg Glu Arg Leu Lys Thr Val Ser Ala Ala Leu Leu Val 1 5 10 15 Cys Leu Asn Ile Gly Val Asp Pro Pro Asp Val Ile Lys Pro Asn Pro 20 25 30 Ala Ala Lys Tyr Glu Cys Trp Ile Asp Pro Phe Ser Leu Pro Ala Ser 35 40 45 Lys Ala Leu Glu Ala Ile Gly Lys Asn Leu Gln Gln Gln Tyr Glu Thr 50 55 60 Leu Ser Met Arg Thr Arg Tyr Arg His Tyr Leu Asp Pro Ala Ile Glu 65 70 75 80 Glu Val Lys Lys Leu Cys Ile Gly Gln Arg Arg Asn Ala Lys Glu Glu 85 90 95 Arg Ile Leu Phe His Tyr Asn Gly His Gly Val Pro Met Pro Thr Ala 100 105 110 Ser Gly Glu Ile Trp Val Phe Asn Lys Asn Tyr Thr Gln Tyr Ile Pro 115 120 125 Val Ser Leu Tyr Asp Leu Gln Ser Trp Leu Gly Ala Pro Cys Ile Tyr 130 135 140 Val Tyr Asp Cys Ser Ala Ala Gly Asn Ile Ile Val Asn Phe 145 150 155 10 125 PRT S. Pombe 10 Cys Ile Gln Leu Ala Ala Cys Gly Pro Lys Glu Thr Leu Pro Met Asn 1 5 10 15 Pro Asp Leu Pro Ala Asp Leu Phe Thr Ser Cys Leu Thr Ser Pro Ile 20 25 30 Ile Pro Gly Arg Leu Gln Asp Arg Arg Thr Pro Leu Gly Glu Leu Asn 35 40 45 Trp Ile Phe Thr Ala Ile Thr Asp Thr Ile Ala Trp Asn Val Phe Pro 50 55 60 Lys His Leu Phe Arg Arg Leu Phe Arg Gln Asp Leu Met Val Ala Ala 65 70 75 80 Leu Phe Arg Asn Phe Leu Leu Ala Glu Arg Ile Met Leu Val His Ser 85 90 95 Cys His Pro Gln Ser Ser Pro Glu Leu Pro Pro Thr His Asp His Pro 100 105 110 Met Trp Asn Ser Trp Asp Leu Ala Ile Asp Asn Cys Leu 115 120 125 11 105 PRT S. Pombe 11 Phe Phe Ser Glu Gln Leu Thr Ala Phe Glu Val Trp Leu Ser Gln Gly 1 5 10 15 Leu Ile Ser Arg Lys Pro Pro Asp Gln Leu Pro Leu Val Leu Gln Val 20 25 30 Leu Leu Ser Gln Val His Arg Leu Arg Ala Leu Ile Leu Leu Ser Lys 35 40 45 Phe Leu Asp Leu Gly Val Trp Ala Val Asp Leu Ala Leu Ser Ile Gly 50 55 60 Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Pro Ala Ile Glu Leu 65 70 75 80 Lys Pro Val Leu Val Phe Ile Trp Ala Arg Ile Leu Ala Val Asp Asp 85 90 95 Ser Cys Gln Ala Asp Leu Leu Lys Asp 100 105 12 160 PRT S. cerevisiae 12 Trp Lys Ile Met Lys Asp Arg Gln Lys Thr Val Ser Ala Ala Leu Leu 1 5 10 15 Leu Cys Leu Asn Leu Gly Val Asp Pro Pro Asp Val Met Lys Thr His 20 25 30 Pro Cys Ala Arg Val Glu Ala Trp Val Asp Pro Leu Asn Phe Gln Asp 35 40 45 Ser Lys Lys Ala Ile Glu Gln Ile Gly Lys Asn Leu Gln Ala Gln Tyr 50 55 60 Glu Thr Leu Ser Leu Arg Thr Arg Tyr Lys Gln Ser Leu Asp Pro Cys 65 70 75 80 Val Glu Asp Val Lys Arg Phe Cys Asn Ser Leu Arg Arg Thr Ser Lys 85 90 95 Glu Asp Arg Ile Leu Phe His Tyr Asn Gly His Gly Val Pro Lys Pro 100 105 110 Thr Lys Ser Gly Glu Ile Trp Val Phe Asn Arg Gly Tyr Thr Gln Tyr 115 120 125 Ile Pro Val Ser Leu Tyr Asp Leu Gln Thr Trp Leu Gly Ala Pro Cys 130 135 140 Ile Phe Val Tyr Asp Cys Asn Ser Ala Glu Asn Ile Leu Ile Asn Phe 145 150 155 160 13 126 PRT S. cerevisiae 13 Cys Phe Gln Leu Ala Ser Cys Thr Ser Asp Glu Leu Leu Leu Met Ser 1 5 10 15 Pro Glu Leu Pro Ala Asp Leu Phe Ser Cys Cys Leu Thr Cys Pro Ile 20 25 30 Ile Pro Gly Met Leu Ser Asp Arg Arg Thr Pro Leu Gly Glu Leu Asn 35 40 45 Trp Ile Phe Thr Ala Ile Thr Asp Thr Ile Ala Trp Thr Ser Leu Pro 50 55 60 Arg Pro Leu Phe Lys Lys Leu Phe Arg His Asp Leu Met Ile Ala Ala 65 70 75 80 Leu Phe Arg Asn Phe Leu Leu Ala Lys Arg Ile Met Pro Trp Tyr Asn 85 90 95 Cys His Pro Val Ser Asp Pro Glu Leu Pro Asp Ser Ile Thr Thr His 100 105 110 Pro Met Trp Lys Ser Trp Asp Leu Ala Met Asp Glu Val Leu 115 120 125 14 106 PRT S. cerevisiae 14 Phe Phe Glu Gln Asn Leu Thr Ala Phe Glu Leu Trp Leu Lys Tyr Ala 1 5 10 15 Ser Asn Val Arg His Pro Pro Glu Gln Leu Pro Ile Val Leu Gln Val 20 25 30 Leu Leu Ser Gln Val His Arg Ile Arg Ala Leu Val Leu Leu Ser Arg 35 40 45 Phe Leu Asp Leu Gly Pro Trp Ala Val Tyr Leu Ser Leu Ser Ile Gly 50 55 60 Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Pro Ala Pro Glu Leu 65 70 75 80 Lys Pro Ile Leu Val Phe Ile Trp Ala Arg Ile Met Ser Ile Asp Tyr 85 90 95 Lys Asn Thr Gln Ser Glu Leu Ile Lys Glu 100 105 15 162 PRT C. elegans 15 Arg Arg Lys Gly Ser Asp Arg Leu Arg Thr Ala Ser Val Ala Ile Ile 1 5 10 15 Met Cys Leu His Leu Gly Val Asp Pro Pro Glu Ala Pro Ser Arg Lys 20 25 30 Asp Ala Pro Ser Arg Leu Leu Ser Trp Val Asp Pro Tyr Lys Cys Gly 35 40 45 Ala His Lys Ala Ala Ile Glu Ile Gly Leu Ser Thr Gln Arg Ala Tyr 50 55 60 Glu Lys Trp Gln Pro Lys Ser Lys Thr Arg Tyr Lys Ile Cys Thr Asp 65 70 75 80 Pro Pro Ile Asp Asp Val Arg Lys Val Ala Thr Asn Leu Arg Arg Ile 85 90 95 Ser Gly Asn Asp Arg Val Leu Phe His Tyr Asn Gly His Gly Val Pro 100 105 110 Lys Pro Thr Asp Asn Gly Glu Ile Trp Val Phe Asn Lys Ser Phe Thr 115 120 125 Gln Tyr Ile Pro Leu Ser Ile Phe Asp Leu Gln Gly Trp Leu Asp Tyr 130 135 140 Pro Thr Ile Tyr Ile Trp Glu Thr His Ser Ala Glu Thr Ile Leu His 145 150 155 160 Asn Tyr 16 126 PRT C. elegans 16 Cys Ile His Leu Ala Ala Cys Ser Ala Gly Gln Trp Leu Pro Met Ser 1 5 10 15 Asn Pro His Leu Pro Ala Asp Leu Phe Thr Ser Cys Leu Thr Thr Pro 20 25 30 Ile Ile Pro Gly Asn Leu Ala Glu Arg Arg Thr Val Leu Gly Glu Leu 35 40 45 Asn Trp Ile Phe Thr Ala Ile Thr Asp Thr Ile Ala Phe Thr Ser Ile 50 55 60 Asp Arg Asp Ser Phe Gln Lys Leu Phe Arg Gln Asp Leu Leu Leu Ala 65 70 75 80 Thr Leu Phe Arg Ser Phe Leu Leu Ala Gln Arg Val Met Ser Lys Phe 85 90 95 Asn Val Leu Pro Val Ser Gln Pro Pro Leu Lys Asp Thr Ser Met His 100 105 110 Asp Leu Trp Arg Cys Trp Asp Gln Thr Met Asp Met Val Leu 115 120 125 17 105 PRT C. elegans 17 Phe Phe Thr Glu Gln Leu Gln Ala Phe Glu Leu Trp Leu Asp Tyr Gly 1 5 10 15 Val Asp Glu Gly Thr Pro Pro Leu Gln Leu Pro Met Val Leu Gln Val 20 25 30 Leu Leu Ser Gln Ala His Arg Val Arg Ala Leu Arg Leu Leu Ala Arg 35 40 45 Phe Leu Asp Phe Gly Arg Trp Ala Ile Gly Tyr Ser Leu Ala Val Gly 50 55 60 Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Asn Ile Ser Asp Leu 65 70 75 80 Lys Ile Ser Leu Ala Phe Ile Trp Ala Lys Ile Leu Ala Val Asp Pro 85 90 95 Ser Cys Gln Met Glu Leu Phe Asn Glu 100 105 18 158 PRT A. thaliana 18 Trp Arg Leu Lys Glu Arg Met Lys Thr Gly Cys Val Ala Leu Val Leu 1 5 10 15 Cys Leu Asn Ile Thr Val Asp Pro Pro Asp Val Ile Lys Ile Ser Pro 20 25 30 Cys Ala Arg Ile Glu Ala Trp Ile Asp Pro Phe Ser Met Ala Pro Pro 35 40 45 Lys Ala Leu Glu Thr Ile Gly Lys Asn Leu Ser Thr Gln Tyr Glu Arg 50 55 60 Trp Gln Pro Arg Ala Arg Tyr Lys Val Gln Leu Asp Pro Thr Val Asp 65 70 75 80 Glu Val Arg Lys Leu Cys Leu Thr Cys Arg Lys Tyr Ala Lys Thr Glu 85 90 95 Arg Val Leu Phe His Tyr Asn Gly His Gly Val Pro Lys Pro Thr Ala 100 105 110 Asn Gly Glu Ile Trp Val Phe Asn Lys Ser Tyr Thr Gln Tyr Ile Pro 115 120 125 Leu Pro Ile Ser Glu Leu Asp Ser Trp Leu Lys Thr Pro Ser Ile Tyr 130 135 140 Val Phe Asp Cys Ser Ala Ala Arg Met Ile Leu Asn Ala Phe 145 150 155 19 125 PRT A. thaliana 19 Cys Ile Leu Leu Ala Ala Cys Asp Val His Glu Thr Leu Pro Gln Ser 1 5 10 15 Val Glu Phe Pro Ala Asp Val Phe Thr Ser Cys Leu Thr Thr Pro Ile 20 25 30 Ile Pro Gly Arg Gln Asn Asp Arg Lys Thr Leu Leu Gly Glu Leu Asn 35 40 45 Trp Ile Phe Thr Ala Val Thr Asp Thr Ile Ala Trp Asn Val Leu Pro 50 55 60 His Glu Leu Phe Gln Arg Leu Phe Arg Gln Asp Leu Leu Val Ala Ser 65 70 75 80 Leu Phe Arg Asn Phe Leu Leu Ala Glu Arg Ile Met Arg Ser Ala Asn 85 90 95 Cys Asn Pro Ile Ser His Pro Met Leu Pro Pro Thr His Gln His His 100 105 110 Met Trp Asp Ala Trp Asp Met Ala Ala Glu Ile Cys Leu 115 120 125 20 105 PRT A. thaliana 20 Phe Phe Thr Glu Gln Leu Thr Ala Phe Glu Val Trp Leu Asp His Gly 1 5 10 15 Ser Glu His Lys Lys Pro Pro Glu Gln Leu Pro Ile Val Leu Gln Val 20 25 30 Leu Leu Ser Gln Cys His Arg Phe Arg Ala Leu Val Leu Leu Gly Arg 35 40 45 Phe Leu Asp Met Gly Ser Trp Ala Val Asp Leu Ala Leu Ser Val Gly 50 55 60 Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Thr Thr Thr Asn Glu Leu 65 70 75 80 Arg Gln Ile Leu Val Phe Ile Trp Thr Lys Ile Leu Ala Leu Asp Lys 85 90 95 Ser Cys Gln Ile Asp Leu Val Lys Asp 100 105 21 1538 PRT D. melanogaster 21 Met Thr Ser Ala Val Arg Ala Lys Ser Ala Ala Ala Ala Val Gly Val 1 5 10 15 Ala Val Asp Glu Ser Ser Gly Ser Ser Asn Gly Ser Val Ala Asp Gln 20 25 30 Glu Val Ala Ser Ser Lys Glu Ala Val Leu Ser Val Leu Ile Arg Lys 35 40 45 Arg Tyr Glu Lys Tyr Val Phe Ala Pro Trp Asp Asp Pro Ile Ile Leu 50 55 60 Ile Met Glu Gln Leu Glu His Ile Glu His Cys Arg Ser Ala Ala Leu 65 70 75 80 Gly Asn Gln Val Ala Ile Gly Arg Asp Pro Ser Gln Asp Ile Arg Asp 85 90 95 Asp Arg Cys Pro Leu Ser Leu Gln Thr Lys Arg His Lys Glu Arg Ile 100 105 110 Gln Ala Met Met Tyr Glu Arg Gln Pro Trp Arg Ile Arg Glu Arg Met 115 120 125 Lys Thr Ala Ser Val Ala Leu Val Leu Cys Leu Asn Ile Gly Val Asp 130 135 140 Pro Pro Asp Val Val Lys Ile Gln Pro Cys Ser Arg Leu Glu Cys Trp 145 150 155 160 Ile Asp Pro Ser Ser Val Ser Pro Pro Lys Ala Met Glu Leu Ile Gly 165 170 175 Ser Asn Leu Gln Met Gln Tyr Glu Arg Trp Gln Pro Arg Ala Arg Tyr 180 185 190 Lys Lys Cys Asn Asp Pro Thr Val Asp Asp Val Lys Lys Leu Cys Thr 195 200 205 Ser Leu Arg Arg Asn Ala Lys Gly Glu Arg Ile Leu Phe His Tyr Asn 210 215 220 Gly His Gly Val Pro Lys Pro Thr Ala Asn Gly Glu Ile Trp Val Phe 225 230 235 240 Asn Lys Thr Phe Thr Gln Tyr Ile Pro Leu Ser Ile Phe Glu Leu Ile 245 250 255 Thr Trp Met Ser Ala Pro Ser Ile Tyr Val Tyr Asp Cys Ser Asn Ala 260 265 270 Gly Ile Ile Ile Asn Ser Phe Gln Pro Tyr Ala Lys Gln His Glu Glu 275 280 285 Glu Leu Gly Lys Ala Leu Ala Ala Ala Gln Gln Arg Gly Gly Val Gln 290 295 300 Gln Leu Val Gly Gly Ser Gly Gly Ala Ala Asn Val Ala Ala Asn Gln 305 310 315 320 Val Gln Leu Thr Asn Gln Met Val Ser Tyr Lys Asn Cys Ile His Leu 325 330 335 Ala Ala Cys Ala Ala Asn Glu Ile Leu Pro Met Asn Ala Gln Leu Pro 340 345 350 Ala Asp Leu Phe Thr Ser Cys Leu Thr Thr Pro Ile Asn Ile Ala Leu 355 360 365 Lys Trp Tyr Ala Met Gln Glu Lys Leu Gly Met Val Pro Arg Ile Gln 370 375 380 Ser Glu Leu Ile Asp Lys Ile Pro Gly Lys Val Asn Asp Arg Arg Thr 385 390 395 400 Met Met Gly Glu Leu Asn Trp Ile Phe Thr Ala Ile Thr Asp Thr Ile 405 410 415 Ala Trp Asn Thr Leu Pro Arg Glu Leu Phe Gln Arg Leu Phe Arg Gln 420 425 430 Asp Leu Leu Val Ala Ser Leu Phe Arg Asn Phe Leu Leu Ala Glu Arg 435 440 445 Ile Leu Arg Ser His Asp Cys Thr Pro Val Ser Leu Pro Ala Leu Pro 450 455 460 Pro Cys Tyr Arg His Pro Met Trp Lys Ala Trp Asp Leu Val Val Asp 465 470 475 480 Leu Ala Leu Gln Gln Leu Pro Glu Ile Leu Asp His Asn Ala Pro Tyr 485 490 495 Arg Gln Leu Pro Phe Phe Glu His Gln Leu Thr Ala Phe Gln Val Trp 500 505 510 Leu Asp Ser Glu Ser Glu Ser Arg Thr Pro Pro Glu Gln Leu Pro Ile 515 520 525 Val Leu Gln Val Leu Leu Ser Gln Val His Arg Leu Arg Ala Leu Glu 530 535 540 Leu Leu Ala Arg Phe Leu Asp Leu Gly Pro Trp Ala Val Asn Leu Ala 545 550 555 560 Leu Gly Val Gly Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Ser 565 570 575 Thr Arg Glu Leu Arg Pro Val Leu Val Phe Ile Trp Ala Lys Ile Leu 580 585 590 Ala Val Asp Pro Ser Cys Gln Val Asp Leu Val Lys Glu Tyr Lys Tyr 595 600 605 Phe Leu Ser Val Leu Gln Asp Thr Ser Val Ser Lys Glu His Arg Thr 610 615 620 Leu Ser Ala Phe Val Leu Ser Ser Ile Val His Asn Phe Leu Leu Gly 625 630 635 640 Gln Thr Ser Ala Leu Gln Gly Pro Leu Leu Ser Ile Cys Leu Glu Gln 645 650 655 Leu Asn Asp Gly Ser Trp Leu Leu Arg Gln Trp Leu Ala Ile Cys Leu 660 665 670 Gly Met Leu Trp Gln Asn Phe Glu Lys Ala Arg Trp Ser Gly Ala Arg 675 680 685 Asp Leu Ala His Glu Lys Leu Tyr Val Leu Leu Arg Asp Ser Ile Pro 690 695 700 Glu Val Arg Ala Ala Ala Val Phe Ala Leu Gly Thr Phe Ile Ser Ser 705 710 715 720 Val Thr Asp Arg Ser Glu Glu Gln Ala Asn Asn Ile Asp Arg Ile Ile 725 730 735 Ala Ile Thr Met Leu Glu Thr Val Gly Glu Asp Met Ser Pro Leu Val 740 745 750 Arg Met Glu Leu Ala Ala Ala Leu Gln Trp Met Val Val Leu Phe Glu 755 760 765 Ser Gln Phe Val Ala Val Tyr Leu Thr Glu His Met Arg Gly His Val 770 775 780 Ser Ala Ala Ser Phe Val Met Cys Pro Asp Pro Arg Asp Leu Thr Ser 785 790 795 800 Ser Thr His Ser Leu Glu Arg His Val Thr Ile Arg Arg Gly Ala Ser 805 810 815 Ser Ser Ser Ile Ser Asn Met Gly Gly Ala Ser Ile Ser Ser Gly Thr 820 825 830 Gly Ser Ser Ser Asn Pro Leu Gly Arg Ser Lys Ser Gly Ser Gly Ser 835 840 845 Ser Gly Gly Arg Gly Ala Ala Gly Ala Ala Gly Gly Thr Asn Ser Ile 850 855 860 Pro Phe Gln Ser Ile Phe Thr Lys Leu Trp Leu Gly Ile Cys Asn Leu 865 870 875 880 Ala Gln Asp Pro Phe Pro Arg Val Ala Ala Ile Ala Gln Glu Ile Val 885 890 895 Glu His Val Arg Asp Thr Ala Leu Cys Pro Ile Met Ala Ala Lys Glu 900 905 910 Ala Thr Met Ala Asn Ala Ser Glu Lys Cys Ser Ser Leu Ser Val Ser 915 920 925 Leu Pro Pro Ser Pro Asn Thr Arg Val Asn Tyr Leu Gly Gly Gly Gly 930 935 940 Ala Pro Gly Gly Ala Gly Ala Thr Ala Glu Ser Pro Pro Val Gly Ala 945 950 955 960 Ala Ala Ser Gly Ala Ser His Trp Ala Gln Lys Leu Arg Leu Ser Gly 965 970 975 Ser Gly Ala Cys Met Gly Ala Gly Asp Pro Gln Thr Ile Leu Gln Arg 980 985 990 Lys Leu Arg Thr Ser Ser Ile Asn Asp Glu Thr Asp Ser Gly Pro Gly 995 1000 1005 Tyr Ser Arg Gly Val Ala Gly Ala Gly Thr Gly Leu Gly Leu Pro Ser 1010 1015 1020 Gly Pro Arg Glu Ser Thr His Ser Ala Arg Ser Ser Gly Ser Glu Ser 1025 1030 1035 1040 Asn His Tyr Leu Glu Pro Gly His Ser Leu Thr Pro Ile Val Leu Thr 1045 1050 1055 Glu Phe Ile Pro Trp Ala Ile Ser Tyr Phe Thr Arg Pro Gly Lys Glu 1060 1065 1070 Arg Tyr Ser Ser Ala Glu Gly Ser Ala Glu Glu Gly Arg Gln Arg Phe 1075 1080 1085 Pro Val Asp Arg Asn Ser Ala Glu Leu Arg Arg Arg Arg Leu Arg Phe 1090 1095 1100 Leu Arg Asn Asp Phe Val Arg Arg His Ala Gly Arg Lys His Gln Glu 1105 1110 1115 1120 Leu Ile Asp Pro Tyr Gly Phe Asp Val Cys Val Trp Asn Arg Lys Thr 1125 1130 1135 Gln Phe Thr Pro Ser Ile Val Lys Leu Leu Pro Tyr Glu Pro Gln Ile 1140 1145 1150 Ala Val Ala Tyr Arg Glu Lys Val Leu Val Tyr Asp Phe Gln Tyr Asn 1155 1160 1165 Ser Val Arg Thr Tyr Gly Ala Glu Ser Leu Gly Gly Asn Phe Thr Gly 1170 1175 1180 Tyr Ser Ser Gly Tyr Gly Ser Gly Ser Gly Ser Ser Ser Ser Leu Asn 1185 1190 1195 1200 Gly Ser Thr Pro Arg Lys Ser Gly Ala His Thr Ser Gly Gly Gly Gly 1205 1210 1215 Gly Gly Phe Ser Ala Ala Pro Phe Ala Arg Val Ser Ser Ile Glu Phe 1220 1225 1230 Ile Asn Ala Gln Asp Met Ala Leu Asn Leu Val Ala His Glu Asp Gly 1235 1240 1245 Val Val Arg Val Trp Gln Ser Ser Pro Pro Ala Ser Ser Ala Thr Ser 1250 1255 1260 Glu Asp Thr Pro Ser Lys Ala Arg Leu Val Thr Ala Trp Thr Ala Leu 1265 1270 1275 1280 Gly Gln Val Asn Gln His Asn His Arg Gln Arg Ser Val Ala Ser Gly 1285 1290 1295 Gly Ser Ile Gly Lys Gly Ala Asp Ala Ser Gly Leu Ser Ser Ala Ala 1300 1305 1310 Ile Gly Ser Gly Gly Ile Val Thr Ala Trp Gln Gln Cys Ser Gln His 1315 1320 1325 Leu Val Ile Gly Gly Gly Gly Cys Arg Phe Ile Arg Ile Trp Asp Val 1330 1335 1340 Glu Arg Glu Leu Lys Leu Ala Asp Ile Pro Leu Gly Arg Ser Glu Thr 1345 1350 1355 1360 Cys Ala Arg Val Leu Ser Pro Tyr Leu Pro Asn Met Arg Ser Asp Val 1365 1370 1375 Ile Leu Ala Gly Cys Asp Asp Gly Ser Val Arg Leu Phe Asp Lys Arg 1380 1385 1390 Cys Ala Pro Gln Asp Ala Gly Ile Ser Val Tyr Arg Arg His Ser Ala 1395 1400 1405 Pro Ile Leu His Ala Ser Leu Arg Ala Asn Gly Leu Val Leu Val Ser 1410 1415 1420 Gly Cys Thr Glu Gly Arg Ile Ser Val Ala Asp Met Arg Gly Ser Gln 1425 1430 1435 1440 Ser Ser Ile Asn Pro Phe Asp Val Val Tyr Glu Trp Asp Ala Gly Gly 1445 1450 1455 Asp Val Thr Ala Ile Thr Ser His Gln Leu Glu Asp Thr Val Ala Cys 1460 1465 1470 Gly Asn Ala Ser Lys Ile Val Ile Tyr Ser Leu Asp Gly Arg Gln Gln 1475 1480 1485 Lys Val Leu Arg Thr Asn Glu Gly Phe Ile Gly Pro Lys Ile Gly His 1490 1495 1500 Pro Thr Tyr Leu Tyr Phe His Pro Tyr Lys Ala Gln Leu Ala Val Gly 1505 1510 1515 1520 Phe Val Asp Asn Thr Val Ala Ile Tyr Ser Pro Thr Pro Gly Arg Glu 1525 1530 1535 Gln Ser 22 1313 PRT S. pombe 22 Met Asn Asp Arg Ile Ser Glu Val Ser Gly Ser Ser Arg Ala Arg Arg 1 5 10 15 Ser Val Leu Ser Tyr Gly Thr Thr Glu Thr Gly Ser Asp Arg Tyr Thr 20 25 30 Glu Asn Ser Asn Ile Ala Thr Glu Asn Gly Val Asp Thr Ala Ser Ser 35 40 45 Met Ile Asp Gly Ile Gln Ser Gly Phe Pro Gln Pro Arg His Gly Phe 50 55 60 Glu Glu Glu Tyr Asn Asn Ala Glu Tyr Ile Asn Met Leu Glu Gln Val 65 70 75 80 Phe Tyr Met Tyr Tyr Thr Asp Lys Arg His Arg Gly Val Ile Ser Lys 85 90 95 Lys Asn Ala Glu Pro Thr Glu Thr Ile His Asp Trp Arg Met Arg Glu 100 105 110 Arg Leu Lys Thr Val Ser Ala Ala Leu Leu Val Cys Leu Asn Ile Gly 115 120 125 Val Asp Pro Pro Asp Val Ile Lys Pro Asn Pro Ala Ala Lys Tyr Glu 130 135 140 Cys Trp Ile Asp Pro Phe Ser Leu Pro Ala Ser Lys Ala Leu Glu Ala 145 150 155 160 Ile Gly Lys Asn Leu Gln Gln Gln Tyr Glu Thr Leu Ser Met Arg Thr 165 170 175 Arg Tyr Arg His Tyr Leu Asp Pro Ala Ile Glu Glu Val Lys Lys Leu 180 185 190 Cys Ile Gly Gln Arg Arg Asn Ala Lys Glu Glu Arg Ile Leu Phe His 195 200 205 Tyr Asn Gly His Gly Val Pro Met Pro Thr Ala Ser Gly Glu Ile Trp 210 215 220 Val Phe Asn Lys Asn Tyr Thr Gln Tyr Ile Pro Val Ser Leu Tyr Asp 225 230 235 240 Leu Gln Ser Trp Leu Gly Ala Pro Cys Ile Tyr Val Tyr Asp Cys Ser 245 250 255 Ala Ala Gly Asn Ile Ile Val Asn Phe Asn Arg Phe Ala Glu Gln Arg 260 265 270 Asp Lys Glu Ala Leu Arg Ile Ala Lys Gln Asn Pro Asn Val Leu Ala 275 280 285 Met Pro Ser His Thr Ser Cys Ile Gln Leu Ala Ala Cys Gly Pro Lys 290 295 300 Glu Thr Leu Pro Met Asn Pro Asp Leu Pro Ala Asp Leu Phe Thr Ser 305 310 315 320 Cys Leu Thr Ser Pro Ile Glu Ile Ser Val Arg Trp Tyr Val Leu Gln 325 330 335 Asn Pro Phe Pro Asn Lys Leu Asn Leu Asn Met Leu Leu Lys Ile Pro 340 345 350 Gly Arg Leu Gln Asp Arg Arg Thr Pro Leu Gly Glu Leu Asn Trp Ile 355 360 365 Phe Thr Ala Ile Thr Asp Thr Ile Ala Trp Asn Val Phe Pro Lys His 370 375 380 Leu Phe Arg Arg Leu Phe Arg Gln Asp Leu Met Val Ala Ala Leu Phe 385 390 395 400 Arg Asn Phe Leu Leu Ala Glu Arg Ile Met Leu Val His Ser Cys His 405 410 415 Pro Gln Ser Ser Pro Glu Leu Pro Pro Thr His Asp His Pro Met Trp 420 425 430 Asn Ser Trp Asp Leu Ala Ile Asp Asn Cys Leu Ser Gln Leu Pro Asp 435 440 445 Met Leu Asp Ala Glu Ser Lys Gly Ile Ala Tyr Glu Tyr Lys His Ser 450 455 460 Thr Phe Phe Ser Glu Gln Leu Thr Ala Phe Glu Val Trp Leu Ser Gln 465 470 475 480 Gly Leu Ile Ser Arg Lys Pro Pro Asp Gln Leu Pro Leu Val Leu Gln 485 490 495 Val Leu Leu Ser Gln Val His Arg Leu Arg Ala Leu Ile Leu Leu Ser 500 505 510 Lys Phe Leu Asp Leu Gly Val Trp Ala Val Asp Leu Ala Leu Ser Ile 515 520 525 Gly Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Pro Ala Ile Glu 530 535 540 Leu Lys Pro Val Leu Val Phe Ile Trp Ala Arg Ile Leu Ala Val Asp 545 550 555 560 Asp Ser Cys Gln Ala Asp Leu Leu Lys Asp Asn Gly Tyr Gly Tyr Phe 565 570 575 Val Gln Ile Leu Asn Pro Asn Ser Ser Ile Phe Pro Ser Ser Asn Ile 580 585 590 Ser Glu His Arg Ala Met Cys Ala Phe Ile Leu Ser Val Phe Cys Arg 595 600 605 Gly Phe Pro Gln Gly Gln Leu Ala Cys Leu Asn Pro Gln Val Leu Ser 610 615 620 His Cys Leu Ser His Leu Asn Ser Pro Asp Ser Leu Leu Arg Gln Trp 625 630 635 640 Ala Cys Leu Cys Ile Ser Gln Leu Trp Glu Asn Tyr Ser Glu Ala Lys 645 650 655 Trp Ser Gly Thr Arg Asp Asn Ala His Val Lys Leu Ala Glu Ile Ile 660 665 670 Val Asp Ser Val Pro Glu Val Arg Ala Ser Val Leu Thr Ala Phe Thr 675 680 685 Thr Phe Leu Gly Phe Pro Glu Lys Thr Glu Glu Val Val Ala Val Glu 690 695 700 Thr Tyr Ile Ala Ile Ala Ala Leu Ala Ala Leu Ser Asp Ala Ser Pro 705 710 715 720 Leu Val Arg His Glu Leu Val Ile Phe Leu Ser His Phe Val Val Asn 725 730 735 Tyr Lys Lys Gln Leu Met Val Val Ala Tyr Glu Ser Ser Leu Ala Asp 740 745 750 Ile Leu Glu Lys Lys Asn His Asn Ser Ile Ser Ala Ser Thr Ile Tyr 755 760 765 Glu Thr Val Trp Gln Ala Val Leu Val Leu Ala Ala Asp Pro Ser Ile 770 775 780 Glu Ile Ser Leu Ala Ala Glu Ala Ile Ile Asn Tyr Val Tyr Gln Ser 785 790 795 800 Met Leu Asn Ser Glu Leu Arg Glu Ser Phe Leu Ala Phe Leu Leu Gln 805 810 815 His Leu Pro Ala Leu His Lys Ala Ser Leu Ser Lys Asp Thr Asp Thr 820 825 830 Asn Ser Val Thr Ser Asp Pro Lys Pro His Pro Phe Val Pro Ser Val 835 840 845 Ser Glu Asn Lys Ile Leu Asn Arg Ser Phe Ser Leu Thr Arg Ser Leu 850 855 860 Lys Gly Leu Ala Leu Ser Leu Ala Gly Ser Asp Arg Ala Ser Glu Leu 865 870 875 880 Leu Ser Leu Asn Gly Glu Asn Lys Pro Ala Glu Ser Asn Leu Asn His 885 890 895 Leu Thr Ser Ala Lys Val Pro Gly Pro Pro Ala Phe Asn Glu Leu Glu 900 905 910 Tyr Gln Ser Glu Leu Asp Met Pro Leu Thr Ser Tyr Leu Phe Asp Trp 915 920 925 Ser Arg Lys Tyr Phe Thr Glu Pro Gln Met Arg Pro Asn Glu Asp Asp 930 935 940 Glu Pro Gly Ser Ile Cys Tyr Asn Gln Arg Leu Trp Arg Arg Asn Arg 945 950 955 960 Asn Glu Lys Leu Ile Tyr Arg Thr Arg Pro Leu Ala Glu Tyr Ser Thr 965 970 975 Asn Gly Arg Trp Asn Gln Gln Leu Met Thr Phe Asn Asn Thr Ile Ala 980 985 990 Pro Arg Lys Leu Met Phe His Gln Phe Glu Asp Gln Leu Ile Thr Leu 995 1000 1005 Gly Asp Lys Asp Ile Ile Gln Val Trp Asp Trp Arg Arg Asn Arg Cys 1010 1015 1020 Leu Asn Ser Phe Lys Thr Ser Ala Ser Ala Thr Thr Asn Val Thr Asp 1025 1030 1035 1040 Met Gln Leu Leu Asn Glu Asp Asp Val Ala Leu Leu Met Thr Gly Ser 1045 1050 1055 Ser Asp Gly Thr Ile Lys Leu Tyr Arg Asp Tyr Glu Asn Glu Lys Val 1060 1065 1070 Glu Leu Val Thr Ser Trp Asn Ser Leu Ser Asp Leu Val Phe Gly Asp 1075 1080 1085 Arg Asn Ala Ser Leu Leu Met Ser Trp Gln Gln Asn Cys Gly His Leu 1090 1095 1100 Leu Val Ala Gly Asp Val Arg Val Ile Arg Ile Trp Asp Ala Ser Lys 1105 1110 1115 1120 Glu Ile Cys Tyr Ala Asn Leu Pro Val Arg Ser Ser Asn Ser Ile Thr 1125 1130 1135 Ser Leu Thr Ser Asp Leu Val Gly Cys Asn Ile Ile Val Ala Gly Phe 1140 1145 1150 Ser Asp Gly Val Leu Arg Val Tyr Asp Lys Arg Leu Pro Ala Arg Asp 1155 1160 1165 Ser Leu Thr Asp Val Trp Lys Glu His Ser Ser Glu Ile Val Asn Val 1170 1175 1180 Glu Met Gln Ser Ser Gly Met Arg Glu Leu Ile Ser Ala Ser Ser Asp 1185 1190 1195 1200 Gly Glu Val Lys Leu Trp Asp Ile Arg Met Asn His Ser Leu Gln Thr 1205 1210 1215 Phe Ser Thr Asp Asn Ser Gly Leu Thr Ser Leu Thr Val His Ser His 1220 1225 1230 Ala Pro Val Tyr Ala Thr Gly Ser Ser Asn Gln Ser Ile Lys Ile Trp 1235 1240 1245 Asp Thr Leu Gly Gln Asn Ile Asn Thr Phe Arg Glu Asn Pro Arg Phe 1250 1255 1260 Leu Asn Gln Pro Lys Pro Ser Ser Leu Met Cys Leu Lys Phe His Pro 1265 1270 1275 1280 His His Leu Leu Leu Ala Cys Gly Asp Asn Thr Asp Ser Arg Val Asn 1285 1290 1295 Leu Tyr Ser Cys Thr Lys Asn Glu Ile His Thr Asp Ser Pro Asn Glu 1300 1305 1310 Phe 23 1527 PRT S. cerevisiae 23 Met Arg His Gly Phe Glu Glu Gln Tyr Gln Ser Asp Gln Leu Leu Gln 1 5 10 15 Ser Leu Ala Asn Asp Phe Ile Phe Tyr Phe Asp Asp Lys Arg His Lys 20 25 30 Thr Asn Gly Asn Pro Ile Pro Glu Glu Asp Lys Gln Arg Asp Val Asn 35 40 45 Arg Tyr Tyr Gln Pro Ile Thr Asp Trp Lys Ile Met Lys Asp Arg Gln 50 55 60 Lys Thr Val Ser Ala Ala Leu Leu Leu Cys Leu Asn Leu Gly Val Asp 65 70 75 80 Pro Pro Asp Val Met Lys Thr His Pro Cys Ala Arg Val Glu Ala Trp 85 90 95 Val Asp Pro Leu Asn Phe Gln Asp Ser Lys Lys Ala Ile Glu Gln Ile 100 105 110 Gly Lys Asn Leu Gln Ala Gln Tyr Glu Thr Leu Ser Leu Arg Thr Arg 115 120 125 Tyr Lys Gln Ser Leu Asp Pro Cys Val Glu Asp Val Lys Arg Phe Cys 130 135 140 Asn Ser Leu Arg Arg Thr Ser Lys Glu Asp Arg Ile Leu Phe His Tyr 145 150 155 160 Asn Gly His Gly Val Pro Lys Pro Thr Lys Ser Gly Glu Ile Trp Val 165 170 175 Phe Asn Arg Gly Tyr Thr Gln Tyr Ile Pro Val Ser Leu Tyr Asp Leu 180 185 190 Gln Thr Trp Leu Gly Ala Pro Cys Ile Phe Val Tyr Asp Cys Asn Ser 195 200 205 Ala Glu Asn Ile Leu Ile Asn Phe Gln Lys Phe Val Gln Lys Arg Ile 210 215 220 Lys Asp Asp Glu Glu Gly Asn His Asp Val Ala Ala Pro Ser Pro Thr 225 230 235 240 Ser Ala Tyr Gln Asp Cys Phe Gln Leu Ala Ser Cys Thr Ser Asp Glu 245 250 255 Leu Leu Leu Met Ser Pro Glu Leu Pro Ala Asp Leu Phe Ser Cys Cys 260 265 270 Leu Thr Cys Pro Ile Glu Ile Ser Ile Arg Ile Phe Leu Met Gln Ser 275 280 285 Pro Leu Lys Asp Ser Lys Tyr Lys Ile Phe Phe Glu Phe Lys Ser Lys 290 295 300 Ile Pro Asn Val Asn Ile Pro Gly Met Leu Ser Asp Arg Arg Thr Pro 305 310 315 320 Leu Gly Glu Leu Asn Trp Ile Phe Thr Ala Ile Thr Asp Thr Ile Ala 325 330 335 Trp Thr Ser Leu Pro Arg Pro Leu Phe Lys Lys Leu Phe Arg His Asp 340 345 350 Leu Met Ile Ala Ala Leu Phe Arg Asn Phe Leu Leu Ala Lys Arg Ile 355 360 365 Met Pro Trp Tyr Asn Cys His Pro Val Ser Asp Pro Glu Leu Pro Asp 370 375 380 Ser Ile Thr Thr His Pro Met Trp Lys Ser Trp Asp Leu Ala Met Asp 385 390 395 400 Glu Val Leu Thr Lys Ile Val Ile Asp Leu Lys Asn Ala Pro Pro Ala 405 410 415 Thr Ala Leu Glu Ser Gln Met Ile Leu Gln Gln Gln Glu Thr Leu Gln 420 425 430 Asn Gly Gly Ser Ser Lys Ser Asn Ala Gln Asp Thr Lys Ala Gly Ser 435 440 445 Ile Gln Thr Gln Ser Arg Phe Ala Val Ala Asn Leu Ser Thr Met Ser 450 455 460 Leu Val Asn Asn Pro Ala Leu Gln Ser Arg Lys Ser Ile Ser Leu Gln 465 470 475 480 Ser Ser Gln Gln Gln Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Phe 485 490 495 Thr Gly Phe Phe Glu Gln Asn Leu Thr Ala Phe Glu Leu Trp Leu Lys 500 505 510 Tyr Ala Ser Asn Val Arg His Pro Pro Glu Gln Leu Pro Ile Val Leu 515 520 525 Gln Val Leu Leu Ser Gln Val His Arg Ile Arg Ala Leu Val Leu Leu 530 535 540 Ser Arg Phe Leu Asp Leu Gly Pro Trp Ala Val Tyr Leu Ser Leu Ser 545 550 555 560 Ile Gly Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Pro Ala Pro 565 570 575 Glu Leu Lys Pro Ile Leu Val Phe Ile Trp Ala Arg Ile Met Ser Ile 580 585 590 Asp Tyr Lys Asn Thr Gln Ser Glu Leu Ile Lys Glu Lys Gly Tyr Met 595 600 605 Tyr Phe Val Thr Val Leu Val Pro Asp Trp Gly Val Asn Gly Met Ser 610 615 620 Ala Thr Asn Gly Ser Ala Met Ile Asn Ser Gly Asn Pro Leu Thr Met 625 630 635 640 Thr Ala Ser Gln Asn Ile Asn Gly Pro Ser Ser Arg Tyr Tyr Glu Arg 645 650 655 Gln Gln Gly Asn Arg Thr Ser Asn Leu Gly His Asn Asn Leu Pro Phe 660 665 670 Tyr His Ser Asn Asp Thr Thr Asp Glu Gln Lys Ala Met Ala Val Phe 675 680 685 Val Leu Ala Ser Phe Val Arg Asn Phe Pro Leu Gly Gln Lys Asn Cys 690 695 700 Phe Ser Leu Glu Leu Val Asn Lys Leu Cys Phe Tyr Ile Asp Asn Ser 705 710 715 720 Glu Ile Pro Leu Leu Arg Gln Trp Cys Val Ile Leu Leu Gly Leu Leu 725 730 735 Phe Ala Asp Asn Pro Leu Asn Arg Phe Val Cys Met Asn Thr Gly Ala 740 745 750 Val Glu Ile Leu Leu Lys Ser Leu Lys Asp Pro Val Pro Glu Val Arg 755 760 765 Thr Ala Ser Ile Phe Ala Leu Lys His Phe Ile Ser Gly Phe Gln Asp 770 775 780 Ala Glu Val Ile Leu Arg Leu Gln Gln Glu Phe Glu Glu Gln Tyr Gln 785 790 795 800 Gln Leu His Ser Gln Leu Gln His Leu Gln Asn Gln Ser His Leu Gln 805 810 815 Gln Gln Gln Ser Gln Gln Gln Gln Gln His Leu Glu Gln Gln Gln Met 820 825 830 Lys Ile Glu Lys Gln Ile Arg His Cys Gln Val Met Gln Asn Gln Leu 835 840 845 Glu Val Ile Asp Leu Arg Lys Leu Lys Arg Gln Glu Ile Gly Asn Leu 850 855 860 Ile Ser Ile Leu Pro Leu Ile Asn Asp Gly Ser Ser Leu Val Arg Lys 865 870 875 880 Glu Leu Val Val Tyr Phe Ser His Ile Val Ser Arg Tyr Ser Asn Phe 885 890 895 Phe Ile Val Val Val Phe Asn Asp Leu Leu Glu Glu Ile Lys Leu Leu 900 905 910 Glu Lys Ser Asp Ile Asn Thr Arg Asn Thr Ser Asp Lys Tyr Ser Val 915 920 925 Ser Gln Gly Ser Ile Phe Tyr Thr Val Trp Lys Ser Leu Leu Ile Leu 930 935 940 Ala Glu Asp Pro Phe Leu Glu Asn Lys Glu Leu Ser Lys Gln Val Ile 945 950 955 960 Asp Tyr Ile Leu Leu Glu Leu Ser Ala His Lys Glu Leu Gly Gly Pro 965 970 975 Phe Ala Val Met Glu Lys Phe Leu Leu Lys Arg Ser Ser Lys Ala His 980 985 990 Gln Thr Gly Lys Phe Gly Phe Asn Ser Ser Gln Val Gln Phe Val Lys 995 1000 1005 Ser Ser Leu Arg Ser Phe Ser Pro Asn Glu Arg Val Asp Asn Asn Ala 1010 1015 1020 Phe Lys Lys Glu Gln Gln Gln His Asp Pro Lys Ile Ser His Pro Met 1025 1030 1035 1040 Arg Thr Ser Leu Ala Lys Leu Phe Gln Ser Leu Gly Phe Ser Glu Ser 1045 1050 1055 Asn Ser Asp Ser Asp Thr Gln Ser Ser Asn Thr Ser Met Lys Ser His 1060 1065 1070 Thr Ser Lys Lys Gly Pro Ser Gly Leu Tyr Leu Leu Asn Gly Asn Asn 1075 1080 1085 Asn Ile Tyr Pro Thr Ala Glu Thr Pro Arg Phe Arg Lys His Thr Glu 1090 1095 1100 Pro Leu Gln Leu Pro Leu Asn Ser Ser Phe Leu Asp Tyr Ser Arg Glu 1105 1110 1115 1120 Tyr Phe Gln Glu Pro Gln Met Lys Lys Gln Glu Ala Asp Glu Pro Gly 1125 1130 1135 Ser Val Glu Tyr Asn Ala Arg Leu Trp Arg Arg Asn Arg Asn Glu Thr 1140 1145 1150 Ile Ile Gln Glu Thr Gln Gly Glu Lys Lys Leu Ser Ile Tyr Gly Asn 1155 1160 1165 Trp Ser Lys Lys Leu Ile Ser Leu Asn Asn Lys Ser Gln Pro Lys Leu 1170 1175 1180 Met Lys Phe Ala Gln Phe Glu Asp Gln Leu Ile Thr Ala Asp Asp Arg 1185 1190 1195 1200 Ser Thr Ile Thr Val Phe Asp Trp Glu Lys Gly Lys Thr Leu Ser Lys 1205 1210 1215 Phe Ser Asn Gly Thr Pro Phe Gly Thr Lys Val Thr Asp Leu Lys Leu 1220 1225 1230 Ile Asn Glu Asp Asp Ser Ala Leu Leu Leu Thr Gly Ser Ser Asp Gly 1235 1240 1245 Val Ile Lys Ile Tyr Arg Asp Tyr Gln Asp Val Asp Thr Phe Lys Ile 1250 1255 1260 Val Ser Ala Trp Arg Gly Leu Thr Asp Met Leu Leu Thr Pro Arg Ser 1265 1270 1275 1280 Thr Gly Leu Leu Thr Glu Trp Leu Gln Ile Arg Gly Ser Leu Leu Thr 1285 1290 1295 Thr Gly Asp Val Lys Val Ile Arg Val Trp Asp Ala His Thr Glu Thr 1300 1305 1310 Val Glu Val Asp Ile Pro Ala Lys Thr Ser Ser Leu Ile Thr Ser Leu 1315 1320 1325 Thr Ala Asp Gln Leu Ala Gly Asn Ile Phe Val Ala Gly Phe Ala Asp 1330 1335 1340 Gly Ser Leu Arg Val Tyr Asp Arg Arg Leu Asp Pro Arg Asp Ser Met 1345 1350 1355 1360 Ile Arg Arg Trp Arg Ala Gly Asn Asp Lys Gln Gly Val Trp Ile Asn 1365 1370 1375 Asn Val His Leu Gln Arg Gly Gly Tyr Arg Glu Leu Val Ser Gly Ala 1380 1385 1390 Thr Asn Gly Val Val Glu Leu Trp Asp Ile Arg Ser Glu Asp Pro Val 1395 1400 1405 Glu Ser Phe Val Asp Gln Asn Val Thr Ser Gln Tyr Gly Ser Gln Gln 1410 1415 1420 Lys Pro Thr Thr Met Thr Cys Met Gln Val His Glu His Ala Pro Ile 1425 1430 1435 1440 Ile Ala Thr Gly Thr Lys Gln Ile Lys Ile Trp Thr Thr Ser Gly Asp 1445 1450 1455 Leu Leu Asn Ser Phe Lys Asn Ser His Asn Asn Gly Val Thr Ser Thr 1460 1465 1470 Leu Ala Ala Thr Gly Ile Pro Lys Ser Leu Ser Tyr Ser Ser Thr Ser 1475 1480 1485 Asp Ala Phe Leu Ser Ser Met Ala Phe His Pro His Arg Met Met Ile 1490 1495 1500 Ala Ala Thr Asn Ser His Asp Ser Ile Val Asn Ile Tyr Lys Cys Glu 1505 1510 1515 1520 Asp Glu Arg Ile Asp Tyr Phe 1525 24 1683 PRT C. elegans 24 Glu Cys Val Asn Arg Arg Lys Gly Ser Asp Arg Leu Arg Thr Ala Ser 1 5 10 15 Val Ala Ile Ile Met Cys Leu His Leu Gly Val Asp Pro Pro Glu Ala 20 25 30 Pro Ser Arg Lys Asp Ala Pro Ser Arg Leu Leu Ser Trp Val Asp Pro 35 40 45 Tyr Lys Cys Gly Ala His Lys Ala Ala Ile Glu Ile Gly Leu Ser Thr 50 55 60 Gln Arg Ala Tyr Glu Lys Trp Gln Pro Lys Ser Lys Thr Arg Tyr Lys 65 70 75 80 Ile Cys Thr Asp Pro Pro Ile Asp Asp Val Arg Lys Val Ala Thr Asn 85 90 95 Leu Arg Arg Ile Ser Gly Asn Asp Arg Val Leu Phe His Tyr Asn Gly 100 105 110 His Gly Val Pro Lys Pro Thr Asp Asn Gly Glu Ile Trp Val Phe Asn 115 120 125 Lys Ser Phe Thr Gln Tyr Ile Pro Leu Ser Ile Phe Asp Leu Gln Gly 130 135 140 Trp Leu Asp Tyr Pro Thr Ile Tyr Ile Trp Glu Thr His Ser Ala Glu 145 150 155 160 Thr Ile Leu His Asn Tyr Arg Arg Phe Gly Glu Asp Gln Lys Ile Ser 165 170 175 Trp Gln Ser Arg Phe Asp Arg Trp Gln Glu Glu Gln Arg Asn Leu Pro 180 185 190 Pro Ile Ser Ser Lys Met Thr Asp Ala Asp Gln Ala Glu Ala Leu Gly 195 200 205 Phe Pro Glu Lys Tyr Pro Arg Tyr Asn Asp Cys Ile His Leu Ala Ala 210 215 220 Cys Ser Ala Gly Gln Trp Leu Pro Met Ser Asn Pro His Leu Pro Ala 225 230 235 240 Asp Leu Phe Thr Ser Cys Leu Thr Thr Pro Ile Arg Thr Ser Leu Ala 245 250 255 Phe His Leu Ser His Ser Glu His Arg Asp Glu Tyr Pro Glu Asn Ile 260 265 270 Leu Asp Met Ile Pro Gly Asn Leu Ala Glu Arg Arg Thr Val Leu Gly 275 280 285 Glu Leu Asn Trp Ile Phe Thr Ala Ile Thr Asp Thr Ile Ala Phe Thr 290 295 300 Ser Ile Asp Arg Asp Ser Phe Gln Lys Leu Phe Arg Gln Asp Leu Leu 305 310 315 320 Leu Ala Thr Leu Phe Arg Ser Phe Leu Leu Ala Gln Arg Val Met Ser 325 330 335 Lys Phe Asn Val Leu Pro Val Ser Gln Pro Pro Leu Lys Asp Thr Ser 340 345 350 Met His Asp Leu Trp Arg Cys Trp Asp Gln Thr Met Asp Met Val Leu 355 360 365 Asp Tyr Cys Arg Glu Leu Ile Tyr Val Lys Glu Cys Glu Arg Ala Leu 370 375 380 Phe Cys Gly Arg Asp Val Ile Asn Arg Ser Asn Val Phe Pro Phe Leu 385 390 395 400 Val Leu Ile Asp Phe Glu His Val Leu Thr Tyr Asn Glu Thr His Gln 405 410 415 Tyr Asn Thr Phe Phe Thr Glu Gln Leu Gln Ala Phe Glu Leu Trp Leu 420 425 430 Asp Tyr Gly Val Asp Glu Gly Thr Pro Pro Leu Gln Leu Pro Met Val 435 440 445 Leu Gln Val Leu Leu Ser Gln Ala His Arg Val Arg Ala Leu Arg Leu 450 455 460 Leu Ala Arg Phe Leu Asp Phe Gly Arg Trp Ala Ile Gly Tyr Ser Leu 465 470 475 480 Ala Val Gly Ile Phe Pro Tyr Val Leu Lys Leu Leu Gln Ser Asn Ile 485 490 495 Ser Asp Leu Lys Ile Ser Leu Ala Phe Ile Trp Ala Lys Ile Leu Ala 500 505 510 Val Asp Pro Ser Cys Gln Met Glu Leu Phe Asn Glu Cys Ile Asp Glu 515 520 525 Ser Ala Thr Gln Gln Gln Ser Pro Ala Gln Ser Gln Gln Pro Ser Val 530 535 540 Ala Leu Lys Pro Ser Ser Met Phe Ala Asn His Pro Pro Pro Ala Gln 545 550 555 560 Leu Thr Met Asp Lys Arg Ala Leu Gln Ile Asn Thr Gln Thr Gly Gly 565 570 575 Arg Phe Gln Thr Asn Gln Asn Gln Ala Leu Asp Met Gln Leu Gln Tyr 580 585 590 Ala Gln Gln Gln Ala His Gln Arg Gln Met Asp Pro Ser Gly Gly Gln 595 600 605 His Ser Pro Asn Ala Gln Pro Arg Gln Pro Gln Pro Gln Gln Pro Gln 610 615 620 Gln Leu Gln Gln Gln Pro Ser Gln Gln Gln Gln Gln Gln Ser Arg Gln 625 630 635 640 Pro Gln Gly Ile Met Lys Pro Thr Asn Ala Gln Pro Gln Glu Lys Lys 645 650 655 Thr Met Leu Arg Phe Gln Tyr Phe Leu Gln Leu Leu Gly Asp Pro Glu 660 665 670 Ile Lys Pro Lys Gln Lys Thr Val Ala Ala Phe Val Leu Ala Gln Leu 675 680 685 Thr Ser Asn Asn Phe Lys Leu Ala Gln Lys Glu Leu Thr Asn Lys Gly 690 695 700 Tyr Met Gly Ile Cys Thr Glu Leu Met Val Asp Asp Thr Ala Arg Ser 705 710 715 720 Val Lys Leu Leu Lys Leu Trp Ile Leu Ile Gly Leu Gly Arg Leu Trp 725 730 735 Ser Asp Tyr Asp Val Ala Arg Trp Gln Gly Ile Arg Leu Met Ala His 740 745 750 Asp Lys Met Ile Leu Glu Leu Ser Asp Glu Ser Ala Glu Val Arg Ala 755 760 765 Ala Ala Val Phe Ala Leu Gly Ser Leu Leu Arg Asn Ser Ser Arg Ser 770 775 780 Asn Glu His Ala Ser Ala Val Glu Glu Asn Leu Ala Gly Glu Leu Cys 785 790 795 800 Ser Gln Cys Val Phe Asp Ser Ser Val Leu Val Arg Glu Glu Leu Ile 805 810 815 Val Ala Leu Gln Trp Phe Val Phe Asp Phe Glu Lys Arg Phe Val Lys 820 825 830 Phe Leu Leu Glu Leu Ser Asn Gln Ile Lys Phe Lys Leu Pro Arg Lys 835 840 845 Arg Asn Ser Ser Glu Asn Leu Ser Glu Gln Gly Leu Asp Ile Ala Ala 850 855 860 Glu Met Pro Ser Asn His Arg Arg Pro Arg Ala Pro Pro Val Asn Glu 865 870 875 880 Phe Met Gln Pro Ser Val Ile Arg Lys Lys Met Ser Thr Ser Val Phe 885 890 895 Ser Thr Ala Val Glu Glu Thr Val His Glu Asp Gly Ile Gly Asn Val 900 905 910 Tyr His Ile Ala His Glu Ala Gly Asn Arg Val Glu Gln Glu Ser Glu 915 920 925 Asp Ile Glu Tyr Arg Glu Arg Ala Lys Val Gln Ile Lys His Leu Glu 930 935 940 Ala Lys Thr Phe Asn Glu Ala Val Ala Lys Thr Trp Leu Ser Leu Leu 945 950 955 960 Ile Leu Ser Leu Asp Pro Ile Glu Arg Val Ala Arg Met Ala Gln Lys 965 970 975 Ile Val His Arg Val Glu Ala Gly Met Pro Glu Met Gln Ala Asn Ile 980 985 990 Asp Asn Thr Met Ala His Leu Ser Arg Lys Met Ile Ala Arg Lys Asn 995 1000 1005 Ser Lys Ala Thr Val Glu Met Pro Arg Lys Pro Val Gly Pro Ser Glu 1010 1015 1020 Glu Val Ser Thr Leu Ser Glu Arg Ile Lys Arg Asn Leu Glu Val Glu 1025 1030 1035 1040 Lys Glu Ser Gly Val Ser Ala Asp Gly Ile Asp Phe Thr Lys Leu Asn 1045 1050 1055 Met Gln Lys Ala Lys Glu Gln Arg Lys Gly Gly Lys Gly Gly Gln Ile 1060 1065 1070 Lys Lys Leu Gln Asp Glu Ser Lys Val Lys Phe Gln Val Gly Ser Pro 1075 1080 1085 Gly Asp Val Asn Gln Gln His Lys Asn Ala Leu Ser Pro Gly Ser Ser 1090 1095 1100 Phe Thr Asp Glu Thr Gln Ser Ser Glu Leu Phe Ser Lys Asp Asp Ser 1105 1110 1115 1120 Ser Asp Asp Asp Glu Asn Lys Ser Ala Asn Asp Glu Lys Asp Arg Ala 1125 1130 1135 Thr Thr Ala Ser Gly Lys Thr Val Lys Gly Ala Phe Arg Lys Ser Asp 1140 1145 1150 Ile Met Asp Ala Ala Leu Met Glu Phe Thr Pro Arg Arg Thr Lys Ala 1155 1160 1165 Val Pro Gly Asp Phe Ala Val Lys Glu Glu Ser Lys Thr Leu Arg Thr 1170 1175 1180 Val Ile Glu Asn Pro Ile Val Ser Thr Gln Phe Val Ala Trp Cys Ser 1185 1190 1195 1200 Lys Val Phe Val Glu Pro Ile Leu His Val Ile Thr Leu Asp Glu Glu 1205 1210 1215 Lys Ser Glu Asp Trp Glu His Glu Asp Glu Asp Glu Phe Met Pro Glu 1220 1225 1230 Gln Phe Arg Glu Lys Ile Lys Asp Ile Gln Lys Gln Asp Arg Lys Met 1235 1240 1245 His Glu Glu Lys Arg Ala Arg Asp Lys Lys Glu Gly Lys Lys Glu Lys 1250 1255 1260 Ser Gln Glu Asp Val Val Val Thr Thr Thr Thr Ile Ser Asp Trp Ala 1265 1270 1275 1280 Ile His Ala Met Glu Gly Met Leu Gln Ser Ala Asp Ile Glu Gly Arg 1285 1290 1295 Asp Phe Gln Ser Cys Lys Tyr Asp Glu Cys Leu Trp Arg Val Lys Leu 1300 1305 1310 Thr His Pro Ala Arg Ser Leu Thr Thr Ser Lys Leu Arg Arg Cys Met 1315 1320 1325 Tyr Ala Ser Asp Gly Arg Leu Met Thr Ile Ile Arg Gln Asp Thr Asp 1330 1335 1340 Thr Arg Ser Phe Arg Arg Phe Asp Leu Thr Ala Ser Asn Pro Phe Cys 1345 1350 1355 1360 Pro Ser Tyr Val Ser Gln Leu Ile Leu Ile Asn Asp Met Ser Arg Glu 1365 1370 1375 Met Val Ile Ala Cys Ser Thr Asn Gly Val Val Arg Ile Trp Asp Pro 1380 1385 1390 Tyr Phe Leu Gly Trp Cys Asp Asp Tyr Glu Lys Pro Pro Glu Leu Val 1395 1400 1405 Ser Ala Ser Phe Pro Leu Glu Ser Gln Met Lys Ile Ser Asp Glu Thr 1410 1415 1420 Asn Arg Cys Leu Phe Asp Trp Asn Gln Gln Asn Gly Lys Leu Leu Cys 1425 1430 1435 1440 Thr Gly Thr Arg Ser Val Arg Ile Trp Asp Ala His Cys Glu Lys Val 1445 1450 1455 Cys His Asp Val Met Tyr Ser Pro Gly Ser Lys Gln Gln Thr Gly Pro 1460 1465 1470 Gly Ala Lys Leu Asn Thr Asn Thr Leu Pro Thr Ala Met Ser Gly Asn 1475 1480 1485 Leu Asp Glu Glu Gly Asn Met Val Ala Val Gly Tyr Ala Asp Gly Arg 1490 1495 1500 Val Asp Tyr Phe Asp Met Arg Met Ala Asn Pro Lys Ala Ala Arg Cys 1505 1510 1515 1520 Asn Leu Ala Pro Val Asn Met Ser Lys Gln Phe Asn Asp Arg Pro Pro 1525 1530 1535 Gly Ile Met His Leu Arg Val Asn Arg Gln Gly Phe Gly Thr Glu Leu 1540 1545 1550 Phe Ala Gly Ser Gln Asp Gly Ser Ile Tyr Lys Leu Gln Leu Arg Met 1555 1560 1565 Phe Lys Glu Ala Thr Pro Ser Ile Val Thr Pro Trp Glu Tyr Gly Ala 1570 1575 1580 His Ser His Met Phe Val His Glu Asp Ser Arg Ile Leu Ala Ser Ala 1585 1590 1595 1600 Gly Gly Gly Glu Leu Ala Ile Tyr Asp Val Ala Gln Asn Lys Leu Met 1605 1610 1615 Ala Arg Ile Leu Pro Pro Gly Glu Pro Asn Ser Met Arg Arg Asp Lys 1620 1625 1630 Asn Ser Ser Thr Phe Gly Leu Phe Ser Ser Gln Arg Lys Leu Ser Gln 1635 1640 1645 Val Gln Met Ser Gly Arg Met Ser Ser Met Ser Ser Ala Thr Val Gly 1650 1655 1660 Glu Asp Arg Gln Pro Lys Val Ile Ser Leu Thr Met His Gln Met Arg 1665 1670 1675 1680 Leu Val Asp 25 1384 PRT A. thaliana 25 Met Ala Leu Gly Asp Leu Met Val Ser Arg Phe Ser Gln Ser Ser Val 1 5 10 15 Ser Leu Val Ser Asn His Arg Tyr Asp Glu Asp Cys Val Ser Ser His 20 25 30 Asp Asp Gly Asp Ser Arg Arg Lys Asp Ser Glu Ala Lys Ser Ser Ser 35 40 45 Ser Tyr Gly Asn Gly Thr Thr Glu Gly Ala Ala Thr Ala Thr Ser Met 50 55 60 Ala Tyr Leu Pro Gln Thr Ile Val Leu Cys Glu Leu Arg His Asp Ala 65 70 75 80 Ser Glu Ala Ser Ala Pro Leu Gly Thr Ser Glu Ile Val Leu Val Pro 85 90 95 Lys Trp Arg Leu Lys Glu Arg Met Lys Thr Gly Cys Val Ala Leu Val 100 105 110 Leu Cys Leu Asn Ile Thr Val Asp Pro Pro Asp Val Ile Lys Ile Ser 115 120 125 Pro Cys Ala Arg Ile Glu Ala Trp Ile Asp Pro Phe Ser Met Ala Pro 130 135 140 Pro Lys Ala Leu Glu Thr Ile Gly Lys Asn Leu Ser Thr Gln Tyr Glu 145 150 155 160 Arg Trp Gln Pro Arg Ala Arg Tyr Lys Val Gln Leu Asp Pro Thr Val 165 170 175 Asp Glu Val Arg Lys Leu Cys Leu Thr Cys Arg Lys Tyr Ala Lys Thr 180 185 190 Glu Arg Val Leu Phe His Tyr Asn Gly His Gly Val Pro Lys Pro Thr 195 200 205 Ala Asn Gly Glu Ile Trp Val Phe Asn Lys Ser Tyr Thr Gln Tyr Ile 210 215 220 Pro Leu Pro Ile Ser Glu Leu Asp Ser Trp Leu Lys Thr Pro Ser Ile 225 230 235 240 Tyr Val Phe Asp Cys Ser Ala Ala Arg Met Ile Leu Asn Ala Phe Ala 245 250 255 Glu Leu His Asp Trp Gly Ser Ser Gly Ser Ser Gly Ser Ser Arg Asp 260 265 270 Cys Ile Leu Leu Ala Ala Cys Asp Val His Glu Thr Leu Pro Gln Ser 275 280 285 Val Glu Phe Pro Ala Asp Val Phe Thr Ser Cys Leu Thr Thr Pro Ile 290 295 300 Lys Met Ala Leu Lys Trp Phe Cys Arg Arg Ser Leu Leu Lys Glu Ile 305 310 315 320 Ile Asp Glu Ser Leu Ile Asp Arg Ile Pro Gly Arg Gln Asn Asp Arg 325 330 335 Lys Thr Leu Leu Gly Glu Leu Asn Trp Ile Phe Thr Ala Val Thr Asp 340 345 350 Thr Ile Ala Trp Asn Val Leu Pro His Glu Leu Phe Gln Arg Leu Phe 355 360 365 Arg Gln Asp Leu Leu Val Ala Ser Leu Phe Arg Asn Phe Leu Leu Ala 370 375 380 Glu Arg Ile Met Arg Ser Ala Asn Cys Asn Pro Ile Ser His Pro Met 385 390 395 400 Leu Pro Pro Thr His Gln His His Met Trp Asp Ala Trp Asp Met Ala 405 410 415 Ala Glu Ile Cys Leu Ser Gln Leu Pro Gln Leu Val Leu Asp Pro Ser 420 425 430 Thr Glu Phe Gln Pro Ser Pro Phe Phe Thr Glu Gln Leu Thr Ala Phe 435 440 445 Glu Val Trp Leu Asp His Gly Ser Glu His Lys Lys Pro Pro Glu Gln 450 455 460 Leu Pro Ile Val Leu Gln Val Leu Leu Ser Gln Cys His Arg Phe Arg 465 470 475 480 Ala Leu Val Leu Leu Gly Arg Phe Leu Asp Met Gly Ser Trp Ala Val 485 490 495 Asp Leu Ala Leu Ser Val Gly Ile Phe Pro Tyr Val Leu Lys Leu Leu 500 505 510 Gln Thr Thr Thr Asn Glu Leu Arg Gln Ile Leu Val Phe Ile Trp Thr 515 520 525 Lys Ile Leu Ala Leu Asp Lys Ser Cys Gln Ile Asp Leu Val Lys Asp 530 535 540 Gly Gly His Thr Tyr Phe Ile Arg Phe Leu Asp Ser Ser Gly Ala Phe 545 550 555 560 Pro Glu Gln Arg Ala Met Ala Ala Phe Val Leu Ala Val Ile Val Asp 565 570 575 Gly His Arg Arg Gly Gln Glu Ala Cys Leu Glu Ala Asn Leu Ile Gly 580 585 590 Val Cys Leu Gly His Leu Glu Ala Ser Arg Pro Ser Asp Pro Gln Pro 595 600 605 Glu Pro Leu Phe Leu Gln Trp Leu Cys Leu Cys Leu Gly Lys Leu Trp 610 615 620 Glu Asp Phe Met Glu Ala Gln Ile Met Gly Arg Glu Ala Asn Ala Phe 625 630 635 640 Glu Lys Leu Ala Pro Leu Leu Ser Glu Pro Gln Pro Glu Val Lys Phe 645 650 655 Arg Phe Leu Tyr Asn Val Leu Arg Val Arg Ala Ala Ala Val Phe Ala 660 665 670 Leu Gly Thr Leu Leu Asp Ile Gly Phe Asp Ser Asn Lys Ser Val Val 675 680 685 Glu Asp Glu Phe Asp Asp Asp Glu Lys Ile Arg Ala Glu Asp Ala Ile 690 695 700 Ile Lys Ser Leu Leu Asp Val Val Ser Asp Gly Ser Pro Leu Val Arg 705 710 715 720 Ala Glu Val Ala Val Ala Leu Ala Arg Phe Ala Phe Gly His Lys Gln 725 730 735 His Leu Lys Leu Ala Ala Ala Ser Tyr Trp Lys Pro Gln Ser Ser Ser 740 745 750 Leu Leu Thr Ser Leu Pro Ser Ile Ala Lys Phe His Asp Pro Gly Ser 755 760 765 Ala Thr Ile Val Ser Leu His Met Ser Pro Leu Thr Arg Ala Ser Thr 770 775 780 Asp Ser Gln Pro Val Ala Arg Glu Ser Arg Ile Ser Ser Ser Pro Leu 785 790 795 800 Gly Ser Ser Gly Leu Met Gln Gly Ser Pro Leu Ser Asp Asp Ser Ser 805 810 815 Leu His Ser Asp Ser Gly Met Met His Asp Ser Val Ser Asn Gly Ala 820 825 830 Val His Gln Pro Arg Leu Leu Asp Asn Ala Val Tyr Ser Gln Cys Val 835 840 845 Arg Ala Met Phe Ala Leu Ala Lys Asp Pro Ser Pro Arg Ile Ala Ser 850 855 860 Leu Gly Arg Arg Val Leu Ser Ile Ile Gly Ile Glu Gln Val Val Ala 865 870 875 880 Lys Pro Ser Lys Pro Thr Gly Arg Pro Gly Glu Ala Ala Thr Thr Ser 885 890 895 His Thr Pro Leu Ala Gly Leu Ala Arg Ser Ser Ser Trp Phe Asp Met 900 905 910 His Ala Glu Phe Phe Lys Val Leu Arg Asn Leu Arg Met Leu Phe Ile 915 920 925 Leu Glu Met Leu Val Asn Ser Asn Leu Thr Leu Leu Leu Thr Thr Gly 930 935 940 Asn Leu Pro Leu Ser Phe Arg Thr Pro Pro Val Ser Pro Pro Arg Thr 945 950 955 960 Asn Tyr Leu Ser Gly Leu Arg Arg Val Cys Ser Leu Glu Phe Arg Pro 965 970 975 His Leu Leu Gly Ser Pro Asp Ser Gly Leu Ala Asp Pro Leu Leu Gly 980 985 990 Ala Ser Gly Ser Glu Arg Ser Leu Leu Pro Leu Ser Thr Ile Tyr Gly 995 1000 1005 Trp Ser Cys Gly His Phe Ser Lys Pro Leu Leu Gly Gly Ala Asp Ala 1010 1015 1020 Ser Gln Glu Ile Ala Ala Lys Arg Glu Glu Lys Glu Lys Phe Ala Leu 1025 1030 1035 1040 Glu His Ile Ala Lys Cys Gln His Ser Ser Ile Ser Lys Leu Asn Asn 1045 1050 1055 Asn Pro Ile Ala Asn Trp Asp Thr Arg Phe Glu Thr Gly Thr Lys Thr 1060 1065 1070 Ala Leu Leu His Pro Phe Ser Pro Ile Val Val Ala Ala Asp Glu Asn 1075 1080 1085 Glu Arg Ile Arg Val Trp Asn Tyr Glu Glu Ala Thr Leu Leu Asn Gly 1090 1095 1100 Phe Asp Asn His Asp Phe Pro Asp Lys Gly Ile Ser Lys Leu Cys Leu 1105 1110 1115 1120 Ile Asn Glu Leu Asp Asp Ser Leu Leu Leu Val Ala Ser Cys Asp Gly 1125 1130 1135 Ser Val Arg Ile Trp Lys Asn Tyr Ala Thr Lys Gly Lys Gln Lys Leu 1140 1145 1150 Val Thr Gly Phe Ser Ser Ile Gln Gly His Lys Pro Gly Ala Arg Asp 1155 1160 1165 Leu Asn Ala Val Val Asp Trp Gln Gln Gln Ser Gly Tyr Leu Tyr Ala 1170 1175 1180 Ser Gly Glu Thr Ser Thr Val Thr Leu Trp Asp Leu Glu Lys Glu Gln 1185 1190 1195 1200 Leu Val Arg Ser Val Pro Ser Glu Ser Glu Cys Gly Val Thr Ala Leu 1205 1210 1215 Ser Ala Ser Gln Val His Gly Gly Gln Leu Ala Ala Gly Phe Ala Asp 1220 1225 1230 Gly Ser Leu Arg Leu Tyr Asp Val Arg Ser Pro Glu Pro Leu Val Cys 1235 1240 1245 Ala Thr Arg Pro His Gln Lys Val Glu Arg Val Val Gly Leu Ser Phe 1250 1255 1260 Gln Pro Gly Leu Asp Pro Ala Lys Val Val Ser Ala Ser Gln Ala Gly 1265 1270 1275 1280 Asp Ile Gln Phe Leu Asp Leu Arg Thr Thr Arg Asp Thr Tyr Leu Thr 1285 1290 1295 Ile Asp Ala His Arg Gly Ser Leu Thr Ala Leu Ala Val His Arg His 1300 1305 1310 Ala Pro Ile Ile Ala Ser Gly Ser Ala Lys Gln Leu Ile Lys Val Phe 1315 1320 1325 Ser Leu Gln Gly Glu Gln Leu Gly Ile Ile Arg Tyr Tyr Pro Ser Phe 1330 1335 1340 Met Ala Gln Lys Ile Gly Ser Val Ser Cys Leu Thr Phe His Pro Tyr 1345 1350 1355 1360 Gln Val Leu Leu Ala Ala Gly Ala Ala Asp Ser Phe Val Ser Ile Tyr 1365 1370 1375 Thr His Asp Asn Ser Gln Ala Arg 1380 26 9 PRT Unknown Peptide within KIAA1303, a partial ORF for a human protein of unknown function. 26 Ser Val Ser Ser Tyr Gly Asn Ile Arg 1 5 27 9 PRT Unknown Peptide within KIAA1303, a partial ORF for a human protein of unknown function. 27 Leu Asp Asp Gln Ile Phe Leu Asn Arg 1 5 28 12 PRT Unknown Peptide within KIAA1303, a partial ORF for a human protein of unknown function. 28 Ile Pro Glu Glu His Asp Leu Glu Ser Gln Ile Arg 1 5 10

Claims

What is claimed is:

1. An isolated nucleic acid molecule comprising SEQ ID NO: 1.

2. An isolated nucleic acid molecule which is the complement of SEQ ID NO: 1.

3. An isolated nucleic acid molecule that encodes an amino acid sequence comprising SEQ ID NO: 2.

4. The isolated nucleic acid of claim 3 which comprises SEQ ID NO: 1.

5. An isolated nucleic acid molecule comprising a sequence that hybridizes under highly stringent conditions to SEQ ID NO: 1 or a complement of SEQ ID NO: 1.

6. An isolated nucleic acid molecule comprising a sequence that hybridizes under highly stringent conditions to a complement of SEQ ID NO: 1 and encodes a mammalian raptor protein.

7. An isolated polypeptide encoded by a nucleic acid comprising SEQ ID NO: 1.

8. An isolated polypeptide having an amino acid sequence comprising SEQ ID NO: 2.

9. An expression construct comprising SEQ ID NO: 1.

10. The expression construct of claim 9 wherein SEQ ID NO: 1 is operably linked to a regulatory sequence.

11. A host cell comprising the isolated nucleic acid of claim 3.

12. The host cell of claim 11 wherein the isolated nucleic acid is operably linked to a regulatory sequence.

13. A method of producing a mammalian raptor polypeptide comprising culturing the host cell of claim 12 under conditions in which the raptor polypeptide is produced.

14. The method of claim 13 further comprising isolating the raptor polypeptide from the cell.

15. An isolated raptor polypeptide produced by the method of claim 14.

16. An antibody or antigen binding fragment thereof that specifically binds to a mammalian raptor polypeptide, wherein the mammalian raptor polypeptide is encoded by an isolated nucleic acid that encodes SEQ ID NO: 2.

17. The antibody of claim 16 wherein the antibody recognizes an epitope from about amino acid 985 to about amino acid 1001 of SEQ ID NO: 2.

18. The antibody of claim 17 wherein the antibody is a polyclonal antibody.

19. A method of identifying a nucleic acid that encodes a mammalian raptor polypeptide in a sample comprising:

a) contacting the sample with a nucleotide sequence comprising SEQ ID NO: 1 under conditions in which hybridization occurs between SEQ ID NO: 1 and nucleic acid in the sample using high stringency conditions;

b) identifying the nucleic acid of a) which hybridizes to SEQ ID NO: 1 under high stringency conditions,

thereby identifying a nucleic acid that encodes a mammalian raptor polypeptide in a sample.

20. A method of identifying a mammalian raptor polypeptide in a sample comprising:

a) contacting the sample with an antibody or antigen binding fragment thereof that specifically binds to a mammalian raptor polypeptide wherein the mammalian raptor polypeptide is encoded by an isolated nucleic acid that encodes SEQ ID NO: 2; and

b) identifying the polypeptide which specifically binds to the antibody, thereby identifying a mammalian raptor polypeptide in a sample.

21. The method of claim 20 wherein the antibody recognizes an epitope from about amino acid 985 to about amino acid 1001 of SEQ ID NO: 2.

22. The method of claim 21 wherein the antibody is a polyclonal antibody.

23. A method of identifying an agent that alters interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising:

a) contacting a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 with mTOR protein under conditions in which the raptor protein interacts with the mTOR protein, with an agent to be assessed;

b) assessing the extent to which raptor interacts with mTOR in the presence of the agent to be assessed,

wherein if the extent to which raptor interacts with mTOR is altered in the presence of the agent compared to the extent to which raptor interacts with mTOR in the absence of the agent, then the agent alters interaction of a mammalian raptor protein with mTOR protein.

24. A method of identifying an agent that alters interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising:

a) contacting a cell which comprises nucleic acid that encodes a raptor protein having an amino acid sequence comprising SEQ ID NO: 2 wherein the raptor protein, when expressed, interacts with mTOR protein in the cell, with an agent to be assessed;

b) assessing growth rate or size of the cell,

wherein an altered growth rate or size of the cell compared to the growth rate or size of a control cell indicates that the agent alters interaction of a mammalian raptor protein with mTOR protein.

25. A method of identifying an agent that inhibits an interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising:

b) assessing growth rate or size of the cell,

wherein a decrease in growth rate or size of the cell compared to growth rate or size of a control cell indicates that the agent inhibits interaction of a mammalian raptor protein with mTOR protein.

26. The method of claim 25 wherein growth rate or size is assessed by measuring phosphorylation of a regulator of protein synthesis selected from the group consisting of: S6 kinase 1, 4E-BP1 and combinations thereof.

27. The method of claim 25 wherein growth rate or size is assessed by measuring binding of the mammalian raptor protein with mTOR protein.

28. A method of identifying an agent that enhances an interaction of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising:

b) assessing growth rate or size of the cell,

wherein an increase in growth rate or size of the cell compared to growth rate and/or size of a control cell indicates that the agent enhances interaction of a mammalian raptor protein with mTOR protein.

29. The method of claim 28 wherein growth rate or size is assessed by measuring phosphorylation of a regulator of protein synthesis selected from the group consisting of: S6 kinase 1, 4E-BP1 and combinations thereof.

30. The method of claim 28 wherein growth rate or size is assessed by measuring binding of the mammalian raptor protein with mTOR protein.

31. A method of altering growth rate or size of a cell comprising introducing into the cell an agent that alters interaction of mammalian raptor protein with mTOR.

32. A method of enhancing growth rate or size of a cell comprising introducing into the cell an agent that inhibits interaction of mammalian raptor protein with mammalian TOR protein.

33. The method of claim 32 wherein the agent is exogenous nucleic acid that inhibits activity of a mammalian raptor protein.

34. The method of claim 33 wherein the exogenous nucleic acid results in overexpression of mammalian raptor protein in the cell.

35. The method of claim 33 wherein the exogenous nucleic acid is mRNA that specifically targets and destroys the mammalian raptor protein.

36. The method of claim 35 wherein the mRNA that specifically targets and destroys the mammalian raptor protein comprises pairs of oligoribonucleotides which correspond to nucleotides from about nucleotide 1531 to about nucleotide 1551 of SEQ ID NO: 1.

37. A method of inhibiting growth rate or size of a cell comprising introducing into the cell an agent that enhances interaction of mammalian raptor protein with mammalian TOR protein.

38. The method of claim 35 wherein the agent is exogenous nucleic acid that enhances activity of a mammalian raptor protein.

39. A method of altering kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that alters interaction of a mammalian raptor protein with the mTOR.

40. A method of enhancing kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that inhibits interaction of a mammalian raptor protein with the mTOR.

41. A method of inhibiting kinase activity of mTOR protein in a cell comprising introducing into the cell an agent that enhances interaction of a mammalian raptor protein with the mTOR.

42. A method of treating cancer in an individual comprising administering to the individual an agent that enhances interaction of raptor protein with mTOR protein.

43. A method of identifying proteins that associate with mTOR within a cell comprising:

a) contacting a lysate of the cell with a reversible crosslinker thereby forming a crosslink between proteins that associate with mTOR within the cell;

b) separating proteins that are associated by the crosslink from the lysate;

thereby identifying proteins that associate with mTOR within a cell.

44. The method of claim 43 further comprising reducing the crosslink between the proteins and isolating the proteins.

45. The method of claim 43 wherein the reversible crosslinker is dithiobis(succinmidylpropionate) (DSP).

46. The method of claim 43 wherein the proteins that are associated by the crosslink are separated from the lysate using an antibody that specifically binds one of the proteins.

47. The method of claim 44 wherein the crosslink between the proteins is reduced using dithiothreitol.

48. A method of identifying proteins that associate with mTOR within a cell, comprising:

a) preparing a lysate of the cell with a buffer comprising a detergent other than Triton, thereby preserving an association between the proteins;

b) separating proteins that are associated from the lysate, thereby identifying proteins that associate with mTOR within a cell.

49. The method of claim 48 further comprising isolating the proteins that are associated from one another.

50. The method of claim 48 wherein the buffer comprises CHAPS.

51. The method of claim 50 wherein the buffer comprises about 0.05% to about 2% CHAPS.

52. The method of claim 51 wherein the buffer comprises about 0.3% CHAPS.

53. The method of claim 52 wherein the buffer further comprises 120 mM NaCl.