WO2011133493A2 - Allosteric regulation of kinase activity - Google Patents

Abstract

The present invention relates to fusion proteins comprising a kinase and a ligand binding domain inserted in the kinase in or near the catalytic domain to produce a ligand-dependent kinase. The invention further relates to polynucleotides and vectors encoding the fusion proteins, cells comprising the fusion proteins, and methods of using the fusion proteins to study kinase function and analyze cellular activity, as well as diagnostic and therapeutic methods.

Description

Allosteric Regulation of Kinase Activity

STATEMENT OF FEDERAL SUPPORT

[0001] This invention was made, in part, with government support under grant numbers GM 82288 and GM 57464 from the National Institutes of Health. The United States government has certain rights to this invention.

FIELD OF THE INVENTION

[0002] The present invention relates to fusion proteins comprising a kinase and a ligand binding domain inserted in the kinase in or near the catalytic domain to produce a ligand-dependent kinase. The invention further relates to polynucleotides and vectors encoding the fusion proteins, cells comprising the fusion proteins, and methods of using the fusion proteins to study kinase function and analyze cellular activity, as well as diagnostic and therapeutic methods.

BACKGROUND OF THE INVENTION

[0003] Current approaches to alter protein function in living cells suffer from critical limitations. Genetic alterations exert their effects slowly, enabling cells to compensate, and preventing control of activity with precise timing during dynamic processes. Small molecule inhibitors have much better kinetic resolution, but often show broad or unknown specificity and are unavailable for many targets. Recent chemical biology approaches provide exciting alternatives, but are often technically challenging and/or applicable only to a limited set of proteins. Development of a more universal method suitable for regulation of a specific domain of a protein will help to overcome many of the limitations of current tools.

[0004] Catalytic function of protein kinases is an essential element of the signaling machinery in a living cell. For example, focal adhesion kinase (FAK) is a tyrosine kinase that has been implicated in a wide variety of cell behaviors, including proliferation, apoptosis, migration and tumorigenesis, but relatively little is known about the specific role of its catalytic activity. FAK is a multidomain protein that functions as both a scaffolding protein and a kinase. Despite a significant amount of data demonstrating the biological importance of FAK, relatively little is known about its role in modulation of cellular events. In part, such a surprising discrepancy can be explained by the lack of reliable tools for regulation of the kinase activity of FAK with high specificity and temporal resolution. [0005] The present invention addresses previous shortcomings in the art by providing a strategy for regulating kinase activation with high specificity and temporal resolution.

SUMMARY OF THE INVENTION

[0006] The present invention is based, in part, on the identification of methods for regulating the catalytic activity of kinases. The insertion of a ligand binding domain (LBD) in or near the catalytic domain of a kinase permits ligand-dependent regulation of kinase activity with high specificity and temporal resolution. The fusion proteins of the invention provide tremendous opportunities to study protein and cell function for research, diagnostic, and therapeutic purposes.

[0007] Accordingly, as one aspect, the invention relates to a fusion protein comprising a kinase and a LBD inserted within the kinase, wherein the kinase is catalytically active when a ligand is bound to the LBD and is less active when no ligand is bound to the LBD. In another aspect, the invention relates to a fusion protein comprising a kinase and a LBD inserted within the kinase, wherein the kinase is catalytically inactive when a ligand is bound to the LBD and is more active when no ligand is bound to the LBD. In certain embodiments, the ligand is a small molecule that readily enters cells. In other embodiments, the ligand is a signal such as illumination.

[0008] In another aspect, the invention relates to polynucleotides and vectors encoding the fusion proteins of the invention.

[0009] In a further aspect, the invention relates to host cells comprising the polynucleotides and vectors of the invention and expressing the fusion proteins of the invention.

[0010] In another aspect, the invention relates to methods of producing the fusion proteins of the invention, comprising expressing the fusion protein encoded by the polynucleotides and/or vectors of the invention.

[0011] In an additional aspect, the invention relates to cells comprising the fusion proteins of the invention.

[0012] In a further aspect, the invention relates to methods of converting a kinase to a ligand-dependent kinase, comprising inserting a LBD into or near the catalytic domain of the kinase.

[0013] In an additional aspect, the invention relates to methods of activating a kinase present in the fusion protein of the invention, comprising contacting the fusion protein with a ligand that binds to the LBD. [0014] In another aspect, the invention relates to methods of inactivating a kinase present in the fusion protein of the invention, comprising contacting the fusion protein with a ligand that binds to the LBD.

[0015] In a further aspect, the invention relates to methods of assessing the function of a kinase present in the fusion protein of the invention, comprising contacting the fusion protein with a ligand that binds to the LBD and assessing one or more activities of the kinase.

[0016] In an additional aspect, the invention relates to methods of assessing the function of a kinase present in the fusion protein of the invention, comprising removing a ligand that binds to the LBD from the fusion protein and assessing one or more activities of the kinase.

[0017] In another aspect, the invention relates to kits comprising the fusion proteins, polynucleotides, vectors, and/or cells of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1A-1D show the design and generation of apR-FAK. Figure 1A is a schematic representation of the approach used to regulate the catalytic activity of FAK. A fragment of FKBP12 is inserted at a position in the catalytic domain where it abrogates catalytic activity. Binding to rapamycin and FRB restores activity. Figure IB shows the truncated fragment of FKBP12 (iFKBP) inserted into the kinase domain. Black and dark gray, full length FKBP12; dark gray, proposed structure of the inserted fragment. The FKBP12 is shown in complex with rapamycin and FRB (light gray). Figure 1C shows an immunoblot analysis of iFKBP interaction with rapamycin and FRB. Myc-tagged FKBP12 and iFKBP constructs were immunoprecipitated from cells treated for 1 hour with either 200 nM rapamycin or ethanol (solvent control). Co- immunoprecipitation of co-expressed GFP-FRB was detected using anti-GFP antibody (example of three independent experiments). Figure ID shows changes in the molecular dynamics of iFKBP upon binding to rapamycin and FRB. Darker colors and thicker tube length indicate increasing root mean square fluctuation (RMSF).

[0019] Figure 2 shows the root mean square fluctuation (RMSF) of each amino acid residue in iFKBP, with and without ligands.

[0020] Figures 3A-3F show development and biochemical characterization of RapR-FAK. Figure 3A shows rapamycin regulation of FAK variants with iFKBP inserted at different positions. HEK293T cells coexpressing myc-tagged FAK constructs and GFP-FRB were treated for one hour with either 200 nM rapamycin or ethanol (solvent control). The activity of immunoprecipitated FAK variants was tested using the N-terminal fragment of paxillin as a kinase substrate. Figure 3B shows sites of iFKBP insertion (light gray) and connecting linkers (bold). Figure 3C shows FAK Y180A and M183A mutations were introduced to eliminate autoinhibitory interactions, thereby generating RapR-FAK-YM, which was tested as in Figure 3A. Figure 3D shows HEK293T cells co-expressing Cherry- FRB, GFP-paxillin and either myc-tagged RapR- FAK-YM or its kinase-inactive mutant (RapR-FAK-YM-KD) were treated with 200 nM rapamycin or ethanol (solvent control) for 1 hour. GFP-paxillin was immunoprecipitated and its phosphorylation was assessed using anti-phosphoTyr31 antibody. Autophosphorylation of FAK on Tyr397 was also analyzed using total cell lysate. Figures 3E and 3F show F1EK293T cells co-expressing RapRFAK and FRB were treated with the indicated amount of rapamycin for 1 hour or with 200 nM rapamycin for the indicated period of time. The kinase was immunoprecipitated and its activity tested as described above (all gels are representative of at least three independent experiments).

[0021] Figure 4 shows the predicted structures of FAK with different iFKBP insertions in the Ala442-Met448 loop of FAK (FAK = light gray, iFKBP insert = dark gray, linkers = black). In FAK-iFKBP445, the iFKBP insert replaces FAK amino acid Glu445. The FAK-.FKBP442-448 construct has an iFKBP insert replacing Ala442- Met448, without any linkers. In the RapR-FAK construct the iFKBP insert and Gly-Pro- Gly linkers replace Ala442-Met448.

[0022] Figures 5A-5B show the interaction of RapR-FAK with binding partners. HEK293T cells were co-transfected with the indicated FAK construct and either Src (A) or paxillin (B). The FAK constructs were immunoprecipitated using anti-myc antibody. Co-immunoprecipitation of Src and paxillin was detected using anti-Src and anti-paxillin antibody.

[0023] Figure 6 shows the co-localization of wild-type FAK and RapR-FAK in HeLa cells. GFP-RapR-FAK and mCherry-FAK were co-expressed in HeLa cells and imaged using total internal reflection fluorescence (TIRF) microscopy.

[0024] Figure 7 shows the co-localization of FRB with RapR-FAK upon treatment with rapamycin. HeLa cells co-expressing mCherry-FRB and GFP-RapR-FAK were imaged using TIRF microscopy before and after addition of rapamycin.

[0025] Figures 8A-8D show activation of FAK catalytic activity initiates large dorsal ruffles via the activation of Src. Figure 8A shows rapamycin treatment of HeLa cells co-expressing RapR-FAK- YM and FRB caused formation of large dorsal ruffles. Figure 8B shows HeLa cells expressing either GFP-RapRFAK-YM (YM, 64 cells), GFP- RapR-FAK kinase-dead mutant (YM-KD, 35 cells) or GFP-tagged Y397F mutant (YM- Y397F, 47 cells) were scored for ruffle induction by rapamycin. No dorsal ruffles were seen before rapamycin addition. Figure 8C shows inhibition of Src family kinases eliminated the FAK-induced ruffles. Cells co-expressing GFP-RapR-FAKYM and Cherry-FRB were treated with rapamycin for 1 hour and imaged before and after addition of Src family kinase inhibitor PP2. PP2 addition stopped dorsal protrusion in all cells analyzed (16 cells). Figure 8D shows activation of FAK leads to activation of Src. HeLa cells co-expressing myc-tagged Src, Cherry-FRB and either GFP -RapR-FAK- YM or its Y397F mutant were treated with rapamycin for 1 hour. Src was immunoprecipitated using anti-myc antibody and its phosphorylation on Tyr418 was assessed by immunoblotting.

[0026] Figures 9A-9E show the mechanism of regulation by iFKBP and Src regulation. Figure 9A shows the portion of the FAK catalytic domain targeted for insertion of iFKBP (dark gray) and the G-loop (black). Figure 9B shows the dynamic correlation analysis of the FAK catalytic domain (black, positive correlation; gray, negative correlation). The circled region indicates strong negative correlation between the movement of the insertion loop and the G-loop. Figure 9C shows a tube representation depicting changes in the dynamics of the FAK catalytic domain's N- terminal lobe, based on molecular dynamics simulations. Darker colors and thicker backbone correspond to higher RMSF values, reflecting the degree of free movement within the structure. The arrow points to the G-loop. Figure 9D shows the root mean square fluctuation (RMSF) of amino acids in FAK and RapR-FAK (arrow indicates G- loop). The break in the wild type FAK graph corresponds to the iFKBP insert in RapR- FAK. Figure 9E shows regulation of Src kinase by insertion of iFKBP. HEK293T cells co-expressing the indicated myc-tagged Src construct and GFP-FRB were treated with either 200 nM rapamycin or ethanol solvent control. The kinase activity of immunoprecipitated Src was tested as in Figure 2A.

[0027] Figure 10 shows the root mean square fluctuation (RMSF) of each amino acid residue in the catalytic domain of FAK with different iFKBP insertions. Breaks in the graphs for RapR-FAK and FAK-iFKBP442-8 are created due to the lack of amino acids present in FAK-iFKBP445. All three constructs are depicted in Fig. 1.

[0028] Figure 11 shows a comparison of catalytic domains structure for FAK (PDB ID: 2J0M) and Src (PDB ID: 1YOJ). The loop where iFKBP is inserted (insertion loop) is depicted in dark gray. The G-loop is depicted in black. [0029] Figure 12 shows the root mean square fluctuation (RMSF) for each amino acid residue of the catalytic domains of wild-type Src and Src with iFKBP insertion (RapR-Src). The break in the graph for wild-type Src corresponds to the iFKBP insert. The arrow indicates the G-loop.

[0030] Figure 13 shows tube representations of the catalytic domain of wild-type Src and RapR-Src. Darker colors and thicker backbone correspond to higher RMSF values, reflecting the degree of free movement within the structure. The arrows point to the G-loop within the catalytic domain.

[0031] Figure 14 shows the rapamycin-dependent activity of RapR-Src fusion proteins in living cells.

[0032] Figure 15 shows the rapamycin-dependent kinase activity of RapR-p38 fusion proteins.

[0033] Figure 16 shows the regulation of RapR-FAK with non- immunosuppressive analogs of rapamycin.

[0034] Figure 17 shows the different behavior of Cos7 cells generated by activation of homologous kinases Src, Fyn and Yes.

[0035] Figures 18A-18B shows the targeted activation of RapR-Src at the plasma membrane. MEF (A) and HeLa (B) cells co-expressing the nonmyristoylated mutant of RapR-Src and myristoylated GFP-FRB were treated with 0.5 μΜ rapamycin.

[0036] Figure 19 shows a schematic diagram of targeted activation of RapR-Src in a complex with FAK using a FAK-FRB chimera.

[0037] Figure 20 shows that RapR-Src activated in a complex with a FAK-FRB chimera induces formation of large dorsal protrusions. HeLa cells co-transfected with RapR-Src and GFP-FAK-FRB were treated with 0.5 μΜ rapamycin to activate RapR-Src. Arrowheads indicate dorsal protrusions.

[0038] Figure 21 shows an amino acid sequence comparison of the Src and p38 catalytic domains. The insertion site for iFKBP in the generated RapR-Src and RapR-p38 is indicated by the black arrow and outlined by the rectangle. The β-sheet strands and the a-helixes are outlined for each kinase.

[0039] Figure 22 shows that the activity of LOV-Src can be inhibited when the LOV domain is in the lit conformation.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

[0042] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. For example, features described in relation to one embodiment may also be applicable to and combinable with other embodiments and aspects of the invention.

[0043] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0044] Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0045] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g. , Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York). I. Definitions

[0046] As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0047] Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0048] The term "about," as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, + 1%, + 0.5%, or even ± 0.1% of the specified amount.

[0049] The term "consists essentially of (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5' and/or 3' or N-terminal and/or C-terminal ends of the recited sequence such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids on both ends added together. The term "materially altered," as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term "materially altered," as applied to polypeptides of the invention, refers to an increase or decrease in an activity (e.g., catalytic activity or ligand binding activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0050] The term "activate," as used herein, refers to an increase in at least one biological activity of a protein of interest of the invention, e.g., an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

[0051] The term "inactivate," as used herein, refers to a decrease in at least one biological activity of a protein of interest of the invention, e.g., a decrease of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0052] As used herein, "nucleic acid," "nucleotide sequence," and "polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide or nucleotide sequence refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid or nucleotide sequence of this invention.

[0053] An "isolated polynucleotide" is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated polynucleotide includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence. An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.

[0054] The term "isolated" also can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated fragment" is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated" does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.

[0055] An "isolated" cell refers to a cell that is separated from other components with which it is normally associated in its natural state. For example, an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention. Thus, an isolated cell can be delivered to and/or introduced into a subject. In some embodiments, an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.

[0056] The term "fragment," as applied to a nucleic acid, nucleotide sequence, or polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention. In other embodiments, such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of less than about 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, or less consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0057] The term "fragment," as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%o, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention. In other embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of less than about 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, or less consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0058] A "vector" is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell. A vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence. A "replicon" can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. For example, the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini. Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker. A "recombinant" vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences.

[0059] Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, expression control sequences, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (e.g., delivery to specific tissues, duration of expression, etc.).

[0060] Vectors may be introduced into the desired cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311 , filed Mar. 15, 1990). [0061] In some embodiments, a polynucleotide of this invention can be delivered to a cell in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a nucleotide sequence of this invention (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413 (1987); Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 55:8027 (1988); and Ulmer et al., Science 259: 1745 (1993)). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al., Science 337:387 (1989)). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications W095/18863 and W096/17823, and in U.S. Patent No. 5,459,127. The use of lipofection to introduce exogenous nucleotide sequences into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. In representative embodiments, transfection is directed to particular cell types in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al , 1988, supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.

[0062] In various embodiments, other molecules can be used for facilitating delivery of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., W095/21931), peptides derived from nucleic acid binding proteins {e.g. , WO96/25508), and/or a cationic polymer {e.g. , W095/21931).

[0063] It is also possible to introduce a vector in vivo as naked nucleic acid (see U.S. Patent Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated nucleic acid delivery approaches can also be used (Curiel et al, Hum. Gene Ther. 3: 147 (1992); Wu et al, J. Biol. Chem. 262:4429 (1987)).

[0064] The term "transfection" or "transduction" means the uptake of exogenous or heterologous nucleic acid (RNA and/or DNA) by a cell. A cell has been "transfected" or "transduced" with an exogenous or heterologous nucleic acid when such nucleic acid has been introduced or delivered inside the cell. A cell has been "transformed" by exogenous or heterologous nucleic acid when the transfected or transduced nucleic acid imparts a phenotypic change in the cell and/or a change in an activity or function of the cell. The transforming nucleic acid can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell or it can be present as a stable plasmid. [0065] As used herein, the terms "protein" and "polypeptide" are used interchangeably and encompass peptides, unless indicated otherwise. In some embodiments, a peptide is a chain of amino acids having a length of about 3 to about 50 residues.

[0066] A "fusion protein" is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides and/or peptides not found fused together in nature are fused together in the correct translational reading frame. In one embodiment, fusion polypeptides include fusions of a polypeptide of the invention (or a fragment thereof) to a polypeptide that is useful for identifying and/or purifying the fusion protein, e.g., all or a portion of glutathione- S- transferase, maltose-binding protein, or a reporter protein (e.g., Green Fluorescent Protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.

[0067] As used herein, a "functional" polypeptide or "functional fragment" is one that substantially retains at least one biological activity normally associated with that polypeptide (e.g. , catalytic activity, ligand binding). In particular embodiments, the "functional" polypeptide or "functional fragment" substantially retains all of the activities possessed by the unmodified peptide. By "substantially retains" biological activity, it is meant that the polypeptide retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). A "non-functional" polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g. , less than about 10% or even 5%). Biological activities such as protein binding and fluorescence can be measured using assays that are well known in the art and as described herein.

[0068] By the term "express" or "expression" of a polynucleotide coding sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present invention, expression of a coding sequence of the invention will result in production of the polypeptide of the invention. The entire expressed polypeptide or fragment can also function in intact cells without purification.

[0069] As used herein, the term "homolog of rapamycin" refers to any molecule that can bind to a rapamycin binding domain and has substantially the same activity as rapamycin when bound to the rapamycin binding domain. The term "has substantially the same activity" refers to a molecule that has at least about 50% of the activity of rapamycin upon binding to a rapamycin binding domain, e.g. , at least about 60, 70, 80, 90, of 95% of the activity of rapamycin. In one embodiment, the activity is the induction of a conformational change in the rapamycin binding domain. Rapamycin homologs are well known in the art and include, without limitation, iRap, AP21967, ILS-920, WYE- 592, CCI-779, meridamycin, temsirolimus, everolimus, and the homologs disclosed in Bayle et al, Chem. Biol. 13:99 (2006) and Grimley et al, Bioorg. Med. Chem. Lett. 75:759 (2008), each of which is hereby incorporated by reference in its entirety.

[0070] As used herein, the term "substantially inactive" in reference to a kinase is defined as a catalytic activity that is less than about 20% of the catalytic activity of a wild-type kinase, e.g. , less than about 10, 5, 4, 3, 2, or 1% of the catalytic activity.

[0071] As used herein, the term "ligand binding domain" refers to a peptide or protein domain that exhibits a change in molecular dynamics {e.g., a change in conformation) when it physically binds a ligand {e.g. , rapamycin) or in response to a direct signal {e.g., illumination).

[0072] As used herein, the term "ligand-dependent kinase" refers to a kinase comprising a LBD and that exhibits a modulation in catalytic activity when a ligand is bound to the LBD. The modulation can be either an increase or a decrease in activity upon ligand binding.

[0073] As used herein, the term "near the catalytic domain" with reference to a kinase is defined as a location within the primary amino acid chain of the kinase that is sufficiently close to the catalytic domain such that insertion of a sequence at the location substantially alters the catalytic activity of the kinase, e.g. , alters the catalytic activity by at least about 10% or more, e.g. , at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

II. Fusion Proteins

[0074] One aspect of the invention relates to fusion proteins that are ligand- dependent kinases. The LBD is inserted into the kinase such that the catalytic activity of the kinase becomes dependent (either positively or negatively) on ligand binding. In one embodiment, the invention relates to fusion proteins comprising, consisting essentially of, or consisting of a kinase or a functional fragment thereof and a LBD inserted within the kinase, wherein the kinase is catalytically active when a ligand is bound to the LBD and is less active when no ligand is bound to the LBD. In one embodiment, the kinase is substantially inactive when no ligand is bound to the LBD. In one embodiment, the invention relates to fusion proteins comprising, consisting essentially of, or consisting of a kinase or a functional fragment thereof and a LBD inserted within the kinase, wherein the kinase is cataiytically inactive when a ligand is bound to the LBD and is more active when no ligand is bound to the LBD. In one embodiment, the kinase is substantially inactive when ligand is bound to the LBD.

[0075] In certain embodiments, insertion of the LBD disrupts kinase catalytic activity without disrupting other activities of the enzyme such as binding to other proteins. In one embodiment, the LBD is inserted within an amino acid loop on the surface of the kinase (referred to herein as the "insertion loop"). In another embodiment, the LBD is inserted within the catalytic domain of the kinase. In certain embodiments, the LBD is inserted into the N-terminal lobe of the catalytic domain. In certain embodiments, the LBD is inserted into a loop between two beta strands. In other embodiments, the loop is near the Gly loop (G loop) of the catalytic domain, e.g. , separated from the G loop by a beta strand. In a further embodiment, the LBD is inserted near the catalytic domain of the kinase, e.g., within 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of the N-terminal and/or C-terminal end of the catalytic domain. In one embodiment, one or more kinase amino acid residues are removed at the site at which the LBD is inserted, e.g. , 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or amino acids are removed.

[0076] The catalytic domains of most known protein kinases are related in amino acid sequence and comprise a superfamily of eukaryotic protein kinases (ePKs). All ePK catalytic domains share a very similar basic structure. The human kinome (the full complement of human protein kinases) counts 478 ePKs out of the 518 identified protein kinases. The rest of the kinases are described as atypical kinases. Despite very little sequence similarity, even some of the atypical kinases display structural similarity to ePKs. The conserved homology among kinases indicates that a suitable insertion location can be identified in most if not all kinases. It further indicates that, even if the structure of the targeted catalytic domain is unavailable, one can identify the insertion loop by comparing its amino acid sequence to the sequence of a non-related protein kinase with known structure. For example, by comparing the amino acid sequences of a tyrosine kinase (Src) and a serine/threonine kinase (p38) the site for insertion of a LBD can be readily identified using the structure of only one kinase as a reference (see Fig. 21). The experiments discussed below demonstrate that insertion of iFKBP at the selected sites leads to successful generation of both RapR-Src and RapR-p38.

[0077] Without being limited to a particular theory, it is thought that insertion of the LBD into an insertion loop in the catalytic domain decreases the rigidity of the insertion loop which influences the conformational mobility of the nearby G loop, a structural feature important for positioning the ATP phosphate groups in the catalytic site. The increase in the conformational dynamics of the G loop interferes with catalytic activity. Binding of the ligand to the LBD (along with the subsequent heterodimerization of the LBD) restores the rigidity of the insertion loop and therefore the G loop back to wild-type conditions. In the case of the light-regulated LOV domain, it is thought that illumination ("ligand binding") leads to decreased rigidity of the insertion loop and inactivation of kinase activity while removal of illumination increases rigidity and restores catalytic activity.

[0078] In one embodiment of the invention, the LBD comprises a linker at one or both termini. The linker(s) may be of a length suitable to provide sufficient flexibility between the domains of the fusion protein to allow changes in conformation upon ligand binding. For example, the linker can comprise, consist essentially of, or consist of a peptide of about 2 to about 12 amino acids or more, e.g., about 3 to about 8 amino acids, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more amino acids. In another embodiment, the linker comprises repeats of the amino acid sequence Gly-Ser, e.g., 2, 3, 4, 5, or 6 repeats or more. In a further embodiment, the linker comprises repeats of a thermostable helix from ribosomal protein L9, e.g., 2, 3, 4, 5, or 6 repeats or more.

[0079] The kinase of the fusion protein can be any kinase known in the art. The kinase can be, for example, a tyrosine kinase or a serine/threonine kinase. Examples of known kinases include, without limitation, Abl, Aktl, Akt2, ΑΜΡΚαΙβΙγΙ ,

ΑΜΡΚα1β1γ2, ΑΜΡΚα2β1γ1, Aurora-A, Aurora-B, Aurora-C, B-RAF, Btk, CaMK2a, CDKl/CycA2, CDKl/CycB, CDK2/CycA, CDK2/CycE, CDK2/Cyclin A2,

CDK4/CycDl, CDK9/CycT, Chkl, Chk2, c-KIT, CLK1, COT/Tpl2, CSF1R, DAPK3, EGFR, EGF-R T790M L858R, Erkl, Erk2, FAK, Fer, FLT3, Fyn, GSK3 , GSK3p, Hck, HER2/ErbB2, ΙΚΚβ, ΙΚΚε, IRAK2, JAK2, J K1, KHS1, LYN, MEK2, Met, MKK3, MKK4, Mkk6 SDTD, MYLK, NEK2, p38a, ρ38β, ρ38δ, p70 S6K, PAK1, PDGFR-β, PDK1 , PKAC-a, PKCa, ΡΚΟβ2, ΡΚΟγ, PKC5, PKCe, ΡΚΟζ, ΡΚΟμ, PKD2,

PKD3 PKCV, PLK1, PLK3, PLK4, ROCK1, ROCK2, Rskl, Rsk2, SNK, Src, Syk, TAOK1, TAOK2, ΤΓΕ2, TrkA, VEGF-R2, VEGF-R3, and Yes. In one embodiment, the kinase is FAK. In another embodiment, the kinase is Src. In a further embodiment, the kinase is a p38 kinase. In another embodiment, the kinase is Pak. In another

embodiment, the kinase is Lyn. In another embodiment, the kinase is Fyn. In another embodiment, the kinase is Yes. In another embodiment, the kinase is CaMKII. In an additional embodiment, the kinase is a human kinase. In one embodiment, the kinase is one that is not naturally ligand-dependent. In another embodiment, the kinase is one that is naturally ligand-dependent and the native LBD is optionally removed.

[0080] In one embodiment of the invention, the kinase is FAK, e.g. , a mammalian FAK such as human FAK or mouse FAK, and the LBD is inserted within the Met442-Ala448 loop, e.g. , before or after Glu445, e.g., immediately before or after Glu445. The numbering of the amino acids is according to the sequence disclosed in GenBank Accession No. NM_153831 for human FAK. The corresponding sequence in mouse FAK is Leu442-Ala448. In another embodiment, the LBD replaces the Met442- Ala448 loop. The LBD replacing the loop can comprise linkers at one or both termini.

[0081] In one embodiment of the invention, the kinase is Src, e.g. , a mammalian Src such as human Src or mouse Src, and the LBD is inserted before or after Gly288, e.g., immediately before or after Gly288. The numbering of the amino acids is according to the sequence disclosed in GenBank Accession No. NM_001025395 for mouse Src.

[0082] In one embodiment of the invention, the kinase is Lyn A, e.g., a mammalian Lyn A such as human Lyn A or mouse Lyn A, and the LBD is inserted in place of Asn268 (numbering according to the mouse sequence).

[0083] In one embodiment of the invention, the kinase is Fyn, e.g. , a mammalian Fyn such as human Fyn or mouse Fyn, and the LBD is inserted in place of Gly292 (numbering according to the human sequence).

[0084] In one embodiment of the invention, the kinase is Yes, e.g., a mammalian Yes such as human Yes or mouse Yes, and the LBD is inserted in place of Gly298 (numbering according to the human sequence).

[0085] In one embodiment of the invention, the kinase is Pakl, e.g., a mammalian Pakl such as human Pakl or mouse Pakl , and the LBD is inserted in place of Ala291 (numbering according to the human sequence).

[0086] The LBD can be any known LBD that causes a change in the catalytic activity of a kinase in which it is inserted upon ligand binding. The ligand can be any molecule or signal that causes a change in the conformation of the LBD when the LBD is contacted with the ligand. In one embodiment, the ligand for the LBD is a small molecule, e.g., having a molecular weight less that about 1000 Da, e.g., less than about 500 Da. In one embodiment, the ligand is a signal such as illumination. In one embodiment, the LBD is a rapamycin binding domain and the ligand is rapamycin or a homolog thereof. In one embodiment, the LBD is a FK506 binding protein and the ligand is FK506 or a homolog thereof. In one embodiment, the LBD is FKBP12 or a functional fragment thereof, i.e. , a fragment that binds rapamycin. In one embodiment, the LBD is iFKBP, which corresponds to Thr22-Glul08 of human FKBP12 (GenBank Accession No. NM_054014.2).

[0087] In one embodiment of the invention, the binding of the ligand to the LBD in the fusion protein promotes binding of the LBD to a second protein. In certain embodiments, the binding of the second protein assists in activation of the kinase through stabilization of the catalytic domain. In certain embodiments, the binding of the second protein permits less ligand to be used to achieve activation of kinase activity (e.g., at least 5, 10, 25, or 50-fold less). In one embodiment, the ligand is rapamycin or a homolog thereof and binding of rapamycin or a homolog to the LBD promotes binding of the fusion protein to FRB. In a further embodiment of the invention, the ligand is a dimeric ligand that binds the LBD in the fusion protein and a second protein, e.g., FK1012. In another embodiment, the ligand is FK506 and the second protein is calcineurin.

[0088] In certain embodiments, the second protein is linked to another moiety (e.g., fused to another protein). In this manner, the kinase can be brought together with a desired protein or other target. Using this technique, not only is the kinase catalytically activated by binding of a ligand in a time-specific manner, the activated kinase can be targeted in a spatially-specific manner. In some embodiments, the moiety may be one that targets the kinase fusion protein to interact with a certain pathway and/or targets it to a specific protein complex. For example, the Src pathway that is stimulated after Src interacts with FAK can be specifically activated by using a Src-LBD fusion protein of the invention and a second protein (e.g., FRB) linked to FAK. Upon activation of Src by delivery of ligand and binding through the second protein to FAK, the Src pathway that is normally stimulated by the interaction between FAK and Src is activated in the absence of any other Src-induced activities.

[0089] In other embodiments, the second protein is linked to a moiety that targets the kinase fusion protein to a particular subcellular location. For example, the moiety can be a nuclear import or export signal, a membrane targeting moiety (such as a myristoylated protein), or a targeting signal for mitochondria, peroxisomes, or other organelles.

[0090] In one particular embodiment of the invention, the LBD can be an amino acid sequence that is responsive to illumination, e.g., that changes structure in response to illumination. In this embodiment, light acts as the ligand, wherein the kinase is inactive in the presence of light (when the structure is in an open and more flexible configuration) and active in the absence of light (when the structure is in a closed configuration). In one embodiment, the LBD comprises, consists essentially of, or consists of a LOV (light, oxygen or voltage) domain or a portion thereof that retains substantially all of the responsiveness to illumination. The LOV domain can be any LOV domain known in the art. In one embodiment, the LOV domain is a LOV2 domain. In another embodiment, the LBD further comprises a Ja domain. In a further embodiment, the LBD comprises, consists essentially of, or consists of a LOV domain and a Ja domain, e.g., a LOV2 domain and a Ja domain. In one embodiment, the LBD comprises, consists essentially of, or consists of one or more fragments from a phototropin, e.g., phototropin I, e.g., phototropin I from Avena sativa (oat). In another embodiment, the LBD comprises, consists essentially of, or consists of the LOV2 domain and/or Ja domain from A. sativa phototropin I. The A. sativa phototropin I nucleotide and amino acid sequences are known in the art and can be found, e.g., in GenBank at accession number AF033096, herein incorporated by reference in its entirety. In one embodiment, the LBD comprises, consists essentially of, or consists of a LOV domain from an aureochrome, e.g. , aureochrome from the stramenopile algae Va cheria frigida. In certain embodiments, the LBD further comprises the coiled-coil domain from aureochrome, which can function as a dimerization domain to bind other fusion proteins comprising a coiled-coil domain. In other embodiments, the LBD comprises, consists essentially of, or consists of one or more fragments from an aureochrome, e.g., amino acids 144-348 of V. frigida aureochrome. The V. frigida aureochrome nucleotide and amino acid sequences are known in the art and can be found, e.g., in GenBank at accession numbers AB272981 and AB252504, herein incorporated by reference in its entirety. Linkers (e.g., 1 , 2, 3, 4, 5, or 6 or more amino acids) can be added to one or both ends of the light-regulated domain to optimize the conformational flexibility of the domain. The fusion protein is illuminated at an appropriate wavelength or wavelength range and for a sufficient period of time to inactivate the kinase. The fusion protein can be repeatedly activated or inactivated by removing and supplying illumination. The level of activation or inactivation can be regulated by controlling the level of illumination. Illumination can be provided by any means known in the art, e.g., by using a broad spectrum white light or a light providing a narrower wavelength range, such as a blue light (e.g. , blue AlGalnP LEDs). Further description of light-regulated domains and their use in fusion proteins is found in International Publication No. WO 2011/002977, herein incorporated by reference in its entirety.

[0091] Fusion proteins of the invention can be modified for use in cells in vitro, ex vivo, or in vivo by the addition, e.g., at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in the cell or in vivo. This can be useful in those situations in which the protein termini tend to be degraded by proteases prior to or after cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the protein to be administered. This can be done either chemically during the synthesis of the fusion protein or by recombinant DNA technology by methods familiar to artisans of average skill. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Likewise, the fusion proteins can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins or other molecules {e.g., PEG) prior to administration, e.g. , for use in animal models of disease.

III. Polynucleotide, Vectors, and Cells

[0092] One aspect of the invention relates to polynucleotides encoding the fusion proteins of the invention. In one embodiment, the polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence that encodes the fusion proteins of the invention. Polynucleotides of this invention include R A, DNA (including cDNAs) and chimeras thereof. The polynucleotides can further comprise modified nucleotides or nucleotide analogs. It will be appreciated by those skilled in the art that there can be variability in the polynucleotides that encode the fusion proteins of the present invention due to the degeneracy of the genetic code. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same polypeptide, is well known in the literature.

[0093] The isolated polynucleotides encoding the fusion proteins of the invention will typically be associated with appropriate expression control sequences, e.g. , promoters, enhancers, transcription/translation control signals and polyadenylation signals.

[0094] A variety of promoter/enhancer elements can be used depending on the level and tissue-specific expression desired. The promoter can be constitutive or inducible, depending on the pattern of expression desired. The promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. The promoter is chosen so that it will function in the target cell(s) of interest. [0095] To illustrate, the polynucleotide encoding the fusion protein can be operatively associated with a cytomegalovirus (CMV) major immediate-early promoter, an albumin promoter, an Elongation Factor 1-a (EFl-a) promoter, a ΡγΚ promoter, a MFG promoter, or a Rous sarcoma virus promoter.

[0096] Inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements, and other promoters regulated by exogenously supplied compounds, including without limitation, the zinc-inducible metallothionein (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (see WO 98/10088); the ecdysone insect promoter (No et al , Proc. Natl. Acad. Set. USA 93:3246 (1996)); the tetracycline- repressible system (Gossen et al, Proc. Natl. Acad. Sci. USA 89:5547 (1992)); the tetracycline-inducible system (Gossen et al, Science 268: 1766 (1995); see also Harvey et al, Curr. Opin. Chem. Biol. 2:512 (1998)); the RU486-inducible system (Wang et al, Nat. Biotech. 15:239 (1997); Wang et al, Gene Ther., 4:432 (1997)); and the rapamycin- inducible system (Magari et al. , J. Clin. Invest. 100:2865 (1997)).

[0097] Moreover, specific initiation signals are generally required for efficient translation of inserted protein coding sequences. These translational control sequences, which can include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

[0098] The present invention further provides cells comprising the polynucleotides and fusion proteins of the invention. The cell may be a cultured cell or a cell ex vivo or in vivo, e.g., for use in therapeutic methods, diagnostic methods, screening methods, methods for studying the biological action of kinases, methods of producing fusion proteins, or methods of maintaining or amplifying the polynucleotides of the invention, etc. The cell can be e.g., a bacterial, fungal (e.g., yeast), plant, insect, avian, mammalian, or human cell.

[0099] The polynucleotide can be incorporated into an expression vector. Expression vectors compatible with various host cells are well known in the art and contain suitable elements for transcription and translation of nucleic acids. Typically, an expression vector contains an "expression cassette," which includes, in the 5' to 3' direction, a promoter, a coding sequence encoding a fusion protein operatively associated with the promoter, and, optionally, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase.

[0100] Non-limiting examples of promoters of this invention include CYC1 , HIS3, GAL1 , GAL4, GAL 10, ADH1 , PGK, PH05, GAPDH, ADC1, TRP1 , URA3, LEU2, ENO, TPI, and alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichia); β-lactamase, lac, ara, tet, trp, IP_L, IPR, T7, tac, and trc promoters (useful for expression in Escherichia coli); light regulated-, seed specific-, pollen specific-, ovary specific-, pathogenesis or disease related-promoters, cauliflower mosaic virus 35S, CMV 35S minimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific promoters, root specific promoters, chitinase, stress inducible promoters, rice tungro bacilliform virus, plant super-promoter, potato leucine aminopeptidase, nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells).

[0101] Further examples of animal and mammalian promoters known in the art include, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the El A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, a-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis and/or disease-related promoters, and promoters that exhibit tissue specificity, such as the elastase I gene control region, which is active in pancreatic acinar cells; the insulin gene control region active in pancreatic beta cells, the immunoglobulin gene control region active in lymphoid cells, the mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; the albumin gene promoter, the Apo AI and Apo All control regions active in liver, the alpha-fetoprotein gene control region active in liver, the alpha 1 -antitrypsin gene control region active in the liver, the beta-globin gene control region active in myeloid cells, the myelin basic protein gene control region active in oligodendrocyte cells in the brain, the myosin light chain-2 gene control region active in skeletal muscle, and the gonadotropic releasing hormone gene control region active in the hypothalamus, the pyruvate kinase promoter, the villin promoter, the promoter of the fatty acid binding intestinal protein, the promoter of smooth muscle cell a-actin, and the like. In addition, any of these expression sequences of this invention can be modified by addition of enhancer and/or regulatory sequences and the like.

[0102] Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor I (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.

[0103] Termination control regions, i.e., terminator or polyadenylation sequences, may be derived from various genes native to the preferred hosts. In some embodiments of the invention, the termination control region may comprise or be derived from a synthetic sequence, a synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.

[0104] Expression vectors can be designed for expression of polypeptides in host cells, e.g., prokaryotic or eukaryotic cells. For example, polypeptides can be expressed in bacterial cells such as E. coli, insect cells {e.g., the baculovirus expression system), yeast cells, plant cells or mammalian cells. Some suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Examples of bacterial vectors include pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia). Examples of vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al, EMBO J. 6:229 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933 (1982)), pJRY88 (Schultz et al, Gene 54:113 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors available for expression of nucleic acids to produce proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al, Mol. Cell. Biol. 3:2156 (1983)) and the pVL series (Lucklow and Summers Virology 170:31 (1989)).

[0105] Examples of mammalian expression vectors include pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed, Nature 329:840 (1987)) and pMT2PC (Kaufman et al, EMBO J. 6:187 (1987)). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.

[0106] Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).

[0107] In addition to the regulatory control sequences discussed above, the recombinant expression vector can contain additional nucleotide sequences. For example, the recombinant expression vector can encode a selectable marker gene to identify host cells that have incorporated the vector.

[0108] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques, including, without limitation, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989), and other laboratory manuals.

[0109] If stable integration is desired, often only a small fraction of cells (in particular, mammalian cells) integrate the foreign DNA into their genome. In order to identify and select integrants, a nucleic acid that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the nucleic acid of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that comprising the nucleic acid of interest or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection {e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0110] The polynucleotide can also be introduced into a plant, plant cell or protoplast and, optionally, the isolated nucleic acid encoding the polypeptide is integrated into the nuclear or plastidic genome. Plant transformation is known in the art. See, in general, Meth, Enzymol. Vol. 153 ("Recombinant DNA Part D") 1987, Wu and Grossman Eds., Academic Press and European Patent Application EP 693554. [0111] According to certain embodiments, the polynucleotides or vectors can be targeted to specific cells or tissues in vivo. Targeting delivery vehicles, including liposomes and viral vector systems are known in the art. For example, a liposome can be directed to a particular target cell or tissue by using a targeting agent, such as an antibody, soluble receptor or ligand, incorporated with the liposome, to target a particular cell or tissue to which the targeting molecule can bind. Targeting liposomes are described, for example, in Ho et al, Biochemistry 25:5500 (1986); Ho et al, J. Biol. Chem. 262: 13979 (1987); Ho et al, J. Biol. Chem. 262: 13973 (1987); and U.S. Pat. No. 4,957,735 to Huang et al. , each of which is incorporated herein by reference in its entirety. Enveloped viral vectors can be modified to deliver a nucleic acid molecule to a target cell by modifying or substituting an envelope protein such that the virus infects a specific cell type. In adenoviral vectors, the gene encoding the attachment fibers can be modified to encode a protein domain that binds to a cell-specific receptor. Herpesvirus vectors naturally target the cells of the central and peripheral nervous system. Alternatively, the route of administration can be used to target a specific cell or tissue. For example, intracoronary administration of an adenoviral vector has been shown to be effective for the delivery of a gene to cardiac myocytes (Maurice et al., J. Clin. Invest. 104:21 (1999)). Intravenous delivery of cholesterol-containing cationic liposomes has been shown to preferentially target pulmonary tissues (Liu et al, Nature Biotechnol. 75: 167 (1997)), and effectively mediate transfer and expression of genes in vivo. Other examples of successful targeted in vivo delivery of nucleic acid molecules are known in the art. Finally, a recombinant nucleic acid molecule can be selectively (i.e. , preferentially, substantially exclusively) expressed in a target cell by selecting a transcription control sequence, and preferably, a promoter, which is selectively induced in the target cell and remains substantially inactive in non-target cells.

[0112] In one aspect, the invention relates to methods of producing the fusion proteins of the invention, comprising expressing the fusion protein encoded by the polynucleotides and/or vectors described above. In one embodiment, the fusion proteins can be expressed in vitro, e.g., by in vitro transcription and/or translation. In another embodiment, the fusion protein can be expressed in a cell, e.g. , an isolated cell, such as a cell line or a primary cell or a cell in an isolated tissue. In some embodiments, the cell can be a bacterial, fungal (e.g., yeast), insect, plant, or animal (e.g. , mammalian) cell. In a different embodiment, the cell can be present in an animal or plant, e.g., for in vivo production of the fusion protein or for therapeutic or diagnostic purposes. [0113] In one aspect, the invention relates to methods of converting a kinase to a ligand-dependent kinase, comprising inserting a LBD into or near the catalytic domain of the kinase. In one embodiment, the fusion protein is produced recombinantly by preparing a polynucleotide encoding the fusion protein. Coding sequences for the fusion proteins of the invention can be prepared using techniques well known in the art, including cutting and splicing polynucleotides encoding domains of the fusion protein or chemically synthesizing all or part of the coding sequence. In another embodiment, the fusion protein can be prepared at the protein level, e.g., by linking peptides or chemically synthesizing all or part of the amino acid sequence.

IV. Methods

[0114] One aspect of the invention relates to methods of activating a kinase present in a fusion protein of the invention, comprising contacting the fusion protein with a ligand that binds to the LBD. A further aspect of the invention relates to methods of inactivating a kinase present in a fusion protein of the invention, comprising contacting the fusion protein with a ligand that binds to the LBD.

[0115] Another aspect of the invention relates to methods for assessing the function of a kinase present in a fusion protein of the invention, comprising contacting the fusion protein with a ligand that binds to the LBD and assessing one or more activities of the kinase. An additional aspect relates to methods for assessing the function of a kinase present in the fusion protein of the invention, comprising removing a ligand that binds to the LBD from the fusion protein and assessing one or more activities of the kinase. In one embodiment, the one or more activities is selected from the group consisting of protein binding, signaling, translocation, and enzymatic activity. The addition and removal of ligand can be carried out once or repeated multiple times to study kinase activity in a temporal fashion. The methods of the invention can also be used advantageously to study kinases in a temporal-spatial manner by controlling both the timing of activation and the location of the activated kinase. For example, activated kinase can be targeted to a specific area of the cell, to a specific signaling pathway, and/or to a specific protein cluster. In other embodiments, a specific isoform of a kinase can be activated without activating other members of the kinase family. In other embodiments, a particular function of a kinase can be activated (e.g., binding to a specific partner) without stimulating other activities of the kinase.

[0116] For each of the methods of the invention, the fusion protein may be present outside a cell or in a cell. The cell may be an isolated cell, a cell in an isolated tissue, or a cell in an animal or other organism. The cell can be on a substrate, in a position to be photographed, filmed, viewed, stained, observed, etc., such as before, during, and/or after addition and/or removal of ligand. The fusion protein may be contacted with the ligand by directly adding the ligand to a fluid comprising the fusion protein, exposing cells comprising the fusion protein to the ligand, or administering the ligand to an animal or other organism. In addition to ligand, the fusion protein and/or cell can be contacted with other agents, e.g. , kinase inhibitors, other research tools, etc.

[0117] The present invention finds use in research applications, as well as diagnostic and medical applications. Suitable subjects include all organisms, e.g. , bacteria, fungi, plants, insects, avians, fish, and mammals. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. The term "mammal" as used herein includes, but is not limited to, humans, bovines, ovines, caprines, equines, felines, canines, murines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults. In other embodiments, the subject is an animal model of disease.

[0118] A further aspect of the invention relates to kits for carrying out the methods of the invention. The kits can comprise the fusion proteins, polynucleotides, vectors, and/or cells of the invention. The kits can comprise further components useful for carry out the methods of the invention, including without limitation, containers, buffers, ligands, reagents, fluorescent dyes, antibodies, cells, probes, primers, vectors, etc.

[0119] The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLE 1

Materials and Methods

Antibodies and reagents:

[0120] Anti-phospho-paxillin (Tyr31), anti-phospho-FAK (Tyr397), anti- phospho-Src (Tyr418) and anti-GFP antibodies were purchased from Invitrogen Co.

Anti-myc antibodies and IgG-coupled agarose beads were purchased from Millipore.

Anti-paxillin antibodies were a gift from Dr. Michael Schaller. Rapamycin was purchased from Sigma. All restriction enzymes were purchased from New England

Biolabs. Molecular biology:

[0121] The construct for myc-tagged mouse FAK was a gift from Dr. Steven K. Hanks. The construct expressing the GST-tagged N-terminal fragment of paxillin was a gift from Dr. Michael Schaller. The mouse Src construct was purchased from Upstate. The FKBP12 and FRB domains of human FRAP1 DNA constructs were a gift from Dr. Gary Johnson. Insertion of wild type FKBP12 or iFKBP at the ends or in the middle of FAK and Src genes was performed using a modification of the QuikChange site-directed mutagenesis kit (Stratagene). The FKBP12 and iFKBP inserts were created by PCR such that their 5'- and 3'- end sequences annealed at the desired insertion site within the Src and FAK genes. The PCR products were used as mega-primers for QuikChange mutagenesis reactions. The FRB domain of human FRAP1 protein was cloned into pmCherry-CI vector using EcoRI/BamHI cloning sites. GFP-tagged FAK variants were created by subcloning the FAK gene into pEGFP-CI vector (Clontech) using Bglll/BamHI cloning sites. The myc-tagged Src gene was constructed by insertion of a myc-tag sequence at the 3 '-end of the Src gene using the QuikChange mutagenesis kit (Stratagene).

Immunoprecipitation and kinase assay:

[0122] Cells expressing FAK or Src were treated with either rapamycin or equivalent volumes of ethanol (solvent control). After treatment, cells were washed with ice-cold PBS and lysed with Lysis Buffer (20 mM Hepes-KOH, pH 7.8, 50 mM KC1, 100 mM NaCl, 1 mM EGTA, 1% NP40, 1 mM NaF, 0.1 mM Na₃V0₄, 0.033% EtOH). Cells treated with rapamycin were lysed with Lysis Buffer containing 200 nM rapamycin.

Cleared lysates were incubated for 2 hours with IgG-linked agarose beads prebound with antibody used for immunoprecipitation. The beads were washed 2 times with Wash

Buffer (20 mM Hepes-KOH, pH 7.8, 50 mM KC1, 100 mM NaCl, 1 mM EGTA, 1%

NP40) and two times with Kinase Reaction Buffer (25 mM HEPES pH 7.5, 5 mM MgCl₂,

5 mM MnCl₂, 0.5 mM EGTA, 0.005% BRIJ-35). No MnCl₂ was used in the Kinase

Reaction Buffer for Src kinase immunoprecipitation and assay. 20 uL of bead suspension were used in kinase assays using the N-terminal fragment of paxillin as previously described (Cai et al, Mo I. Cell. Biol. 28:201 (2008)). No kinase reaction was performed for co-immunoprecipitation experiments.

Cell imaging:

[0123] Cells were plated on fibronectin-coated coverslips (10 mg/L fibronectin) 2 hours prior to imaging, then transferred into L-15 imaging medium (Invitrogen) supplemented with 5% fetal bovine serum. Live cell imaging was performed in an open heated chamber (Warner Instruments) using an Olympus IX-81 microscope equipped with an objective-based total internal reflection fluorescence (TIRF) system and a PlanApo N 60x TIRFM objective (NA 1.45). All images were collected using a Photometrix CoolSnap ES CCD camera controlled by Metamorph software. The 468 nm and 568 nm lines from an omnichrome series 43 Ar/Kr laser were used for TIRF imaging. Epifluorescence images were taken using a high pressure mercury arc light source. Cells expressing GFP-RapRFAK constructs and mCherry-FRB were selected using epifluorescence imaging. Time-lapse movies were taken at 1 min time intervals. GFP- RapR-FAK expression level quantification and other image analysis were performed using Metamorph software.

Thermodynamics study of FKBP, FKBP deletion mutant with and without binding partners:

[0124] We performed replica exchange discrete molecular dynamics (DMD) simulations of various molecular systems to estimate the thermo-stabilities and to study the conformational dynamics of FKBP and its deletion mutant, dFKBP. Details of the DMD method and simulation protocols can be found in previous studies (Ding et al., Proc. Natl. Acad. Sci. U S A 105: 19696 (2008); Ding et al, Structure 16:1010 (2008)). Briefly, DMD is an efficient conformational sampling algorithm and an all-atom DMD model has been shown to fold several small proteins to their native states ab initio (Ding et al., Structure 16:1010 (2008)). Using replica exchange DMD simulations, the folding thermodynamics of superoxide dismutase (SOD1) and its variants were computationally characterized in agreement with experiments (Ding et al, Proc. Natl. Acad. Sci. U S A 105:19696 (2008)). We applied a similar method to study the folding thermodynamics and conformational dynamics of FKBP and dFKBP bound to either rapamycin or both rapamycin and FRB. The x-ray crystal structure of FKBP, FRB, and rapamycin was used to set up the simulations (PDB code: 3FAP).

Model construction of chimeric kinase:

[0125] To model FAK with dFKBP insertion, we first manually positioned the dFKBP with various linkers in the proximity of insertion loci of FAK (PDB code: 2J0J) using PyMol (www.pymol.org). To model the relative orientation of dFKBP with respect to FAK, we performed all-atom discrete molecular dynamics simulations at room temperature (Ding et al., Structure 16: 1010 (2008)) with the FAK molecule kept static while dFKBP and linkers were allowed to move. Since the simulation temperature is below the folding transition temperature of dFKBP, the inserted domain stays folded while the DMD simulation optimizes its relative orientation with respect to FAK. By clustering the snapshot conformations from equilibrium DMD simulations, the centroid structure was identified. We modeled the chimera in complex with rapamycin and FRB by aligning the corresponding FKBP domains in the chimera and in the complex structure of FKBP, rapamycin, and FRB. Similarly, we also constructed the model of FKBP insertion into Src kinase (PDB code: 1Y57).

DMD simulations of chimeric kinases:

[0126] We performed equilibrium DMD simulations of FKBP-dFKBP chimera with different linkers at room temperature. We also studied wild type FAK, FAK-dFKBP chimera, and FAK-dFKBP chimera in complex with rapamycin and FRB. To reduce the computational overhead, we kept the distal FERM domain of FAK and alpha-helical subdomain of the catalytic domain fixed. We allowed the inserted FKBP and the directly modified catalytic subdomain to sample their conformational space. Similarly, we also studied the SrcdFKBP chimera. The dynamic coupling of the wild-type FAK was obtained by computing the normalized correlation matrix (Sharma et al, Biophys J

92: 1457 (2007); Teotico et al, PLoS Compiit. Biol. 4:el 0001 1 1 (2008)) from DMD simulation trajectories. In the calculation of the dynamics coupling and root-mean-square fluctuation, the translational and rotational freedom was reduced by translating the center of mass to the origin and then aligning each snapshot with respect to the average structure.

EXAMPLE 2

Development of Allosterically Regulated FAK

[0127] We describe here a new, broadly applicable approach to activate kinases in living cells with absolute specificity and within minutes (Fig. 1A). A protein cassette introduced into a conserved portion of the kinase catalytic domain confers allosteric regulation by the membrane-permeable molecule rapamycin. The inserted domain, based on a fragment of FK506 binding protein (FKBP12), substantially decreases the rigidity of a critical loop in the kinase active site, disrupting kinase activity while not affecting other normal binding interactions. Upon binding to rapamycin, the inserted FKBP fragment heterodimerizes with co-expressed FRB protein (FKBP12-rapamycin binding domain), reducing the mobility of the active site loop and restoring catalytic activity.

[0128] A previous study had shown that the secondary structure elements of FKBP 12 were not greatly altered upon ligand binding, but that binding did greatly increase conformational rigidity (Marquis-Omer et al, Biochem. Biophys. Res. Commun. 179:741 (1991)). This suggested that insertion of FKBP12 near the catalytic site of kinases could be used to control the conformational mobility of the kinase active site, thereby controlling its activity. It was, however, unclear that FKBP12 could be inserted into the middle of the kinase sequence without severely disrupting kinase structure or FKBP12 binding interactions. We therefore created a truncated form of FKBP12 named iFKBP (insertable FKBP, Fig. IB). In iFKBP, the N and C termini are positioned near one another for minimal perturbation of the kinase secondary structure (Fig IB). Co- immunoprecipitation experiments showed that iFKBP interacts with rapamycin and FRB as efficiently as does wild type FKBP 12, even when inserted in the middle of the FAK molecule (Fig. 1C). Computational analysis of iFKBP indicated that it is stabilized by interaction with rapamycin and FRB, as is FKBP 12 (Fig. ID and Fig. 2). Changes in mobility were especially pronounced at the N and C termini where iFKBP would be linked to FAK, suggesting that the effects of rapamycin/FRB binding could be communicated to FAK,

[0129] Optimization of the insertion site and the linkers connecting iFKBP to FAK led to a version of FAK that was susceptible to regulation by rapamycin and FRB. With insertion of iFKBP at Glu445 (FAK-iFKBP445 construct), FAK catalytic activity was dramatically reduced, but addition of rapamycin and binding to FRB restored activity (Fig. 3A). In control studies, treatment with rapamycin did not affect the activity of wild- type FAK (FAKwt) or a construct with iFKBP attached to the FAK N-terminus, demonstrating that regulation of catalytic activity is dependent on specific placement of the insert in the catalytic subunit. To optimize regulation of FAK-iFKBP445 by rapamycin, several modifications were introduced into the regions where iFKBP was connected to FAK (Fig. 3A). iFKBP had been positioned within the FAK loop Met442- Ala448, between two β-strands in the N-terminal lobe of the FAK catalytic domain. We tested whether placing iFKBP closer to the β-strands will result in tighter control of catalytic activity (Fig. 3B). Replacing Met442-Ala448 in FAK with iFKBP without any linkers (Fig. 3B, construct FAK- iFKBP 442-448) negated the effect of iFKBP on FAK activity and dramatically reduced interaction with rapamycin and FRB. Computational analysis revealed that the construct without linkers is locked in a distorted conformation that prevents ligand binding (Fig. 4). In contrast, introduction of short linkers to connect iFKBP with the β- strands of the FAK catalytic domain led to an optimized structure (RapR-FAK, for Rapamycin regulated FAK). The activity of RapR-FAK in the absence of rapamycin was significantly lower then that of FAK-iFKBP445. Treatment with rapamycin restored activity to near wild type level. [0130] The effects of iFKBP insertion on normal FAK binding interactions and FAK regulation were important; we wanted to specifically control catalytic activity without perturbing other interactions. FAK catalytic activity is regulated by an autoinhibitory interaction between the N-terminal FERM domain and the catalytic domain (Lietha et al, Cell 129: 1 177 (2007)). Two amino acids known to be involved in this interaction were mutated to alanines (Y180A and M183A (Lietha et al, Cell 129: 1177 (2007))) to test if RapR-FAK remains regulated by the autoinhibitory mechanism (construct called RapR-FAK-YM). When activated by rapamycin, RapR- FAK- YM demonstrates significantly higher activity than RapR-FAK (Fig. 3C),

consistent with published results for the constitutively active FAK (Lietha et al , Cell 129: 1 177 (2007)) and demonstrating that RapR-FAK is still regulated by auto inhibition. Importantly, RapR-FAK- YM is regulated solely by rapamycin, and not by endogenous mechanisms. To confirm that RapR-FAK phosphorylates substrates in a rapamycin- dependent manner in cells, phosphorylation of two known FAK substrates was tested before and after addition of rapamycin. Upon activation of RapR-FAK- YM, phosphorylation of paxillin on residue Tyr31 and autophosphorylation of FAK on residue Tyr397 are significantly increased (Fig. 3D). A control construct lacking catalytic activity (RapR-FAK- YM with additional mutation D546R) failed to demonstrate any change in phosphorylation. RapR-FAK and wtFAK showed similar binding to paxillin and Src in coimmunoprecipitation assays (Fig. 5), indicating that introduction of iFKBP into the catalytic domain of FAK does not affect interaction with binding partners. Also, iFKBP insertion did not perturb the intracellular distribution of RapR-FAK as its localization was identical to that of wild-type FAK (Fig. 6). Activation of RapR-FAK catalytic activity was achieved in living cells within 2 minutes and with 50 nM rapamycin (Fig. 3E and 3F). Activation was accompanied by translocation of fluorescently labeled FRB into focal adhesions and co-localization with fluorescent RapR-FAK (Fig. 7). The translocation of fluorescent FRB into adhesions, clearly visible 5 minutes after rapamycin addition, served as useful markers of FAK activation in live cell studies. Thus, RapR- FAK enables robust and specific re-activation of FAK catalytic activity in living cells without perturbation of other properties of FAK.

EXAMPLE 3

Regulation of FAK

[0131] FAK is known to be overexpressed and activated in human tumors (Golubovskaya et al , Histol. Histopathol. 24:503 (2009); Chatzizacharias et al , Histol Histopathol. 23 :629 (2008); Sood et al. , Am. J. Pathol. 165 : 1087 (2004)), but the specific role of its catalytic activity in tumorigenesis remains unclear. RapR-FAK provided an unprecedented opportunity to examine the role of increased FAK activity in cancer cells. To identify processes affected specifically by the catalytic function of FAK we examined activation of RapR-FAK- YM in HeLa cells. HeLa cells normally show small peripheral ruffles that remain near the cell border. Upon addition of rapamycin, very large and highly dynamic ruffles appeared on the dorsal surface within 10-15 minutes, and the extent of ruffling greatly increased (Fig. 8A, 8B, 36/64 analyzed cells). Control studies demonstrated that cells expressing similar levels of catalytically inactive RapR-FAK- YM-KD did not show the ruffling phenotype upon addition of rapamycin (Fig. 8B, 34/35 analyzed cells), indicating that the formation of dorsal ruffles was stimulated by activation of FAK. Published work has demonstrated that FAK autophosphorylation of Tyr397 plays an important role in FAK-mediated signaling and that its phosphorylation level correlates with the activation of FAK (Schaller et al, Mol. Cell. Biol. 14: 1680 (1994)). Mutation of Tyr397 to phenylalanine in RapR-FAK completely abolished formation of dorsal protrusions (Fig. 8B). Autophosphorylation of FAK on Tyr397 creates a binding site for Src family kinases (Schaller et al, Mol. Cell. Biol. 14: 1680 (1994); Xing et al, Mol. Biol. Cell 5:413 (1994)). It was therefore proposed that interaction of FAK with Src leads to Src activation (Schaller et al , Mol. Cell. Biol.

14: 1680 (1994)). Furthermore, Src is involved in the formation of dorsal protrusions stimulated by platelet derived growth factor (PDGF) (Veracini et al. , J Cell Sci.

119:2921 (2006)). Together these observations led us to hypothesize that the FAK- stimulated formation of dorsal protrusions occurs via activation of Src. Indeed, treatment of cells with PP2, an inhibitor of Src family kinases, stopped FAK-induced dorsal protrusions (Fig 8C). In contrast, control compound PP3, an inactive PP2 stereoisomer, or imatinib, an inhibitor of Abl kinase, had no effect. Phosphorylation of Src Tyr418 (Tyr416 in avian Src) is known to occur upon Src activation (Smart et al , Proc. Natl. Acad. Sci. USA 78:6013 (1981); Playford et al , Oncogene 23 :7928 (2004)). Rapamycin addition to cells transfected with RapR-FAK- YM led to increased Src Tyr418 phosphorylation, while cells expressing RapR-FAK- YM with an additional mutation that abolishes Src binding (Y397F mutation) showed no effect (Fig. 8D). These data demonstrate that specific activation of FAK catalytic function activates Src, and that this in turn leads to formation of dorsal protrusions. This novel function suggests that overexpression and activation of FAK in cancer cells stimulates formation of specific protrusive structures affecting cell invasiveness and tumorigenicity. [0132] Molecular dynamics simulations showed that iFKBP insertion did in fact affect the dynamics of the catalytic subunit, suggesting a potential mechanism for the iFKBP effect. The iFKBP insertion point is connected via a β strand to FAK's Gly loop (G-loop), a structural feature critical for positioning the ATP phosphate groups in the catalytic site (Fig 9A) (Krupa et al. , J. Mol. Biol. 339: 1025 (2004)). Analysis of the dynamic coupling of amino acid residues within the catalytic domain of wt FAK clearly demonstrated that the conformational mobility of the FAK G-loop is correlated with that of the FAK region where iFKBP is inserted (the 'insertion loop', Fig. 9B). This suggested that changes in the dynamics of the insertion loop could affect the dynamics of the G-loop and hence change the catalytic activity. Dynamics simulations clearly indicated that movement of the inserted iFKBP correlates with movement of the Gly- loop. Comparison of wtFAK and RapRFAK dynamics revealed that the amplitude of G- loop conformational dynamics is dramatically increased in RapR-FAK, and that this was decreased back to wild-type levels upon binding to rapamycin/FRB (Fig. 9C, 9D).

Increased conformational mobility of the G-loop will reduce its effectiveness in the phosphate transfer reaction, whereas interaction with rapamycin and FRB will stabilize it and rescue FAK catalytic activity. Analysis of the FAK-iFKBP445 variant suggested that its longer linkers decreased coupling between the iFKBP insert and G-loop dynamics (Fig. 10), resulting in less effective FAK inhibition (Fig. 3A, FAK-iFKBP445 construct). In contrast, insertion of iFKBP without any linkers restricted the structural dynamics of iFKBP, negating its effect on the G-loop (Fig. 10, FAK-iFKBP442-8 construct). This computational analysis indicates that the allosteric modulation of RapR-FAK activity results from dynamic coupling of the optimized iFKBP insertion and the kinase G-loop.

EXAMPLE 4

Development of Allosterically Regulated SRC

[0133] The mechanism of the iFKBP effect results from coupling of highly conserved structural elements found in all known protein kinases (Krupa et ctl., J. Mol. Biol, 339: 1025 (2004)). We explored the generalizability of the approach by examining the effect of iFKBP insertion into Src kinase at a site analogous to that used in FAK (Gly288) (Fig. 11). In Src as in FAK, insertion of iFKBP strongly inhibited Src activity, and activity was rescued by interaction with rapamycin/FRB (Fig. 9E, RapR-Src). Treatment with rapamycin did not affect wild-type Src or control Src constructs in which iFKBP was added to the C-terminus. Molecular dynamics simulations of Src showed the same coupling between iFKBP and the Gly-loop as was observed for FAK (Figs. 12, 13). These data demonstrate that the iFKBP cassette can be used not only for allosteric regulation of FAK, but also for Src and likely for other kinases as well.

[0134] To test the regulation of Src in living cells, HeLa cells were co-transfected with Cherry-FRB and either RapR-Src-GFP or a kinase inactive mutant. Exposure of the cells to rapamycin activated the RapR-Src fusion protein, resulting in cell spreading (Fig. 14). In contrast, rapamycin had no effect on cells containing the kinase inactive mutant (Fig. 14).

EXAMPLE 5

Development of Allosterically Regulated p38

[0135] The general applicability of the present invention was further tested using the kinase p38. To create a RapR-p38 kinase, iFKBP was inserted at and replaced amino acid Lys45 (mouse p38, protein Accession No. NP_036081) within a loop similar to the insertion position for the FAK and Src catalytic domains. Flag-tagged wild type p38 and RapR-p38 were coexpressed with GFP-FRB in HEK293T cells. The cells were treated with 200 nM rapamycin for 1 hour. The Flag-tagged kinase was immunoprecipitated and its activity was assessed in an in vitro kinase assay using ATF2 as a substrate. Phosphorylation level of ATF2 correlates with the activity of p38 kinase. As shown in Fig. 15, the fusion of iFKBP with p38 resulted in rapamycin-dependent kinase activity.

EXAMPLE 6

Regulation of RapR-FAK by Non-immunosuppressive Analogs of Rapamycin

[0136] Although no effects of rapamycin were seen in the absence of RapR kinases, there was concern that some potential studies could be complicated by the known immunosuppressive effects of rapamycin. The ability to regulate RapR kinases using known non-immunosuppressive analogs of rapamycin, iRap and AP21967, was therefore tested. HEK293 cells co-transfected with myc-RapR-FAK-YM and GFP-FRB constructs were treated with the indicated amount of iRap (a gift from Dr. Thomas Wandless) or AP21967 (Ariad Pharmaceuticals, Inc) for 1 hour. RapR-FAK- YM kinase was immunoprecipitated and tested in an in vitro kinase assay using N-terminus of paxillin as a substrate. As shown in Fig. 16, both compounds regulated RapR-FAK activity at concentrations comparable to those reported previously for dimerization of proteins in living cells (Inoue et al , Nature Meth. 2:415 (2005)). Importantly, AP21967 and a similar analog of rapamycin (C20-MaRap) have been successfully used for experiments in animals, indicating that the RapR method can be applied in live animal studies (Stankunas et al, Mol. Cell 72: 1615 (2003); Vogel et al, Hum Gene Ther. 19: 161 (2008)).

EXAMPLE 7

Development of Other Allosterically Regulated Kinases

[0137] We have successfully created rapamycin-regulated (RapR) Src family tyrosine kinases Yes, Fyn and LynA, and a serine/threonine kinase Paid . To generate RapR-kinases the iFKBP domain was inserted at the following positions: replacing Asn268 in Lyn A (mouse), replacing Gly292 in Fyn (human), replacing Gly298 in Yes (human) and replacing Ala291 in Pakl (human).

[0138] We have used the RapR method to regulate highly homologous kinases and to identify functional differences for these homologs. Fyn, Yes and LynA are close homologs of Src kinase. They have multiple overlapping substrates and can partially substitute each other's function in the cell. To identify the differences in the function of these kinases we have tested the effect of activation of each individual kinase on the behavior of Cos7 cells. Striking differences were observed in the initial phenotypes stimulated by Rap-Src, RapR-Fyn and RapR-LynA (Fig. 17): activation of Src stimulated polarized cell protrusions; activation of Fyn led to uniform spreading of cells; and activation of LynA initiated formation of large dorsal protrusions. Thus, the present methods can be used to distinguish the activities of even closely-related kinases.

EXAMPLE 8

Localization of Allosterically Regulated Kinases

[0139] Localization of a kinase is critical for regulation of different signaling pathways. We have developed a novel strategy for targeted activation of kinases in specific subcellular locations. One of the key localization factors for Src is myristoylation of its N-terminus. This modification is required for association of Src with the plasma membrane. To achieve selective regulation of Src at the plasma membrane or in the cytoplasm we used a mutant RapR-Src construct that cannot be myristoylated (G2A mutation). Activation of this form of RapR-Src by rapamycin and cytoplasmic FRB was limited only to the cell cytoplasm. However, activation with a myristoylated form of FRB led to targeted activation of RapR-Src(G2A) at the plasma membrane. Using this system we discovered that activation of Src in the cell cytoplasm is not sufficient for stimulation of cell spreading. In contrast, activation of Src at the plasma membrane can successfully initiate cell spreading (Figs. 18A-18B).

EXAMPLE 9

Targeted Activation of Kinases

[0140] Activation of kinases in specific protein complexes often defines regulation of distinct downstream pathways. We have developed a new method for targeted activation of kinases in specific protein complexes. Focal adhesion kinase (FAK) is a very prominent Src binding partner. We have demonstrated that FAK interaction with Src lead to Src activation and formation of dorsal protrusions in HeLa cells. To achieve targeted activation of Src only in complex with FAK we created a FAK-FRB chimera where FRB is inserted in FAK near the known Src binding site (Tyr397 in FAK). Src interacts with FAK and many other proteins through its SH2 domain. To prevent Src interaction with endogenous FAK and other binding partners we used a RapR-Src construct with a mutated SH2 domain (R175L mutation in mouse Src). Thus, co-expression of the RapR-Src(R175L) and FAK-FRB allows for a specific rapamycin-mediated activation of RapR-Src only in a complex with FAK (Fig. 19). Our experiments demonstrate that targeted activation of Src in complex with FAK stimulates formation of dorsal protrusions in HeLa cells, which is in agreement with our previous observations (Fig. 20).

EXAMPLE 10

Development of Light-Regulated Src

[0141] In order to develop a light-regulated kinase, the LOV2 domain, (including the Ja domain) of phototropin I from Avena sativa (oat) was inserted into Src at the same site as the iFKBP of RapR-Src (Gly288). Cos7 cells were transfected with the following DNA constructs: constitutively active Src mutant (P; Y529F mutation), kinase-inactive mutant (N; D388R mutation), a Src construct with an insertion of a LOV domain in its "lit" (open) state (L; I510E/I539E mutations in LOV domain), and a Src construct with an insertion of a LOV domain in its "dark" (closed) state (D; C450M mutation in LOV). Two different linkers were used to connect the LOV domain to the catalytic domain of Src: single Gly linkers (G link) and Gly-Pro-Gly linkers (GPG link). The top blot in Fig. 22 demonstrates the expression level of Src (kinase-GFP, anti-GFP antibody JL8) compared to a loading control (actin, anti-actin antibody). Transfected cells were incubated overnight at 37°C (5% C0₂) and lysed. Cell lysates were analyzed by Western Blot analysis using anti-phospho-tyrosine antibody (4G10, Fig. 22 bottom blot). An increased level of phosphorylation indicates elevated Src kinase activity. As shown in Fig. 22, LOV-Src is catalytically active when the LOV domain is in the dark state and essentially inactive when the LOV domain is in the lit state. This demonstrates that any LBD that changes conformation upon ligand binding or another signal may function to regulate kinase activity.

[0142] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

That which is claimed is:

1. A fusion protein comprising a kinase and a ligand binding domain inserted within the kinase such that the catalytic activity of the kinase is dependent on ligand binding.

2. The fusion protein of claim 1, wherein the kinase is catalytically active when a ligand is bound to the ligand binding domain and is less active when no ligand is bound to the ligand binding domain.

3. The fusion protein of claim 1, wherein the kinase is substantially inactive when no ligand is bound to the ligand binding domain.

4. The fusion protein of any one of claims 1 -3, wherein the ligand binding domain is inserted within an amino acid loop on the surface of the kinase.

5. The fusion protein of any one of claims 1-4, wherein the ligand binding domain is inserted within the catalytic domain of the kinase.

6. The fusion protein of any one of claims 1-4, wherein the ligand binding domain is inserted near the catalytic domain of the kinase.

7. The fusion protein of any one of claims 1-6, wherein one or more kinase amino acid residues are removed at the site at which the ligand binding domain is inserted.

8. The fusion protein of any one of claims 1-7, wherein the ligand binding domain comprises a linker at one or both termini.

9. The fusion protein of any one of claims 1-8, wherein the ligand binding domain comprises a linker at both termini.

10. The fusion protein of claim 8 or 9, wherein each linker is two to eight amino acid residues in length.

11. The fusion protein of any one of claims 1-10, wherein the kinase is focal adhesion kinase (FAK).

12. The fusion protein of claim 1 1, wherein the FAK is human FAK.

13. The fusion protein of claim 12, wherein the ligand binding domain is inserted within the Met442-Ala448 loop.

14. The fusion protein of claim 13, wherein the ligand binding domain is inserted before or after Glu445.

15. The fusion protein of claim 12, wherein the ligand binding domain comprises linkers at one or both termini and replaces the Met442-Ala448 loop.

16. The fusion protein of any one of claims 1-10, wherein the kinase is Src.

17. The fusion protein of claim 16, wherein the Src is human Src.

18. The fusion protein of claim 16, wherein the ligand binding domain is inserted before or after Gly288.

19. The fusion protein of any one of claims 1-10, wherein the kinase is p38.

20. The fusion protein of any one of claims 1-19, wherein the ligand binding domain binds a ligand having a molecular weight less than about 1000 Da.

21. The fusion protein of any one of claims 1-20, wherein the ligand binding domain binds a ligand having a molecular weight less than about 500 Da.

22. The fusion protein of any one of claims 1-21 , wherein the ligand binding domain binds rapamycin or a homolog thereof.

23. The fusion protein of any one of claims 1-22, wherein the ligand binding domain is a FK506 binding protein.

24. The fusion protein of any one of claims 1 -23, wherein the ligand binding domain is FKBP12 or a functional fragment thereof.

25. The fusion protein of any one of claims 1-24, wherein the ligand binding domain is iFKBP.

26. The fusion protein of any one of claims 1-25, wherein the ligand links the fusion protein to a second protein.

27. The fusion protein of claim 26, wherein the ligand is rapamycin or a homolog thereof and the ligand links the fusion protein to FRB.

28. The fusion protein of claim 26, wherein the ligand is a dimeric ligand that binds the ligand binding domain in the fusion protein and a second protein.

29. The fusion protein of claim 28, wherein the dimeric ligand is FK1012.

30. The fusion protein of claim 1, wherein the kinase is catalytically inactive when a ligand is bound to the ligand binding domain and is more active when no ligand is bound to the ligand binding domain.

31. The fusion protein of claim 30, wherein the kinase is substantially inactive when ligand is bound to the ligand binding domain.

32. The fusion protein of claim 30, wherein the ligand binding domain changes conformation in response to illumination.

33. The fusion protein of claim 32, wherein the ligand binding domain is a LOV domain.

34. A polynucleotide encoding the fusion protein of any one of claims 1-33.

35. The polynucleotide of claim 34, further comprising a promoter operably linked thereto.

36. A vector comprising the polynucleotide of claim 34 or 35.

37. The vector of claim 36, further comprising one or more transcription and/or translation regulatory elements.

38. A host cell comprising the polynucleotide of claim 34 or 35 or the vector of claim 36 or 37.

39. A method of producing a fusion protein, comprising expressing the fusion protein encoded by the polynucleotide of claim 34 or 35 or the vector of claim 36 or 37.

40. The method of claim 39, wherein the expressing occurs in vitro.

41. The method of claim 39, wherein the expressing occurs in a cell.

42. A cell comprising the fusion protein of any one of claims 1 -33.

43. The cell of claim 42, wherein the cell is an isolated cell or a cell in an isolated tissue.

44. The cell of claim 42, wherein the cell is in an animal.

45. A method of converting a kinase to a ligand-dependent kinase, comprising inserting a ligand binding domain into or near the catalytic domain of the kinase.

46. A method of activating a kinase present in the fusion protein of any one of claims 1-29, comprising contacting the fusion protein with a ligand that binds to the ligand binding domain.

47. A method of inactivating a kinase present in the fusion protein of any one of claims 30-33, comprising contacting the fusion protein with a ligand that binds to the ligand binding domain.

48. The method of claim 46 or 47, wherein the fusion protein is present in a cell.

49. The method of claim 48, wherein the cell is an isolated cell.

50. The method of claim 48, wherein the cell is in an animal.

51. A method for assessing the function of a kinase present in the fusion protein of any one of claims 1-29, comprising contacting the fusion protein with a ligand that binds to the ligand binding domain and assessing one or more activities of the kinase.

52. A method for assessing the function of a kinase present in the fusion protein of any one of claims 30-33, comprising removing from the fusion protein a ligand that binds to the ligand binding domain and assessing one or more activities of the kinase.

53. The method of claim 51 or 52, wherein the one or more activities is selected from the group consisting of protein binding, signaling, translocation, and enzymatic activity.

54. The method of claim 51 or 52, wherein the fusion protein is present in a cell.

55. The method of claim 54, wherein the cell is an isolated cell.

56. The method of claim 54, wherein the cell is in an animal.

57. A kit comprising the fusion protein of any one of claims 1-33, the polynucleotide of claim 34 or 35, the vector of claim 36 or 37, and/or the cell of claim 38.