METHODS AND COMPOSITIONS FOR MODULATING CELL DEATH WITH SURVIVAL- OR DEATH KINASES OR PHOSPHATASES

Related Applications

This application claims the benefit of U.S. provisional application no. 60/655,134, filed February 22, 2005, the contents of which is incorporated herein in its entirety by this reference. Government Support This invention was made with government support under Grant numbers

RO1CA46595 and GM51405 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background

Programmed cell death or apoptosis is an evolutionarily conserved process that contributes to the development and maintenance of virtually all cell types and organisms.

Apoptosis is implicated in the progression of many autoimmune and neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. Moreover, inappropriate control of cell death and survival can lead to the development of cancer (Hanahan and

Weinberg, (2000) Cell 100:57-70). Thus, the identification of survival and apoptotic proteins activated or down-regulated in a cancer cell is of paramount importance to improve cancer therapy.

Kinases and phosphatases control the reversible process of phosphorylation and are dysregulated in many diseases, such as cancer. Given that protein and lipid phosphorylation controls cell survival signaling, strategies for targeting kinases and phosphatases are needed for improved therapeutic intervention. For example, drugs such as Gleevec and Iressa inhibit tumor growth by specifically antagonizing specific survival kinases, thus reversing the malignant phenotype. Such "targeted" therapies offer enormous advantages over conventional broad-spectrum anti-cancer agents, which are associated with non-specific toxic side effects. Moreover, the combination of targeted and conventional therapies has the potential to maximize efficacy, while minimizing toxicity (MacKeigan et al, J Biol Chem 275, 38953-6 (2000); Slamon et al, N Engl J Med 344, 783-92 (2001); MacKeigan et al., Clin Cancer Res 8, 2091-9 (2002); Pietras et al., Cancer Res 62, 5476-84 (2002).

Numerous research efforts have focused on the Ras/MAPK cell survival pathway.

In the Ras/MAPK cascade, growth factors activate the small G protein Ras, which recruits

Raf to the plasma membrane where it is activated and phosphorylates MEKl /2, which in turn phosphorylates ERKl/2-MAPKs. Activated ERK1/2 phosphorylates additional kinases, such as RSK, and specific transcription factors, such as c-Fos and EIk-I, which are important in cellular proliferation and survival (Ballif and Blenis, (2001) Cell Growth

Differ 12:397-408; Robinson and Cobb, (1997) Curr Opin Cell Biol 9:180-6; Murphy et al,

(2002) Nat Cell Biol 4:556-64). Mutations in Ras have been identified in 30% of all human cancers and mutations in downstream targets, such as Raf occur in 66% of malignant melanomas (Davies et al., (2002) Nature 417:949-54).

A second cell survival cascade in cancer cells is the PI3K-Akt pathway. In response to extracellular stimuli, PI3K can phosphorylate membrane lipids to form the second messengers PI(3,4,5)P ₃ and PI(3,4)P ₂ , which bind to the PH domain of Akt. This results in the translocation of Akt to the plasma membrane, where it becomes activated and subsequently inactivates proapoptotic molecules BAD, pro-caspase-9, and the Forkhead transcription factor (Datta et al., (1997) Cell 91 :231-41; del Peso et al., (1997) Science 278:687-9; Cardone et al., (1998) Science 282:1318-21; Brunet et al., (1999) Cell 96:857- 68). The tumor-suppressor phosphatase PTEN, which negatively regulates Akt by removing the D-3 position phosphate of phosphoinositide lipids, is mutated in many cancers, such as prostate, ovarian, melanoma, lung, and breast carcinomas. The PI3K-Akt pathway also promotes activation of the nutrient sensing kinase mTOR (the mammalian target of rapamycin) and its downstream effectors S6 kinase (S6K1 and S6K2). mTOR activity is normally suppressed by the TSCl and TSC2 proteins. Akt can phosphorylate and inactivate these tumor suppressors, which leads to activation of mTOR. mTOR, and it downstream target S6K promote the phosphorylation of S6, a component of the 4OS ribosome, and enhance ribosome biogenesis and overall translational capacity.

Taken together, the PI3K-Akt and mT0R/S6K pathways cooperate with the ERK1/2-MAPK pathway to coordinate cell growth, cell proliferation, and cell survival. Overactivation of the MAPK, PI3K, and mTOR pathways in cancer and the use of selective small molecule inhibitors that block these signaling systems have confirmed the MAPK, PI3K, and mTOR cascades as valuable molecular targets in cancer (Sebolt-Leopold et al., (1999) Nat Med 5:810-6; Sivaraman et al., (1997) J Clin Invest 99:1478-83; Mandell et al.,

(1998) Am J. Pathol 153:1411-1423; Mansour et al, (1994) Science 265:966-70; Dancey & Sausville, (2003) Nat Rev Drag Discov 2:296-313).

While progress has been made in identifying the major cell death and cell survival kinases and phosphatases, detailed mechanisms of kinase and phosphatase regulation still need to be determined. The identification of both major and minor kinases and phosphatases involved in cell survival and apoptosis and a subsequent understanding of their roles in cell survival and apoptosis may provide novel therapeutic targets for the treatment of numerous human diseases, including cancer.

Summary The present disclosure is based, at least in part, on the identification of kinases and phosphatases that potentiate or prevent apoptosis and cellular proliferation. Expression of the kinases and phosphatases described herein are important for the modulation of cell death and proliferation, and, as such may provide novel therapeutic targets in the treatment of cancer, neurodegenerative diseases and cardiovascular disease. Further, gaining an understanding of the kinases and phsophatases that when inhibited contribute to apoptosis should enable a more rational approach to drag design and allow for the control of various diseases affected by this critcal process.

In one aspect, the disclosure features nucleic acids encoding cell survival and cell death kinases and phosphatases of the invention, recombinant vectors containing kinase and phosphatase genes described herein, host cells containing the recombinant vectors, and methods of producing the encoded polypeptides.

In another aspect, the disclosure features cell survival and cell death kinase and phosphatase polypeptides. Each ldnase and phosphatase polypeptide described herein plays a role in promoting cell survival or apoptosis. In another aspect, the disclosure features a method for sensitizing a cell to apoptosis comprising, e.g., administering one or more agents, e.g., siRNAs, targeting a cell survival kinase or phosphatase, described herein, in the cell. Also disclosed is a method for rendering a cell resistant to apoptosis comprising administering one or more agents, e.g., siRNAs targeting a cell death kinase or phosphatase, described herein, in the cell. In a further aspect, the disclosure features screening assays for identifying test compounds that modulate the expression level and/or function of any of the kinase or

phosphatases described herein. In one embodiment, the assay is a kinase or phosphatase assay to identify a test compound(s) that modulates the activity of a cell survival or cell death kinase or phosphatase polypeptide of the invention. In another embodiment, the assay is a binding assay to identify a test compound(s) that modulates protein-protein interactions between a particular kinase or phosphatase of the invention and an interacting protein, hi a further embodiment, the assay is an expression assay to identify a test compound(s) that reduces the expression level of a kinase or phosphatase polypeptide of the invention.

Further features and advantages of the disclosed inventions will now be discussed in conjunction with the following Detailed Description and Claims.

Brief Description of the Drawings

Figure 1 are graphs showing flow cytometric analysis of siRNA transfection efficiency. Alexa488-conjugated siRNA was transfected into (A) HeLa, (B) Hl 57, and (C)

BT474 cells. Cells were collected 24 hours post transfection for detection of Alexa488- conjugated siRNA (gray) relative to control (black) cells using a FACScan flow cytometer

(Bectin Dickinson).

Figure 2 shows results from a human siRNA kinase library screen. (A) HeLa cells seeded in 96-well plates were transfected with siRNAs directed against all known and putative human kinases. Cells were incubated for 72 hours to allow target knockdown and apoptosis was measured by a DNA-fragmentation ELISA. A graph shows the relative apoptosis for all 650 kinase siRNA targets and basal cell survival is set at 1. (B) Apoptosis was confirmed by replicate siRNA transfections with CDK6, RPS6KL1, RORl and NLK kinase targets. (C) Cleavage of caspase 9 and the caspase substrate poly (ADP-ribosyl) polymerase (PARP), biochemical features of apoptosis, were detected by western blot analysis. (D) Cell viability was measured at 72 hours after introduction of individual siRNA duplexes, cells were fixed and stained with crystal violet to visualize viable cells. Darker staining represents more viable cells. (E) A replicate siRNA transfection experiment using positively scoring survival kinase siRNAs. (F) Crystal violet cell viability of FER, JIK, and ACKl . (G) Positively scoring siRNA duplexes led to apoptosis in Hl 57 lung carcinoma cells. Hl 57 cells were transfected with siRNA duplexes and cleavage of the caspase substrate PARP was detected by western blot analysis.

Figure 3 are graphs showing individually validated siRNA duplexes directed against survival kinases. (A) A replicate siRNA transfection experiment using 21 positively scoring survival kinase siRNAs. HeLa cells were transfected with control siRNA or siRNA directed against (B) CDK6 or (C) IHPK3, and treated with the indicated concentrations of Taxol.

Figure 4 are graphs showing RT-PCR analysis of selected siRNA duplexes in HeLa cells. Total RNA was isolated 36 hours after transfection from HeLa cells using RNeasy (Qiagen) and primers designed to specifically amplify the indicated kinases and phosphatases. The RT-PCR reactions were performed using QuantiTect SYBR Greeen RT- PCR (Qiagen) and comparative C _T method used for relative expression. N.D. indicates not determined. mRNA levels were measured at 36 hours after introduction of individual siRNA duplexes (25 nM) or two siRNA duplexes (25 nM per duplex) pooled. Expression of nonsilencing scrambled siRNA controls did not result in mRNA decreases.

Figure 5 shows results from a human siRNA phosphatase library screen. (A) HeLa cells seeded in 96-well plates were transfected with siRNAs directed against all known and putative human phosphatases. Cells were incubated for 72 hours to allow target knockdown and apoptosis was measured by a DNA-fragmentation ELISA. A graph shows the relative apoptosis for all 222 phosphatase siRNA targets. (B) Cell viability was measured at 72 hours after introduction of individual siRNA duplexes, cells were fixed and stained with crystal violet to visualize viable cells. Darker staining represents more viable cells. (C) HeLa cells were transfected with 15 positively scoring phosphatase siRNA duplexes and cleavage of caspase 9 and PARP detected by western blot analysis.

Figure 6 shows results from a human siRNA phosphatase library screen identifmg cell death phosphatases and resistance to apoptosis. (A) Control or MK-STYX siRNA transfected HeLa cells were treated with 50 μM Cisplatin, 10 nM paclitaxel, or 100 μM Etoposide for 24 hours, and apoptosis assayed by ELISA that measures DNA-histone fragments. (B) RT-PCR analysis of MK-STYX individual and pooled (1+2) siRNA duplexes. Total RNA was isolated 36 hours after transfection and mRNA levels measured after introduction of individual siRNA duplexes (25 nM) or two siRNA duplexes (25 nM per duplex) pooled. (C) Cells were transfected with control siRNA or MK-STYX siRNA for 48 hours, treated for an additional 24 hours with solvent control (-) or 50 μM Cisplatin (+). Cell viability was visualized by crystal violet stain and cleavage of full length PARP

measured by western blot analysis. (D) Cells were incubated with control siRNA or two siRNA duplexes targeting each cell death phosphatase for 48 hours, treated for an additional 24 hours with 50 μM Cisplatin, and cell viability measure by crystal violet stain.

Figure 7 shows that a low dose (nanomolar range) of paclitaxel and siRNAs directed against survival kinases cause enhanced apoptosis. HeLa cells were transfected with control siRNA or survival kinase siRNA directed against (A) SGK, (B) mTOR, (C) CDK8, and treated with the indicated concentrations of paclitaxel (Taxol). The survival kinase FER is an example of a target that enhances apoptosis with low nanomolar doses of the conventional chemotherapeutic agents (D) paclitaxel, (E) etoposide, and (F) cisplatin. Figure 8 show synergistic chemotherapy-induced apoptosis in BT474 breast carcinoma cells with survival kinase siRNAs. BT474 cells were transfected with control siRNA or survival kinase siRNA directed against (A) FER, (B) JIK, (C) PLK2, and treated with the low dose, nanomolar concentrations of paclitaxel. (D) Synergistic apoptosis in HeLa cells with the combination of siRNA directed against PINKl . (E) The combination of paclitaxel and PINKl siRNA sensitizes BT474 cells to apoptosis. (F) RT-PCR analysis of PINKl siRNA duplexes. Total RNA was isolated 36 hours after transfection and mRNA levels measured after introduction of individual siRNA duplexes (25 nM) or two siRNA duplexes (25 nM per duplex) pooled. (G) HeLa cells were transfected with control or PINKl siRNA and cleavage of caspase 9 and PARP detected by western blot analysis. Detailed Description

1. General

The present disclosure is based, at least in part, on the identification of previously unrecognized kinases and phophatases that are involved in cell survival and apoptosis. As described herein, the cell survival and cell death kinases and phosphatases of the invention were identified in a small-interfering RNA (siRNA)-based screen that was developed to systematically examine the function of all known and putative kinases and phosphatases. Further described are methods for screening compounds to identify compounds that modulate the expression or activity of a cell survival kinase or phosphatase polypeptide or a cell death kinase or phosphatase polypeptide and thereby affect cell proliferation or apoptosis.

2. Definitions

For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. Unless defined otherwise, 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 term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a "therapeutic agent" which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

The terms "agonist" or "activator" refer to an agent that upregulates (e.g., activates or enhances) at least one bioactivity of a protein. An agonist may be a compound which increases the interaction between a protein and another molecule, e.g., a target peptide or enzyme substrate. An agonist may also be a compound that increases expression of a gene or which increases the amount of protein expressed.

The terms "antagonist" or "inhibitor" refer to an agent that downregulates (e.g., suppresses or inhibits) at least one bioactivity of a protein. An antagonist may be a compound which inhibits or decreases the interaction between a protein and another molecule, e.g., a target peptide or enzyme substrate. An antagonist may also be a compound that downregulates expression of a gene or which reduces the amount of protein expressed.

The term "binding" refers to an association, which may be a stable association, between two molecules, e.g., between a polypeptide of the invention and a binding partner, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.

The term "cell death kinase" refers to any kinase that is involved either directly (e.g., the kinase itself is a member of the signal transduction pathway) or indirectly (e.g., the kinase interacts with, or regulates, a member of the signal transduction pathway) in promoting or maintaining a cell death and/or apoptotic signal transduction pathway. Accordingly, an antagonist or inhibitor of a cell death kinase will lead to a reduction in cell death and/or apoptosis (as compared to a similar cell under similar conditions in the absence of the antagonist or inhibitor) whereas an agonist or activator of a cell death kinase

will lead to an increase of cell death and/or apoptosis (as compared to a similar cell under similar conditions in the absence of the agonist or activator). In one embodiment, a cell death kinase may be involved in drug-induced apoptosis, e.g., the activity of the kinase is involved in promoting apoptosis in the presence of a particular drug. Exemplary cell death promoting kinases are shown in Table 2.

The term "cell death phosphatase" refers to any phosphatase that is involved either directly (e.g., the phosphatase itself) or indirectly (e.g., the phosphatase interacts with, or regulates, another polypeptide) in promoting or maintaining a cell death and/or apoptotic signal transduction pathway. Accordingly, an antagonist or inhibitor of a cell death phosphatse will lead to a reduction in cell death and/or apoptosis (as compared to a similar cell under similar conditions in the absence of the antagonist or inhibitor) whereas an agonist or activator of a cell death phosphatase will lead to an increase in cell death and/or apoptosis (as compared to a similar cell under similar conditions in the absence of the agonist or activator). In one embodiment, a cell death phophatase may be a candidate tumor suppressor protein, wherein the expression and/or activity of the cell death phosphatase inhibits cell proliferation. Specific examples of cell death phosphatases are shown in Table 4.

The term "cell survival kinase" refers to any kinase that is involved either directly (e.g., the kinase itself) or indirectly (e.g., the kinase interacts with, or regulates, another polypeptide) in promoting or maintaining cell survival. Accordingly, an antagonist or inhibitor of a cell survival kinase will lead to an increase in cell death and/or apoptosis (as compared to a similar cell under similar conditions in the absence of the antagonist or inhibitor) whereas an agonist or activator of a cell survival kinase will lead to a decrease in cell death and/or apoptosis (as compared to a similar cell under similar conditions in the absence of the agonist or activator). In certain embodiments, cell survival kinases may have a role in one or more of the following: cell cycle control, calcium signaling, AGC kinase signalling, lipid signalling, MAPK signalling, tyrosine kinase signalling, TGFβ signaling, or metabollic regulation. Inhibition of a cell survival kinase may lead to at least about a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or greater fold, increase in cell death. Exemplary cell survival kinases are shown in Table 1 and Table 5.

The term "cell survival phophatase" refers to any phosphatase that is either directly (e.g., the phophatase itself) or indirectly (e.g., the phophatase interacts with, or regulates,

another polypeptide) in promoting or maintaining cell survival. Accordingly, an antagonist or inhibitor of a cell survival phosphatase will lead to an increase in cell death and/apoptosis (as compared to a similar cell under similar conditions in the absence of the antagonist or inhibitor) wherease an agonist or activator of a cell survival kinase will lead to a decrease in cell death and/or apoptosis (as compared to a similar cell under similar conditions in the absence of the agonist or activator). In certain embodiments, cell survival phosphatases may function as protein phosphatases, such as protein tyrosine phophatases or dual specificity phosphatases. Cell survival phophatases may also have a role in lipid signalling. Inhibition of a cell survival phosphatase may lead to at least about a 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, or greater fold, increase in cell death. Exemplary cell survival phosphatases are shown in Table 3 and Table 6.

The term "complex" refers to an association between at least two moieties (e.g. chemical or biochemical) that have an affinity for one another. Examples of complexes include associations between antigen/antibodies, lectin/avidin, target polynucleotide/probe oligonucleotide, antibody/anti-antibody, receptor/ligand, enzyme/ligand, polypeptide/ polypeptide, polypeptide/polynucleotide, polypeptide/co-factor, polypeptide/substrate, polypeptide/inhibitor, polypeptide/small molecule, and the like. "Member of a complex" refers to one moiety of the complex, such as an antigen or ligand. "Protein complex" or "polypeptide complex" refers to a complex comprising at least one polypeptide. A "delivery complex" shall mean a targeting means (e.g. a molecule that results in higher affinity binding of a gene, protein, polypeptide or peptide to a target cell surface and/or increased cellular or nuclear uptake by a target cell). Examples of targeting means include: sterols (e.g. cholesterol), lipids (e.g. a cationic lipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associated virus, and retrovirus) or target cell specific binding agents (e.g. ligands recognized by target cell specific receptors). Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the nucleic acid, e.g., siRNA or nucleic acid encoding such, is released in a functional form. The term "domain", when used in connection with a polypeptide, refers to a specific region within such polypeptide that comprises a particular structure or mediates a particular function. In the typical case, a domain of a polypeptide of the invention is a fragment of the polypeptide. In certain instances, a domain is a structurally stable domain, as evidenced,

for example, by mass spectroscopy, or by the fact that a modulator may bind to a draggable region of the domain.

The term "kinase" refers to an enzyme that is capable of transferring a terminal phosphate group, also known as a gamma-phosphate group, from ATP to a substrate. Kinases phosphorylate specific serine, threonine or tyrosine residues on their target substrates and are typically classified as either serine/threonine kinases or tyrosine ldnases.

The term "modulation", when used in reference to a functional property or biological activity or process (e.g., enzyme activity or receptor binding), refers to the capacity to either up regulate (e.g., activate or stimulate), down regulate (e.g., inhibit or suppress) or otherwise change a quality of such property, activity or process. In certain instances, such regulation may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.

The term "phosphatase" refers to an enzyme that removes a phosphate group from a substrate by hydrolysis.

The term "purified" refers to an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). A "purified fraction" is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all species present. In making the determination of the purity of a species in solution or dispersion, the solvent or matrix in which the species is dissolved or dispersed is usually not included in such determination; instead, only the species (including the one of interest) dissolved or dispersed are taken into account. Generally, a purified composition will have one species that comprises more than about 80 percent of all species present in the composition, more than about 85%, 90%, 95%, 99% or more of all species present. The object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species. A skilled artisan may purify a polypeptide of the invention using standard techniques for protein purification in light of the teachings herein. Purity of a polypeptide may be determined by a number of methods known to those of skill in the art, including for example, amino-terminal amino acid sequence analysis, gel electrophoresis, mass- spectrometry analysis and the methods described in the Exemplification section herein.

"siRNA" stands for short (or small) interfering RNA. siRNAs comprise two sequences that are essentially complementary to each other so that they can hybridize under the desired conditions. The two sequences may be present on one strand or on two strands of nucleic acid. For example, the two sequences may be on one nucleic acid and separated by a spacer sequence that may form a loop when the two sequences interact.

The term "test compound" refers to a molecule to be tested by one or more screening method(s) as a putative modulator of a polypeptide of the invention or other biological entity or process. A test compound is usually not known to bind to a target of interest. The term "control test compound" refers to a compound known to bind to the target (e.g., a known agonist, antagonist, partial agonist or inverse agonist). The term "test compound" does not include a chemical added as a control condition that alters the function of the target to determine signal specificity in an assay. Such control chemicals or conditions include chemicals that 1) nonspecifically or substantially disrupt protein structure (e.g., denaturing agents (e.g., urea or guanidinium), chaotropic agents, sulfhydryl reagents (e.g., dithiothreitol and β-mercaptoethanoi), and proteases), 2) generally inhibit cell metabolism (e.g., mitochondrial uncouplers) and 3) non-specifically disrupt electrostatic or hydrophobic interactions of a protein (e.g., high salt concentrations, or detergents at concentrations sufficient to non-specifically disrupt hydrophobic interactions). In certain embodiments, various predetermined concentrations of test compounds are used for screening such as 0.01 μM, 0.1 μM, 1.0 μM, and 10.0 μM. Examples of test compounds include, but are not limited to, antibodies, peptides, nucleic acids, carbohydrates, and small molecules. The term "novel test compound" refers to a test compound that is not in existence as of the filing date of this application. In certain assays using novel test compounds, the novel test compounds comprise at least about 50%, 75%, 85%, 90%, 95% or more of the test compounds used in the assay or in any particular trial of the assay. Further, the activity of a test compound may render it suitable as a "therapeutic agent" which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject. Thus, a therapeutic agent refers to any substance that intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and/or conditions in an animal or human.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as

being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. 3. Kinase and Phosphatase Nucleic Acids

Provided herein are nucleic acids that encode human kinase and phosphatase polypeptides or variants thereof. Exemplary kinases and phosphatases include cell death kinases, cell death phosphatases, cell survivial kinases, and cell survival phosphatases. Specific examples of kinases and phosphatases disclosed herein, as well as GenBank accession numbers for nucleic acid sequences that encode the polypeptides, are shown in Tables 1-6.

Nucleic acids of the present invention may also comprise, consist of or consist essentially of any of the kinases and phosphatases nucleotide sequences described herein. Yet other nucleic acids comprise, consist of or consist essentially of a nucleotide sequence that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity or homology with a kinases and phosphatases gene described herein. Substantially homologous sequences may be identified using stringent hybridization conditions.

Isolated nucleic acids which differ from the nucleic acids of the invention due to degeneracy in the genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the polypeptides of the invention will exist. One skilled in the art will appreciate that these variations in one or more nucleotides (from less than 1% up to about 3 or 5% or possibly more of the nucleotides) of the nucleic acids encoding a particular protein of the invention may exist among a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. Bias in codon choice within genes in a single species appears related to the level of expression of the protein encoded by that gene. Accordingly, the invention encompasses nucleic acid sequences which have been optimized for improved expression in a host cell

by altering the frequency of codon usage in the nucleic acid sequence to approach the frequency of preferred codon usage of the host cell. Due to codon degeneracy, it is possible to optimize the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, any nucleotide sequence that encodes all or a substantial portion of the amino acid sequence of a polypeptides of the invention is within the scope of the invention.

Nucleic acids encoding proteins which have amino acid sequences evolutionarily related to a polypeptide disclosed herein are provided, wherein "evolutionarily related to", refers to proteins having different amino acid sequences which have arisen naturally (e.g. by allelic variance or by differential splicing), as well as mutational variants of the proteins of the invention which are derived, for example, by combinatorial mutagenesis.

Fragments of the polynucleotides of the invention encoding a biologically active portion of the subject polypeptides are also provided. As used herein, a fragment of a nucleic acid encoding an active portion of a polypeptide disclosed herein refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length amino acid sequence of a polypeptide of the invention, and which encodes a given polypeptide that retains at least a portion of a biological activity of the full-length kinase or phosphatase protein as defined herein, or alternatively, which is functional as a modulator of the biological activity of the full-length protein. For example, such fragments include a polypeptide containing a domain of the full-length protein from which the polypeptide is derived that mediates the interaction of the protein with another molecule (e.g., polypeptide, DNA, RNA, etc.).

Nucleic acids provided herein may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant polypeptides.

A nucleic acid encoding a kinase or phopshatase polypeptide provided herein may be obtained from mRNA or genomic DNA from any organism in accordance with protocols described herein, as well as those generally known to those skilled in the art. A cDNA encoding a polypeptide of the invention, for example, may be obtained by isolating total mRNA from an organism, for example, a bacteria, virus, mammal, etc. Double stranded cDNAs may then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.

A gene encoding a polypeptide of the invention may also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. In one aspect, methods for amplification of a nucleic acid of the invention, or a fragment thereof may comprise: (a) providing a pair of single stranded oligonucleotides, each of which is at least eight nucleotides in length, complementary to sequences of a nucleic acid of the invention, and wherein the sequences to which the oligonucleotides are complementary are at least ten nucleotides apart; and (b) contacting the oligonucleotides with a sample comprising a nucleic acid comprising the nucleic acid of the invention under conditions which permit amplification of the region located between the pair of oligonucleotides, thereby amplifying the nucleic acid.

In another aspect, double stranded small interfering RNAs (siRNAs), and methods for administering the same are provided. (See Tables 7-12 for target DNA and antisense and sense siRNA sequences). siRNAs decrease or block gene expression. While not wishing to be bound by theory, it is generally thought that siRNAs inhibit gene expression by mediating sequence specific mRNA degradation. RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing, particularly in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene (Elbashir et al. Nature 2001; 411(6836): 494-8). Accordingly, it is understood that siRNAs and long dsRNAs having substantial sequence identity to all or a portion of a polynucleotide of the present invention may be used to inhibit the expression of a nucleic acid of the invention, and particularly when the polynucleotide is expressed in a mammalian or plant cell. Exemplary siRNAs to the kinases and phosphatases described herein may be the siRNAs used in the large scale RNA interference screen described below.

Alternatively, siRNAs that decrease or block the expression the kinases and phosphatases described herein may be determined by testing a plurality of siRNA constructs against the target gene. Such siRNAs against a target gene may be chemically synthesized. The nucleotide sequences of the individual RNA strands are selected such that the strand has a region of complementarity to the target gene to be inhibited (i.e., the complementary RNA strand comprises a nucleotide sequence that is complementary to a region of an mRNA transcript that is formed during expression of the target gene, or its processing products, or a region of a (+) strand virus). The step of synthesizing the RNA strand may involve solid-phase synthesis, wherein individual nucleotides are joined end to

end through the formation of internucleotide 3 '-5' phosphodiester bonds in consecutive synthesis cycles.

Provided herein are siRNA molecules comprising a nucleotide sequence consisting essentially of a sequence of a target gene described herein, e.g., a gene encoding a survival kinase. An siRNA molecule may comprise two strands, each strand comprising a nucleotide sequence that is at least essentially complementary to each other, one of which corresponds essentially to a sequence of a target gene. The sequence that corresponds essentially to a sequence of a target gene is referred to as the "sense target sequence" and the sequence that is essentially complementary thereto is referred to as the "antisense target sequence" of the siRNA. The sense and antisense target sequences may be from about 15 to about 30 consecutive nucleotides long; from about 19 to about 25 consecutive nucleotides; from about 19 to 23 consecutive nucleotides or about 19, 20, 21, 22 or 23 nucleotides long. The length of the sense and antisense sequences is determined so that an siRNA having sense and antisense target sequences of that length is capable of inhibiting expression of a target gene, preferably without significantly inducing a host interferon response.

The sense and antisense target sequences are preferably sufficiently complimentary, such that an siRNA comprising both sequences is able to inhibit expression of the target gene, i.e., to mediate RNA interference. For example, the sequences may be sufficiently complementary to permit hybridization under the desired conditions, e.g., in a cell. Accordingly, the sense and antisense target sequences may be at least about 95%, 97%, 98%, 99% or 100% identical and may, e.g., differ in at most 5, 4, 3, 2, 1 or 0 nucleotides.

The sequences of exemplary siRNAs are provided in Tables 7-12. Other siRNAs may comprise a sequence consisting essentially of a sequence disclosed in Tables 7-12 with one or more or one or less nucleotides at one or both ends.

Also provided are recombinant vectors, which include an isolated kinase or phosphatase nucleic acid as disclosed herein, host cells containing the recombinant vectors, and methods of making such vectors and host cells as well as using them for the production of the encoded polypeptides by recombinant techniques. The nucleic acids described herein may be provided in an expression vector comprising a nucleotide sequence encoding a kinase or phopshatase polypeptide that is operably linked to at least one regulatory sequence. It should be understood that the design

of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. The vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered. The subject nucleic acids may be used to cause expression and over-expression of a kinase or phosphatase polypeptide in cells propagated in culture, e.g. to produce proteins or polypeptides, including fusion proteins or polypeptides.

Host cells may be transfected with a recombinant gene in order to express a desired kinase or phosphatase polypeptide. The host cell may be any prokaryotic or eukaryotic cell. For example, a polypeptides may be expressed in bacterial cells, such as E. coli, insect cells (baculovirus), yeast, or mammalian cells. In those instances when the host cell is human, it may or may not be in a live subject. Other suitable host cells are known to those skilled in the art. Additionally, the host cell may be supplemented with tRNA molecules not typically found in the host so as to optimize expression of the polypeptide. Other methods suitable for maximizing expression of the polypeptide will be known to those in the art.

Methods of producing polypeptides are also provided. For example, a host cell transfected with an expression vector encoding a kinase or phosphatase polypeptide may be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.

A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The polypeptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, gel filtration chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, ultrafiltration, electrophoresis, immunoaffϊnity purification with antibodies specific for particular epitopes of a polypeptide of the invention, and high performance liquid chromatography ("HPLC") is employed for purification.

Thus, a nucleotide sequence encoding all or a selected portion of a kinase or phophatase polypeptide, such as for example, those described in Tables 1-6, may be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant polypeptides of the invention by microbial means or tissue-culture technology.

Expression vehicles for production of a recombinant protein include plasmids and other vectors. For instance, suitable vectors for the expression of a polypeptide of the invention include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, ρYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example,

Broach et al., (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye

Academic Press, p. 83). These vectors may replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin may be used.

In certain embodiments, mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-I), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory

Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant protein by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III).

In another variation, protein production may be achieved using in vitro translation systems. In vitro translation systems are, generally, a translation system which is a cell-free extract containing at least the minimum elements necessary for translation of an RNA molecule into a protein. An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in vitro translation systems are well known in the art and include commercially available kits. Examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, IU.; and GIBCO/BRL, Grand Island, N. Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used. Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors. In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs. When expression of a carboxy terminal fragment of a polypeptide is desired, i.e. a truncation mutant, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position may be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben- Bassat et al., (1987) J Bacteriol. 169:751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al., (1987) PNAS USA 54:2718-1722). Therefore, removal of an N-terminal methionine, if desired, may be achieved either in vivo by expressing such recombinant polypeptides in a host which

produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al).

Coding sequences for a polypeptide of interest may be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. The present invention contemplates an isolated nucleic acid comprising a nucleic acid of the invention and at least one heterologous sequence encoding a heterologous peptide linked in frame to the nucleotide sequence of the nucleic acid of the invention so as to encode a fusion protein comprising the heterologous polypeptide. The heterologous polypeptide may be fused to (a) the C-terminus of the polypeptide encoded by the nucleic acid of the invention, (b) the N-terminus of the polypeptide, or (c) the C-terminus and the N-terminus of the polypeptide. In certain instances, the heterologous sequence encodes a polypeptide permitting the detection, isolation, solubilization and/or stabilization of the polypeptide to which it is fused. In still other embodiments, the heterologous sequence encodes a polypeptide selected from the group consisting of a polyHis tag, myc, HA, GST, protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose-binding protein, poly arginine, poly His- Asp, FLAG, a portion of an immunoglobulin protein, and a transcytosis peptide.

Fusion expression systems can be useful when it is desirable to produce an immunogenic fragment of a polypeptide of the invention. For example, the VP6 capsid protein of rotavirus may be used as an immunologic carrier protein for portions of polypeptide, either in the monomeric form or in the form of a viral particle. The nucleic acid sequences corresponding to the portion of a polypeptide of the invention to which antibodies are to be raised may be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising a portion of the protein as part of the virion. The Hepatitis B surface antigen may also be utilized in this role as well. Similarly, chimeric constructs coding for fusion proteins containing a portion of a polypeptide of the invention and the poliovirus capsid protein may be created to enhance immunogenicity (see, for example, EP Publication NO: 0259149; and Evans et al., (1989) Nature 339:385; Huang et al., (1988) J. Virol. 62:3855; and Schlienger et al., (1992) J Virol. 66:2). Fusion proteins may facilitate the expression and/or purification of proteins. For example, a polypeptide of the invention may be generated as a glutathione-S-transferase (GST) fusion protein. Such GST fusion proteins may be used to simplify purification of a polypeptide of the invention, such as through the use of glutathione-derivatized matrices

(see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., (N. Y.: John Wiley & Sons, 1991)). In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, may allow purification of the expressed fusion protein by affinity chromatography using a Ni ²⁺ metal resin. The purification leader sequence may then be subsequently removed by treatment with enterokinase to provide the purified protein (e.g., see Hochuli et al., (1987) J. Chromatography 411: 177; and Janknecht et al., PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments may be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992). In other embodiments, nucleic acids of the invention may be immobilized onto a solid surface, including, plates, microtiter plates, slides, beads, particles, spheres, films, strands, precipitates, gels, sheets, tubing, containers, capillaries, pads, slices, etc. The nucleic acids of the invention may be immobilized onto a chip as part of an array. The array may comprise one or more polynucleotides of the invention as described herein. In one embodiment, the chip comprises one or more polynucleotides of the invention as part of an array of polynucleotide sequences.

4. Kinase and Phosphatase Polypeptides

Kinase and phopshatase polypeptides described herein include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.

Polypeptides may also comprise, consist of or consist essentially of an amino acid sequence encoded by a nucleotide sequence having an accession number shown in Tables 1-6. Yet other polypeptides comprise, consist of or consist essentially of an amino acid sequence that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity or homology with the kinase or phosphatase polypeptides shown in Tables 1-6. For example, polypeptides that differ from a sequence in a naturally occurring protein in about 1, 2, 3, 4, 5 or more amino acids are also contemplated. The differences may be substitutions, e.g., conservative substitutions, deletions or additions. The differences are preferably in regions that are not significantly conserved among different species. Such regions can be identified by aligning the amino acid sequences from various species. These amino acids can be substituted, e.g., with those found in another species. Other amino acids that may be substituted, inserted or deleted at these or other locations can be identified by mutagenesis studies coupled with biological assays.

Proteins may be used as a substantially pure preparation, e.g., wherein at least about 90% of the protein in the preparation are the desired protein. Compositions comprising at least about 50%, 60%, 70%, or 80% of the desired protein may also be used.

Other proteins that are encompassed herein are those that comprise modified amino acids. Exemplary proteins are derivative proteins that may be one modified by glycosylation, pegylation, phosphorylation or any similar process that retains at least one biological function of the protein from which it was derived.

Proteins may also comprise one or more non-naturally occurring amino acids. For example, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into proteins. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary). hi certain embodiments, a kinase or phosphatase polypeptide described herein may be a fusion protein containing a domain which increases its solubility and/or facilitates its

purification, identification, detection, and/or structural characterization. Exemplary domains, include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His-Asp or FLAG fusion proteins and tags. Additional exemplary domains include domains that alter protein localization in vivo, such as signal peptides, type III secretion system-targeting peptides, transcytosis domains, nuclear localization signals, etc. hi various embodiments, a polypeptide of the invention may comprise one or more heterologous fusions. Polypeptides may contain multiple copies of the same fusion domain or may contain fusions to two or more different domains. The fusions may occur at the N- terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C- terminus of the polypeptide. It is also within the scope of the invention to include linker sequences between a polypeptide of the invention and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein. In another embodiment, the polypeptide may be constructed so as to contain protease cleavage sites between the fusion polypeptide and polypeptide of the invention in order to remove the tag after protein expression or thereafter. Examples of suitable endoproteases, include, for example, Factor Xa and TEV proteases.

In another embodiment, kinase or phopshatase polypeptides may be modified so that their rate of traversing the cellular membrane is increased. For example, the polypeptide may be fused to a second peptide which promotes "transcytosis," e.g., uptake of the peptide by cells. The peptide may be a portion of the HIV transactivator (TAT) protein, such as the fragment corresponding to residues 37-62 or 48-60 of TAT, portions which have been observed to be rapidly taken up by a cell in vitro (Green and Loewenstein, (1989) Cell 55:1179-1188). Alternatively, the internalizing peptide may be derived from the

Drosophila antennapedia protein, or homologs thereof. The 60 amino acid long homeodomain of the homeo-protein antennapedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled. Thus, polypeptides may be fused to a peptide consisting of about amino acids 42-58 of Drosophila antennapedia or shorter fragments for transcytosis (Derossi et al. (1996) J. Biol. Chem. 271:18188-18193; Derossi et al. (1994) J.

Biol. Chem. 269:10444-10450; and Perez et al. (1992) J. Cell Sci. 102:717-722). The

transcytosis polypeptide may also be a non-naturally-occurring membrane-translocating sequence (MTS), such as the peptide sequences disclosed in U.S. Patent No.6,248,558.

Polypeptides can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography and high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.

In certain embodiments, polypeptides of the invention may be synthesized chemically, ribosomally in a cell free system, or ribosomally within a cell. Chemical synthesis of polypeptides of the invention may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules, (see e.g., U.S. Patent Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; M. Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol. 264, p 90; R. E. Offord, "Chemical Approaches to Protein Engineering", in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl. Acad. Sci. USA

(1994) 91: 12544-12548; M. Schnlzer, et al., Science (1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).

In certain embodiments, it may be advantageous to provide naturally-occurring or experimentally-derived homo logs of a polypeptide of the invention. Such homologs may function in a limited capacity as a modulator to promote or inhibit a subset of the biological activities of the naturally-occurring form of the polypeptide. Thus, specific biological effects may be elicited by treatment with a homolog of limited function, and with fewer side effects relative to treatment with agonists or antagonists which are directed to all of the biological activities of a polypeptide of the invention. For instance, antagonistic homologs may be generated which interfere with the ability of the wild-type polypeptide of the invention to associate with certain proteins, but which do not substantially interfere with the formation of complexes between the native polypeptide and other cellular proteins.

Polypeptides may be derived from the full-length polypeptides of the invention. Isolated peptidyl portions of those polypeptides may be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such polypeptides. In addition, fragments may be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, proteins may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or may be divided into overlapping fragments of a desired length. The fragments may be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments having a desired property, for example, the capability of functioning as a modulator of the polypeptides of the invention. In an illustrative embodiment, peptidyl portions of a protein of the invention may be tested for binding activity, as well as inhibitory ability, by expression as, for example, thioredoxin fusion proteins, each of which contains a discrete fragment of a protein of the invention (see, for example, U.S. Patents 5,270,181 and 5,292,646; and PCT publication WO94/02502).

In another embodiment, truncated polypeptides may be prepared. Truncated polypeptides have from 1 to 20 or more amino acid residues removed from either or both the N- and C-termini. Such truncated polypeptides may prove more amenable to expression, purification or characterization than the full-length polypeptide. For example, truncated polypeptides may prove more amenable than the full-length polypeptide to crystallization, to yielding high quality diffracting crystals or to yielding an HSQC spectrum with high intensity peaks and minimally overlapping peaks. In addition, the use

of truncated polypeptides may also identify stable and active domains of the full-length polypeptide that may be more amenable to characterization.

It is also possible to modify the structure of the polypeptides of the invention for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life, resistance to proteolytic degradation in vivo, etc.). Such modified polypeptides, when designed to retain at least one activity of the naturally-occurring form of the protein, are considered "functional equivalents" of the polypeptides described in more detail herein.

Such modified polypeptides may be produced, for instance, by amino acid substitution, deletion, or addition, which substitutions may consist in whole or part by conservative amino acid substitutions.

For instance, it is reasonable to expect that an isolated conservative amino acid substitution, such as replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, will not have a major affect on the biological activity of the resulting molecule. Whether a change in the amino acid sequence of a polypeptide results in a functional homolog may be readily determined by assessing the ability of the variant polypeptide to produce a response similar to that of the wild-type protein. Polypeptides in which more than one replacement has taken place may readily be tested in the same manner.

Methods of generating sets of combinatorial mutants of polypeptides of the invention are provided, as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g. homologs). The purpose of screening such combinatorial libraries is to generate, for example, homologs which may modulate the activity of a polypeptide of the invention, or alternatively, which possess novel activities altogether. Combinatorially-derived homologs may be generated which have a selective potency relative to a naturally-occurring protein. Such homologs may be used in the development of therapeutics.

Likewise, mutagenesis may give rise to homologs which have intracellular half- lives dramatically different than the corresponding wild-type protein. For example, the altered protein may be rendered either more stable or less stable to proteolytic degradation or other cellular process which result in destruction of, or otherwise inactivation of the protein. Such homologs, and the genes which encode them, may be utilized to alter protein

expression by modulating the half-life of the protein. As above, such proteins may be used for the development of therapeutics or treatment.

In similar fashion, protein homologs may be generated by the present combinatorial approach to act as antagonists, in that they are able to interfere with the activity of the corresponding wild-type protein.

In a representative embodiment of this method, the amino acid sequences for a population of protein homologs are aligned, preferably to promote the highest homology possible. Such a population of variants may include, for example, homologs from one or more species, or homologs from the same species but which differ due to mutation. Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences. In certain embodiments, the combinatorial library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential protein sequences. For instance, a mixture of synthetic oligonucleotides may be enzymatically ligated into gene sequences such that the degenerate set of potential nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display).

There are many ways by which the library of potential homologs may be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence may be carried out in an automatic DNA synthesizer, and the synthetic genes may then be ligated into an appropriate vector for expression. One purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential protein sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp. 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Patent Nos: 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis may be utilized to generate a combinatorial library. For example, protein homologs (both agonist and antagonist forms) may be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al, (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al., (1992) MoI. Cell Biol. 12:2644- 2652; McKnight et al., (1982) Science 232:316); by saturation mutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell MoI Biol 1:11-19); or by random mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in MoI Biol 7:32-34). Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated forms of proteins that are bioactive.

A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of protein homologs. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the illustrative assays described below are amenable to high throughput analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.

In an illustrative embodiment of a screening assay, candidate combinatorial gene products are displayed on the surface of a cell and the ability of particular cells or viral particles to bind to the combinatorial gene product is detected in a "panning assay". For instance, the gene library may be cloned into the gene for a surface membrane protein of a bacterial cell (Ladner et al., WO 88/06630; Fuchs et al., (1991) Bio/Technology 9:1370- 1371; and Goward et al., (1992) TIBS 18:136-140), and the resulting fusion protein

detected by panning, e.g. using a fluorescently labeled molecule which binds the cell surface protein, e.g. FITC-substrate, to score for potentially functional homologs. Cells may be visually inspected and separated under a fluorescence microscope, or, when the morphology of the cell permits, separated by a fluorescence-activated cell sorter. This method may be used to identify substrates or other polypeptides that can interact with a polypeptide of the invention.

In similar fashion, the gene library may be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences may be expressed on the surface of infectious phage, thereby conferring two benefits. First, because these phage may be applied to affinity matrices at very high concentrations, a large number of phage may be screened at one time. Second, because each infectious phage displays the combinatorial gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage may be amplified by another round of infection. The group of almost identical E. coli filamentous phages Ml 3, fd, and fl are most often used in phage display libraries, as either of the phage gill or gVIII coat proteins may be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al., PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al., (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al., (1993) EMBO J. 12:725-734; Clackson et al., (1991) Nature 352:624-628; and Barbas et al., (1992) PNAS USA 89:4457-4461). Other phage coat proteins may be used as appropriate.

The polypeptides disclosed herein may be reduced to generate mimetics, e.g. peptide or non-peptide agents, which are able to mimic binding of the authentic protein to another cellular partner. Such mutagenic techniques as described above, as well as the thioredoxin system, are also particularly useful for mapping the determinants of a protein which participates in a protein-protein interaction with another protein. To illustrate, the critical residues of a protein which are involved in molecular recognition of a substrate protein may be determined and used to generate peptidomimetics that may bind to the substrate protein. The peptidomimetic may then be used as an inhibitor of the wild-type protein by binding to the substrate and covering up the critical residues needed for interaction with the wild-type protein, thereby preventing interaction of the protein and the substrate. By employing, for example, scanning mutagenesis to map the amino acid

residues of a protein which are involved in binding a substrate polypeptide, peptidoniimetic compounds may be generated which mimic those residues in binding to the substrate.

For instance, derivatives of the kinases and phosphatases described herein may be chemically modified peptides and peptidomimetics. Peptidomimetics are compounds based on, or derived from, peptides and proteins. Peptidomimetics can be obtained by structural modification of known peptide sequences using unnatural amino acids, conformational restraints, isosteric replacement, and the like. The subject peptidomimetics constitute the continum of structural space between peptides and non-peptide synthetic structures; peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity of the parent peptides.

The activity of a kinase or phosphatase protein, fragment, or variant thereof may be assayed using an appropriate substrate or binding partner or other reagent suitable to test for the suspected activity. For catalytic activity, the assay is typically designed so that the enzymatic reaction produces a detectable signal. For example, mixture of a kinase with a substrate in the presence Of ³² P will result in incorporation of the ³² P into the substrate. The labeled substrate may then be separated from the free ³² P and the presence and/or amount of radiolabeled substrate may be detected using a scintillation counter or a phosphorimager. Similar assays may be designed to identify and/or assay the activity of a wide variety of enzymatic activities. Based on the teachings herein, the skilled artisan would readily be able to develop an appropriate assay for a polypeptide of the invention.

In another embodiment, the activity of a polypeptide may be determined by assaying for the level of expression of RNA and/or protein molecules. Transcription levels may be determined, for example, using Northern blots, hybridization to an oligonucleotide array or by assaying for the level of a resulting protein product. Translation levels may be determined, for example, using Western blotting or by identifying a detectable signal produced by a protein product (e.g., fluorescence, luminescence, enzymatic activity, etc.). Depending on the particular situation, it may be desirable to detect the level of transcription and/or translation of a single gene or of multiple genes. Alternatively, it may be desirable to measure the overall rate of DNA replication, transcription and/or translation in a cell. In general this may be accomplished by growing the cell in the presence of a detectable metabolite which is incorporated into the resultant DNA, RNA, or protein product. For example, the rate of DNA synthesis may be

determined by growing cells in the presence of BrdU which is incorporated into the newly synthesized DNA. The amount of BrdU may then be determined histochemically using an anti-BrdU antibody.

IQ other embodiments, polypeptides of the invention may be immobilized onto a solid surface, including, microtiter plates, slides, beads, films, etc. The polypeptides of the invention may be immobilized onto a "chip" as part of an array. An array, having a plurality of addresses, may comprise one or more polypeptides of the invention in one or more of those addresses. In one embodiment, the chip comprises one or more polypeptides of the invention as part of an array of polypeptide sequences. 5. Kinase and Phosphatase Antibodies and Uses Thereof

To produce antibodies against the cell survival and cell death kinases and phosphatases described herein, host animals may be injected with full-length kinase or phosphatase polypeptides or with kinase or phosphatase peptides. Hosts may be injected with peptides of different lengths encompassing a desired target sequence. For example, peptide antigens that are at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 amino acids may be used. Alternatively, if a portion of a protein defines an epitope, but is too short to be antigenic, it may be conjugated to a carrier molecule in order to produce antibodies. Some suitable carrier molecules include keyhole limpet hemocyanin, Ig sequences, TrpE, and human or bovine serum albumen. Conjugation may be carried out by methods known in the art. One such method is to combine a cysteine residue of the fragments with a cysteine residue on the carrier molecule.

In addition, antibodies to three-dimensional epitopes, i.e., non-linear epitopes, may also be prepared, based on, e.g., crystallographic data of proteins. Antibodies obtained from that injection may be screened against the short antigens of proteins described herein. Antibodies prepared against a kinase or phsophatase peptide may be tested for activity against that peptide as well as the full length kinase or phosphatase protein. Antibodies may have affinities of at least about 10 ^"6 M, 10 ^"7 M, 10 ^"8 M, 10 ^'9 M, 10 ^"10 M, 10 ^"11 M or 10 ^"12 M or higher toward the kinase or phosphatase peptide and/or the full length kinase or phosphatase protein described herein.

Suitable cells for the DNA sequences and host cells for antibody expression and secretion can be obtained from a number of sources, including the American Type Culture Collection {"Catalogue of Cell Lines and Hybridomas" 5 ^th edition (1985) Rockville, Md.,

U.S.A.).

Methods of antibody purification are well known in the art. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N. Y. Purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti- antibody. Antibodies may also be purified on affinity columns according to methods known in the art.

Antibodies to kinases or phosphatases described herein may be prepared as described above to induce cell death and potentiate chemotherapy. In a further embodiment, the antibodies to kinases or phosphatases described herein (whole antibodies or antibody fragments) may be conjugated to a biocompatible material, such as polyethylene glycol molecules (PEG) according to methods well known to persons of skill in the art to increase the antibody's half-life. See for example, U.S. Patent No. 6,468,532. Functionalized PEG polymers are available, for example, from Nektar Therapeutics. Commercially available PEG derivatives include, but are not limited to, amino-PEG, PEG amino acid esters, PEG- hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG amino acids, PEG succinimidyl succinate, PEG succinimidyl propionate, succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidyl esters of amino acid PEGs, PEG-oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tresylate, PEG- glycidyl ether, PEG-aldehyde, PEG vinylsulfone, PEG-maleimide, PEG-orthopyridyl- disulfide, heterofunctional PEGs, PEG vinyl derivatives, PEG silanes, and PEG phospholides. The reaction conditions for coupling these PEG derivatives will vary depending on the polypeptide, the desired degree of PEGylation, and the PEG derivative utilized. Some factors involved in the choice of PEG derivatives include: the desired point of attachment (such as lysine or cysteine R-groups), hydrolytic stability and reactivity of the derivatives, stability, toxicity and antigenicity of the linkage, suitability for analysis, etc. 6. Diagnostic Markers and Assays

Under-expression and/or mutation of a cell death kinase or phosphatase may be used as a biomarker for diagnosis of cancer. Alternatively, over-expression and/or mutation of a cell survival kinase or phosphatase may also be used as a biomarker for diagnosis of cancer.

Individual kinase or phosphatase proteins may be used as a marker for diagnosis, or alternatively, a combination of kinases and/or phosphatases may be monitored and used to diagnosis cancer. In certain embodiments, a combination of 2, 3, 4, 5 or more kinases or phosphastases may be monitored and used as a marker for cancer. In exemplary embodiments, suppression of MK-STYZ, PP3CB, ACP6, PPP4R1L, PTPRS, PTPRD and PPP 1R7 may serve as a marker for cancer.

Kinases and phosphatases described herein may also serve as a marker for neurodegenerative and/or cardiovascular disease. Neuronal loss, which is a hallmark of neurodegenerative diseases, is mediated by activation of apoptotic pathways. Massive apoptosis also occurs in acute pathologies, including ischemia, stroke, spinal cord injuries. Further, increased levels of apoptosis are observed in various neuropathologies, including Parkinson's disease, Alzheimer's disease, amyothrophic lateral sclerosis (ALS), denervation atrophy, otosclerosis, stroke, dementia, multiple sclerosis, Huntington's disease and encephalopathy associated with aquired immunodeficiency disease (AIDS). Since nerve cells generally do not divide in adults and, therefore, new cells are not available to replace the dying cells, the nerve cell death occuring in such diseases results in the progressively deteriorating condition of patients suffering from the condition. Over- expression and/or mutation of cell death kinases and phophatases as well as the under- expression and/or mutation of cell survival kinases and phosphatases may serve as markers of acute and/or chronic neuropathologies.

Similarily, cell death is a critical step in the pathogenesis of several cardiovascular diseases, including, but not limited to myocardial infarction, heart failure, and atherosclerosis as well as other diseases including muscular dystrophy, inflammatory bowel disease, Crohn's disease, autoimmune hepatitis, hemochromatosis, Wilson disease, viral hepatitis, alcoholic hepatitis, glomerulosclerosis, and Monckeberg's medical syndrome. Thus, over-expression and/or mutation of cell death kinases and phophatases as well as the under-expression and/or mutation of cell survival kinases and phosphatases may serve as markers of cardiovascular diseases as well as other diseases.

Further, the under- or over-expression and/or mutation of a cell death kinase or phosphastase may be used to identify patient populations for clinical trials related to cancer, neurodegenerative disease, and/or cardiovascular disease. As such, this information may be used to enable clinicians to determine the most appropriate therapies for each patient, thus improving patient quality of life and increasing and survival.

Expression of a marker for cancer, neurodegenerative disease, and/or cardiovascular may be determined from a biological sample from a patient using a variety of assays known in the art. Exemplary assays to monitor expression of a marker may include, but are not limited to, immunoassays, Northern blot, and in situ hybribridization. Biological samples that may be obtained from a patient include, but are not limited to, tissue (e.g., healthy, diseased, and/or tumor tissue), whole blood, plasma, urine, interstitial fluid, lymph, gastric juices, bile, serum, saliva, sweat, and spinal and brain fluids. Furthermore, a biological sample may be either processed (e.g., serum) or present in its natural form.

Tumors that may be diagnosed with the present invention include, but are not limited to, tumors of the breast, colon, lung, liver, lymph node, kidney, pancreas, prostate, ovary, endometrium, spleen, small intestine, stomach, skin, testes, head and neck, esophagus, brain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas), blood cells, bone marrow, blood cells, blood or other tissue. The tumor may be distinguished as metastatic or non-metastatic. The methods and combinations of the present invention may also be used for the diagnosis of neoplasia disorders selected from the group consisting of acral lentiginous melanoma, actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous, cholangiocarcinoma, chondrosarcoma, choriod plexus papilloma/carcinoma, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing's sarcoma, fibrolamellar, focal nodular hyperplasia, gastrinoma, germ cell tumors, glioblastoma, glucagonoma, hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intaepithelial neoplasia, interepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, lentigo maligna melanomas, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial, metastatic carcinoma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat cell carcinoma, oligodendroglial, osteosarcoma, pancreatic polypeptide, papillary serous adenocarcinoma, pineal cell, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastema, renal cell carcinoma, retinoblastoma,

rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinomas, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, submesothelial, superficial spreading melanoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, well differentiated carcinoma, and Wilm's tumor. 7. Therapeutic methods

Provided herein are methods for treating or preventing diseases that can benefit from modulation of the level or activity of a survival kinase, survival phosphatase, death kinase or death phosphatase. An illustrative method comprises administering to a subject in need thereof a therapeutically effective amount of an agent that modulates a kinase or phosphatase. A method may comprise administering two or more agents. An agent may be any agent described herein or an agent identified by a screening method, e.g., those described herein. For example, an agent may be an siRNA or a small molecule that modulates the activity or protein level of a kinase or phosphatase.

Diseases that can be treated or prevented include those that are associated with abnormal cell survival or cell death, e.g., apoptosis. For example, diseases in which cell death is desired can be treated or prevented with agents that induce cell death, e.g., inhibitors of survival kinases or phosphatases or activators of death kinases or phosphatases. Such diseases include those in which excessive cell proliferation occurs, such as those associated with the formation of tumors, e.g., cancer, warts, or other growths. Autoimmune diseases could also be targeted. Exemplary cancers that can be treated are further described herein.

Other diseases that can be treated or prevented include those in which excessive cell death occurs, such as neurodegenerative diseases. Such diseases can be treated or prevented with agents that inhibit cell death, e.g., inhibitors of death kinases or phosphatases or activators of survival kinases or phosphatases.

8. Exemplary Screening Assays to Identify Modulators of Cell Survival and/or Cell Death Kinases and Phosphatases

The identification of agents or compounds capable of modulating the activity or expression of cell survival and/or cell death kinases and phosphatases or, alternatively, the identification of proteins and/or signaling molecules that physically bind to any of the targets mentioned in Tables 1 through 6 and disrupt protein-protein interactions, may be important for killing cancer cells and potentiating chemotherapy. Therefore, it is desirable to identify modulators of cell survival and cell death kinases and phosphatases for future

therapeutic use.

In general, agents or compounds capable of modulating kinase and phosphatase activity may be identified from large libraries of both natural product or synthetic (or semisynthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drag discovery and development will understand that the precise source of agents (e.g. test extracts or compounds) is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the methods described herein. Examples of such agents, extracts, or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FIa.), and PharmnaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their anti-pathogenic activity should be employed whenever possible.

When a crude extract is found to modulate the activity, or a binding activity, of a cell survival ldnase or phosphatase or a cell death kinase or phosphatase further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity

within the crude extract having anti-pathogenic activity. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful agents for the treatment of pathogenicity are chemically modified according to methods known in the art. Potential modulators of the cell survival and cell death kinases and phosphatases disclosed herein may include organic molecules, nucleic acids, peptides, peptide mimetics, polypeptides, and antibodies that bind to a nucleic acid sequence or polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also include small molecules that bind to and occupy the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Other potential antagonists include antisense molecules (e.g., siRNAs).

As described herein, a method for identifying a test compound that modulates cell survival comprises contacting one or more of the following: a cell survival kinase, a cell survival phosphatase, a cell death kinase or a cell death phosphatase with a test compound, and monitoring the activity of the cell survival kinase, cell survival phophatase, cell death kinase or cell death phosphatase in the presence of the test compound as compared to the activity of the cell survival kinase, cell survival phophatase, cell death kinase or cell death phosphatase in the absence of the test compound, wherein a change in the activity of the cell survival kinase, cell survival phophatase, cell death kinase or cell death phosphatase in the presence of the test compound is indicative of a test compound that modulates cell survival. The method may further comprise an additional step of testing for apoptosis or cell survival or increased sensitivity to the effect of other agents, e.g., agents that increase cell death.

Alternatively, a method for identifying a test compound that modulates cell survival comprises contacting a cell comprising a nucleic acid that encodes one or more of the following: a cell survival kinase, a cell survival phopshatase, a cell death ldnase, or a cell death phosphatase with a test compound, and monitoring the expression of the cell survival kinase, cell survival phophatase, cell death ldnase or cell death phosphatase in the presence of the test compound as compared to the expression of the cell survival ldnase, cell survival phophatase, cell death ldnase or cell death phosphatase in the absence of the test compound, wherein a change in the activity of the cell survival ldnase, cell survival phophatase, cell death ldnase or cell death phosphatase in the presence of the test compound is indicative of a test compound that modulates cell survival. The method may further comprise an

additional step of testing for apoptosis or cell survival or increased sensitivity to the effect of other agents, e.g., agents that increase cell death..

8.1 Kinase Assays

High-throughput assays for cell survival or cell death kinase inhibitors may measure, for example, the concentration of ATP in each well using a luciferase assay (luciferase requires ATP for activity). Since kinases hydrolyze ATP, luciferase activity is decreased in the presence of an active kinase, but remains high if the kinase is inhibited by a library compound. In an exemplary assay, the recombinant active kinase may be mixed with assay buffer (e.g., 20 mM Hepes, pH7.2), and dispensed into individual wells of a microtiter plate, such as a 384-well plate. Library compounds may then be pin-transferred along with solvent controls. To initiate the kinase reaction, substrate (peptide, protein or lipid as determined for each kinase target) and ATP (100 μM) is dispensed into each well. Typically, ldnase assays are performed at about 3O ⁰ C for 15-30 minutes but optimal incubation times and temperatures may be determined in pilot experiments so as to be under linear assay conditions. At the end of reactions, luciferin and luciferase may be dispensed into each well and incubated for 10 minutes at room temperature. The luminescence in each well may be measured using a plate reader (Perkin Elmer).

8.2 Phosphatase Assays

The ProFluor™ Ser/Thr Phosphatase Assay (Promega Corporation) may be used to measure purified serine/threonine protein phosphatase activity in a multiwell plate format.

The assay works with protein phosphatase 1 (PPl), PP2A, PP2B, and PP2C enzymes. The assay is initiated with a standard phosphatase reaction performed in the reaction buffer with the provided phosphorylated bisamide rhodamine 110 peptide substrate (S/T PPase RI lO

Substrate) and Control AMC Substrate that serves as a control for compounds that may inhibit the protease. In this configuration, both the S/T PPase RI lO Substrate and Control

AMC Substrate are nonfluorescent. Following the phosphatase reaction, protease solution is added to the mixture to simultaneously stop the phosphatase reaction and completely digest the dephosphorylated S/T PPase RI lO Substrate and the Control AMC substrate, producing highly fluorescent rhodamine 110 and AMC. Phosphorylated S/T PPase RI lO Substrate, however, is resistant to protease digestion and remains nonfluorescent. Thus, the

Rl 10 fluorescence intensity measured in the assay is correlated with phosphatase activity in

the presence of active protease, and the AMC fluorescence intensity is an indication of protease activity.

The assay produces Z' values greater than about 0.8 in both 96- and 384-well plate formats and produces IC ₅₀ values for known inhibitors that are comparable to those currently reported in the literature. The amount of phosphatase used per well is low

(ng/well), and the fluorescence signal is stable (approximately 10 percent change of fluorescence intensity in 4 hours), allowing batch-plate reading.

The ProFluor™ Tyrosine Phosphatase Assay (Promega Corporation) may be used to measure the enzyme activity of tyrosine phosphatases using purified enzymes. The assay may be initiated with a standard phosphatase reaction performed in the provided reaction buffer that contains a bisamide rhodamine 110 phosphopeptide substrate (PTPase RIlO Substrate) and a Control AMC Substrate that serves as a control for compounds that may inhibit the protease. In this configuration, both the PTPase RI lO Substrate and Control AMC Substrate are nonfluorescent. Following the phosphatase reaction, addition of a protease solution simultaneously stops the phosphatase reaction and completely digests the nonphosphorylated PTPase RI lO Substrate and the Control AMC substrate, producing highly fluorescent rhodamine 110 and AMC. The phosphorylated substrate, however, is resistant to digestion by the Protease Reagent and remains nonfluorescent. Thus, the measured fluorescence intensity in the assay correlates with phosphatase activity. The fluorescent signal is very stable (<20% change of fluorescence intensity over 4 hours), allowing batch-plate reading. The assay produces Z'-factor values greater than 0.7 in either 96-well (data not shown) or 384-well plate formats, and it identifies known phosphatase inhibitors and may be used to identify inhibitors in a screen of library compounds. The assay produces IC ₅₀ values for known inhibitors that are comparable to those reported in literature.

8.3 Assays to measure protein-protein interactions

Interaction-Trap Assays

A standard yeast two-hybrid assay may be used to assess the effect of a test compound on the kinase/phosphatase-partner interaction (Mendelsohn and Brent, Curr. Opin. Biotechnol. 5:482-486, 1994). Typically, a vector encoding a synthetic or naturally occurring peptide containing the binding region of the kinase or phosphatase, covalently bound to a DNA binding domain {e.g., GAL4), is transfected into yeast cells containing a

reporter gene operably linked to a binding site for the DNA binding domain. Further, a vector encoding either the native partner protein or corresponding binding domain/motif from the binding partner covalently bound to a transcriptional activator (e.g., GaIAD) is also transfected. The effectiveness of a test compound is then assessed by growing the yeast in the presence of the compound and measuring the level of reporter gene expression.

GST Pulldown Assays

The interaction of the kinase/phosphatase with the partner(s) of interest may be examined using a GST-fusion protein binding study. A vector encoding a naturally- occurring or synthetic polypeptide containing the partner or fragment thereof is fused to GST and expressed in a host cell (e.g., E. coli or Saccharomyces spp.). The GST fusion protein is then contacted with the kinase or phosphatase target in the presence and absence of a test compound. The kinase or phosphatase may be naturally expressed by the host cell or may be expressed from a second vector inserted into the host cell. Following incubation with the test compound, the host cells are lysed and the GST fusion proteins are recovered using glutathione-Sepharose (GSH-Seph) beads. Typically, the GST fusion proteins are released from the GSH-Seph by boiling and the proteins visualized by electrophoretic separation on an SDS-PAGE gel. A skilled artisan will readily understand that the GST- Pulldown assay described here can be readily adapted to a cell-free assay by incubating the purified GST fusion protein with a purified recombinant kinase or phosphatase. Fluorescence Polarization Assay

A variety of well known cell-free techniques may be used to assess the effects of a test compound on the interaction between a kinase or phosphatase and a partner of interest. Fluorescence polarization assays are particularly useful for this purpose. In this assay, a peptide (about 6-12 amino acids) containing the binding motif found in the partner(s) has a fluorophore (e.g., fluorescein, BODIPY) conjugated to its N-terminus is incubated in the presence and absence of increasing amounts of recombinant kinase/phosphatase (e.g., GST- PINKl; 0.01-1 μM) for 10 minutes at room temperature. Aliquots from each reaction are placed in a plate black- walled microtiter (e.g., 384-well) plate and polarization measured using an Analyst plate reader. Increasing concentrations of the kinase/phosphatase causes an increase in polarization. Titrating in the "free" binding motif peptide (i.e., unconjugated) inhibits the change in polarization, whereas a mutated version of the binding peptide does not. The appearance of low polarization, even in the presence of high

concentrations of kinase, indicates flexible binding of the binding peptide to the kinase/phosphatase and suggests the presence of the propeller effect. Designing shorter dye-conjugated binding peptides usually alleviates this problem. The effect of standard assay variables, including incubation time, temperature, pH (7.2-8.5), and buffers, on polarization is readily controlled during routine assay optimization. This assay is readily adaptable for identifying test compounds that inhibit binding of a kinase/phosphatase to partner(s). The use of automated liquid handling systems and plate readers makes this assay readily adaptable to a high-throughput format for screening large numbers of test compounds. For compound screening, the test compound is added to a mixture of the fluorescently labeled binding peptide and the target ldnase or phosphatase. Compounds that inhibit the polarization increase (or cause a decrease in polarization) resulting from increasing amounts of the recombinant kinase or phosphatase are therapeutic candidates.

8.4 Expression Assays

In certain embodiments, modulators of cell survival kinases and phosphatases and cell death kinases and phosphatases disclosed herein may affect the expression of nucleic acid or protein corresponding to each, hi an exemplary assay, cells expressing a cell survival kinase or phosphatase or a cell death kinase or phosphatase of interest may be treated with a compound(s) of interest, and then assayed for the effect of the compound(s) on a particular kinase or phosphatase nucleic acid or protein expression. Alternatively, a plurality of kinases or phosphatases of the invention may be tested using a combinatorial approach.

For example, total RNA may be isolated from cells cultured in the presence or absence of a test compound, using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski et al. (1987) Anal. Biochem. 162:156-159. The expression of a particular kinase or phosphotase of invention may then be assayed by any appropriate method such as Northern blot analysis, polymerase chain reaction (PCR), reverse transcription in combination with polymerase chain reaction (RT-PCR), and reverse transcription in combination with ligase chain reaction (RT-LCR). Northern blot analysis may be performed as described in Harada et al. (1990) Cell

63:303-312. Briefly, total RNA is prepared from cells cultured in the presence of a test compound. For the Northern blot, the RNA is denatured in an appropriate buffer (such as

glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected to agarose gel electrophoresis, and transferred onto a nitrocellulose filter. After the RNAs have been linked to the filter by a UV linker, the filter is prehybridized in a solution containing formamide, SSC, Denhardt's solution, denatured salmon sperm, SDS, and sodium phosphate buffer. A DNA sequence encoding a kinase or phosphatase of the present invention may be labeled according to any appropriate method (such as the ³² P-multiprimed DNA labeling system (Amersham)) and used as probe. After hybridization overnight, the filter is washed and exposed to x-ray film. Moreover, a control can also be performed to provide a baseline for comparison. In the control, the expression of kinase or phosphatase of the invention may be quantitated in the absence of the test compound.

Alternatively, the levels of mRNA encoding kinase or phosphatase polypeptides of the invention may also be assayed, for example, using the RT-PCR method described in Makino et al. (1990) Technique 2:295-301. Briefly, this method involves adding total RNA isolated from cells cultured in the presence of a test agent, in a reaction mixture containing a RT primer and appropriate buffer. After incubating for primer annealing, the mixture may be supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve reverse transcription of the RNA, the RT products are then subject to PCR using labeled primers. Alternatively, rather than labeling the primers, a labeled dNTP can be included in the PCR reaction mixture. PCR amplification may be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity of the appropriate bands may be quantified using an imaging analyzer. RT and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art. Variations on the RT-PCR method will be apparent to the skilled artisan. Other PCR methods that can detect the nucleic acid of the present invention can be found in PCR Primer: A Laboratory Manual (Dieffenbach et al. eds., Cold Spring Harbor Lab Press, 1995). A control can also be performed to provide a baseline for comparison. In the control, the expression of mRNA encoding kinase or phosphatase polypeptides may be quantitated in the absence of the test compound.

Alternatively, the expression of kinase or phosphatase polypeptides described herein may be quantitated following the treatment of cells with a test compound using antibody- based methods such as immunoassays. Any suitable immunoassay can be used, including,

without limitation, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.

For example, cell survival kinase or phosphatase or cell death kinase or phosphatase polypeptides described herein may be detected in a sample obtained from cells treated with a test compound, by means of a two-step sandwich assay. In the first step, a capture reagent (e.g., either an antibody directed to a cell survival or cell death kinase or phosphatase described herein) is used to capture the specific polypeptide. The capture reagent can optionally be immobilized on a solid phase. In the second step, a directly or indirectly labeled detection reagent is used to detect the captured marker. In one embodiment, the detection reagent is an antibody. The amount of a particular kinase or phosphatase polypeptide present in cells treated with a test agent can be calculated by reference to the amount present in untreated cells.

Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody/substrate reaction. Besides enzymes, other suitable labels include radioisotopes, such as iodine ( ¹²⁵ 1, ¹²¹ I), carbon ( ¹⁴ C), sulphur ( ³⁵ S), tritium ( ³ H).

Examples of suitable fluorescent labels include a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.

Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase. Examples of chemiluminescent labels include a luminol label, an isoluminol label, an aromatic acridinium ester label, an

imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.

As will be appreciated by those in the art, the type of host cells used in the present invention can vary widely. Basically, any mammalian cells may be used, with mouse, rat, primate and human cells being particularly preferred, although as will be appreciated by those in the art, modifications of the system by pseudotyping allows all eukaryotic cells to be used, preferably higher eukaryotes. Cell types implicated in a wide variety of disease conditions are particularly useful. Accordingly, suitable cell types include, but are not limited to, tumor cells of all types (particularly melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, lddney, prostate, pancreas and testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell), mast cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes. Suitable cells also include known research cells, including, but not limited to, HeLa cells, Jurkat T cells, NIH3T3 cells, CHO, Cos, etc. See the ATCC cell line catalog, hereby expressly incorporated by reference.

Exemplification The invention, having been generally described, may be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, are not intended to limit the invention in any way.

Example 1 : Identification of Kinase and Phosphatase Targets in Cell Proliferation and Apoptosis

We developed a large scale RNA interference (RNAi) and quantitative apoptosis assay to systematically screen all human kinases and phosphatases involved in cell proliferation and apoptosis. Human kinases and phosphatases were screened in cells senstitized with apoptosis-inducing chemo therapeutic agents. We identified several kinases, including those regulating the cell cycle or involved in calcium, lipid TGFβ, tyrosine kinase, and MAPK signaling pathways. Interestingly, a large percentage of phosphatases contribute to cell survival, revealing a previously unrecognized global role for this gene

family as negative regulators of apoptosis. We also identified a novel subset of tumor suppressor-like phosphatases involved in chemosensitivity. Finally, RNAi-mediated down- regulation of individual survival kinases in the presence of submaximal concentrations of paclitaxel (Taxol) potentiates apoptosis. Importantly, this same approach in a resistant breast cancer cell line restores sensitivity to paclitaxel and leads to potent apoptosis. The future development of inhibitors for the kinases identified may lead to novel cancer treatments and facilitate the use of low chemotherapy that minimizes toxicity.

Experimental Methods siRNA libraries. Custom siRNA human kinase and phosphatase library sets were designed and synthesized with two siRNA duplexes for each gene target (Qiagen) (See

Tables 7-12 for DNA target sequences and antisense and sense siRNA sequences). The two siRNA duplexes for each target were combined and arrayed in a 96-well format, hi total, the kinase library targets 650 genes and the phosphatase library 222 genes. Based on validation data, greater than 98% transfection efficiency and greater than 70% 'knockdown' for each mRNA target species is expected. The selected siRNA sequences were BLAST searched against the human genome sequence to ensure only one gene was targeted, whereas the control (nonsilencing) siRNA used has no known overlap. Critical for efficient gene silencing is the design of the siRNA sequence. The siRNA libraries are designed with a novel informatics algorithm, against all known and putative human kinases and phosphatases. This algorithm takes into account siRNA sequence base composition

(G+C/A+T content), secondary structure of target mRNA, positional effects within the mRNA, and chooses the best sequence motifs ensuring that the siRNA sequence targets a single kinase or phosphatase.

Apoptosis ELISA. siRNAs (50 nM siRNA duplex 1 and siRNA duplex 2) were transfected into HeLa cells seeded 24 hours earlier in 96-well plates (2500 cells/well). In each siRNA screen, negative control scrambled siRNAs were used as siRNA controls.

After 48 hours, the cells were treated with paclitaxel (Taxol), cisplatin (CPDC), or etoposide (VP 16). Stock solutions of paclitaxel, cisplatin, and etoposide were dissolved in

Me ₂ SO ₄ and Me ₂ SO ₄ used as vehicle control. Quantitation of apoptotic cell death was determined by an ELISA that measures cytoplasmic histone-DNA fragments produced during apoptosis (Roche). For relative cytotoxicity, 24 hours after drug treatment, alamar blue reagent was added, incubated for 3 hours and fluorescent intensity measured. Next, the 96-well plates were centrifuged (200 x g) for 10 minutes, supernatant discarded, and

lysis buffer added. Following lysis, the samples were centrifuged and 20 μl of the supernatant transferred to a strepavidin-coated microtiter plate. Anti-histone biotin and anti-DNA peroxidase antibodies were added to each well, and the plate incubated at room temperature for 2 hours. After three washes with buffer, the peroxidase substrate was added to each well. Following five minute incubation, the plates were read at 405 nm in a microplate reader. The enrichment of histone-DNA fragments is expressed as fold-increase in absorbance as compared to control (nonsilencing) siRNA treated with vehicle control (Me ₂ SO ₄ ). The survival kinases and survival phosphatases were filtered by a > 2-fold or a > 3-fold increase in apoptosis over scrambled siRNA controls. The pro-apoptotic phosphatases were selected by protection from drug-induced apoptosis by > 2-fold.

Proliferation assay. After siRNA transfections of 48 and 72 hours, cells were incubated with alamar blue reagent for 3 hours and fluorescent intensity measured at 530- 560nm excitation and 590nm emission wavelength. The alamar blue reagent quantitatively measures proliferation and relative cytotoxicity by detecting metabolic activity of cells and proliferative state of the cell. Specifically, the alamar blue reagent provides a fluorescent readout for the mitochondrial dehydrogenase complex.

Cell lysis and immunoblotting. Cell extracts were prepared by collecting and washing cells in ice-cold PBS and harvesting in lysis buffer (pH 7.2; 10 mM KPO ₄ , 1 mM EDTA, 10 mM MgCl ₂ , 50 mM β-glycerophosphate, 5 mM EGTA, 0.5% NP-40, 0.1% Brij- 35, 1 mM sodium ortho vanadate, 40 μg phenylmethylsulfonyl fluoride/ml, 10 μg leupeptin/ml, 5 μg pepstatin A/ml). Extracts were centrifuged at 15,000 r.p.m. for 10 min at 4 ⁰ C and cell lysates immunoblotted using anti-cleaved-caspase 9 and anti-PARP antibodies (Cell Signaling).

Viability Stain. HeLa cells were plated (85,000/35 mm dish) and transfected with siRNA target duplex 1 or 2 (25 nM) or a combination of 1 and 2 (50 nM). After 72 hours, cells were fixed with 3.7% paraformaldehyde for 15 minutes, washed and then stored in PBS. Visualization of fixed cells was achieved by incubating the cells with 1.5% (w/v) crystal violet for 5 minutes. Excess crystal violet was removed by washing with purified water. Results

Kinases regulating cell survival

We performed a large-scale RNAi approach in order to identify kinases that regulate cell survival and apoptosis. HeLa cervical carcinoma cells were transfected with two short interfering RNAs (siRNAs) targeting each of the 650 known and putative kinases. Under these conditions greater than 98% of cells are transfected with siRNA (Figure 1). After 72 hours, apoptosis was measured using a histone-DNA fragmentation ELISA (Figure 2A). In the screen, siRNAs that increased the level of apoptosis by > 2-fold over control (scrambled) siRNA were defined as survival kinases (Table 5). Overall, 83 kinases were identified as survival kinases, of these CDK6, RPS6KL1, RORl, and NLK were the most potent. A number of survival kinases were identified by this approach and were validated by re-assaying the siRNAs. As expected greater than 3-fold increase in apoptosis was observed following knock-down of CDK6, RPS6KL1, RORl and NLK (Figure 2B). Of the four top survival kinases, two are associated with cell cycle regulation (CDK6 and NLK), and two kinases (RPS6KL1 and RORl) of unknown function.

A critical step in canonical apoptosis is the activation of caspases and the cleavage of ρoly(ADP-ribose) polymerase (PARP). Down-regulation of CDK6, NLK, RPS6KL1, and RORl resulted in an increase in the amount of activated caspase 9 and cleaved PARP

(Figure 2C). To exclude the possibility of "off-target" effects contributing to these observations (Harmon and Rossi, Nature 431:371-8 (2004)), cells were transfected with each siRNA individually, and assayed for cell death (Figure 2D). The individual siRNAs still exhibited potent effects on cell survival and pooling did not result in an averaged effect or dampen the effect of each individual siRNA duplex (Figure 2D). Of the original 83 survival kinases identified, an additional 21 were individually validated (Figure 2E-F, 3) and were shown to increase apoptosis, further validating the apoptosis screen. In addition, the identification of essential survival kinases was confirmed in Hl 57 lung carcinoma cells (Figure 2G). The survival kinases above, following siRNAs introduction, were shown to reduce target mRNA levels by greater than 85 % (Figure 4).

We conclude that in HeLa cells approximately 13% of all kinases positively control cell survival. In addition to previously characterized survival kinases, such as Akt2, SGK, and PKCδ, several novel and/or uncharacterized survival kinases have been identified (Table 5). We do not exclude the possibility that additional survival kinases exist that were not detected to compensatory and/or redundant pathways or non-functional siRNAs.

Phosphatases regulating cell survival

In contrast to kinases, the complexity of protein and lipid phosphatases and lack of available reagents has made the identification and assignment of phosphatase function more difficult. A siRNA library consisting of two siRNAs per gene was used to determine the role of 222 known and putative phosphatases in apoptosis (Figure 5A, Table 6). The most potent survival phosphatases, phosphatases that when suppressed by RNAi led to cell death, were validated by transfection of individual and pooled siRNAs (Figure 5B) and by analyzing the production of cleaved caspase 9 and cleaved PARP (Figure 5C). siRNA- mediated target mRNA reduction was readily observed for selected phosphatases (Figure 4). The survival phosphatases included catalytic and regulatory subunits of serine/threonine protein phosphatases (PPP) and protein tyrosine phosphatases (PTP), and knock-down of each gave rise to apoptosis as measured by cleaved caspase 9 and cleaved PARP (Figure 5C). In addition, we identified several dual specificity phosphatases (DUSP5, DUSP 12, CDC25C) and lipid phosphatases such as myotubularin related phosphatases (e.g. MTMR7). Notably, this analysis identified ninety-three phosphatases, the majority of which were not previously known to be involved in survival signaling (Table 6).

Cell death phosphatases and tumor suppressors

We wished to determine if phosphatases exist that normally sensitize cells to apoptosis perhaps by attenuating cell survival pathways. Loss of function of such cell death phosphatases would therefore confer resistance to apoptosis-inducing agents by activating cell survival pathways. HeLa cells were transfected with the human phosphatase siRNA library and then treated with cisplatin, paclitaxel, or etoposide to induce cell death. Down- regulation of a large number of phosphatases, some of which are known tumor suppressors, indeed conferred dramatic resistance to apoptosis (Table 4). This group of apoptosis sensitizers is typified by the dramatic effect of MK-STYX loss of function which results in a 3-fold increase in basal cell survival and complete resistance to drug-induced apoptosis assayed using DNA fragmentation (Figure 6A). Interestingly, sequence analysis of MK- STYX shows similarity to the dual specificity protein phosphatases such as MAPK phosphatase- 1 (MKP-I), but is catalytically inactive due to a naturally occurring cysteine to serine mutation (Wishart et al., J Biol Chem 270, 26782-5 (1995); Wishart, M. J. & Dixon, J. E., Trends Biochem Sd 23, 301-6 (1998). RT-PCR analysis showed that two pooled siRNAs (1+2) reduced MK-STYX RNA levels upto 95% (Figure 6B). To further examine the ability of MK-STYX down-regulation to protect from apoptosis, HeLa cells were transfected with siRNA for 48 hours and then treated with 50 μM cisplatin for an additional

24 hours. Cisplatin induces apoptosis in many different cell types and 50 μM cisplatin resulted in cell death in greater than 90% of the HeLa cell population after 24 hours of treatment and induced PARP cleavage (Figure 6C). In contrast, MK-STYX siRNA led to complete resistance from cisplatin induced cell death with no visible cell death or PARP cleavage relative to control cells. Similar to MK-STYX, down-regulation of PPP3CB (calcineurin A), ACP6 (lysophosphatidic acid phosphatase), PPP4R1L, PTPRS, and PTPRD also resulted in increased basal cell survival (data not shown) and provided significant protection from cisplatin-induced apoptosis (Figure 6D).

Identification of novel anti-cancer targets Conventional chemotherapuetic agents with differing modes of action trigger apoptosis. The chemotherapeutic agent paclitaxel induces apoptosis by binding and stabilizing microtubules (Schiff et al., Nature 277, 665-7 (1979); Schiff, P. B. & Horwitz, S. B., Proc Natl Acad Sci U S A ll, 1561-5 (1980), cisplatin induces DNA-damage known to activate apoptosis (Sorenson et al., J Natl Cancer Inst 82, 749-55 (1990)), and etoposide inhibits topoisomerases resulting in apoptosis (van Maanen et al., J Natl Cancer Inst 80, 1526-33 (1988)). Given the adaptability of tumor cells however, drug resistance is a major cause of failure to conventional chemotherapy. The potential use of low dose chemotherapy is important, because lower dosages are more attainable during cancer therapy and less likely to cause toxicity in patients. To gather evidence that the survival kinases may provide novel drug combinations, we re-screened the siRNA kinase library in the absence or presence of low dose paclitaxel, cisplatin, and etoposide. We identified kinase targets that when down-regulated increased cellular sensitivity to apoptosis-inducing stimuli. These kinases included validated survival kinases (Figure 2 above), but also kinases that truly sensitize drug-induced apoptosis (see below). Low doses of paclitaxel (5- 10 nM), combined with SGK siRNA (Figure 7A) enhanced apoptosis over control siRNA. SGK is closely related to Akt (PKB), an important survival serine/threonine kinase activated by growth factors. Similar to Akt, SGK activation is dependent on PI3K activity and it controls the phosphorylation of survival proteins such as Forkhead transcription factors (Brunet et al., MoI Cell Biol 21, 952-65 (2001)). Our results emphasize the importance of targeting SGK as it mediates important PI3K-dependent cell survival effects. These results also demonstrate the essential role of lipid signaling in regulating cell survival targets (Table 5). Enhanced apoptosis was not limited to SGK; we also observed apoptosis with siRNA directed against the mammalian target of rapamycin, mTOR. The combination

of low dose paclitaxel (5 nM or 10 nM) and mTOR siRNA tilted the balance to apoptosis (Figure 7B). Knowing the crucial role mTOR plays in coupling cell growth and cell cycle progression (Fingar, D. C. & Blenis, J., Oncogene 23, 3151-71 (2004)), the combination of rapamycin to inhibit mTOR function and paclitaxel may prove to be an effective anti- cancer strategy.

Cyclin-dependent protein kinases (CDKs) have essential roles in cell proliferation and considerable interest has been directed to developing specific CDK inhibitors for cancer therapy (Senderowicz, A. M., Oncogene 22, 6609-20 (2003)). We identified CDK6 and CDK8 as sensitizers for apoptosis. ha combination with paclitaxel, CDK6 siRNA- mediated downregulation increased apoptosis over and above the potent apoptosis with CDK6 siRNA alone (Figure 3). In addition, CDK8 siRNA-mediated downregulation when combined with nanomolar doses of paclitaxel resulted in a dose dependent enhancement of apoptosis (Figure 7C). As more potent CDK inhibitors are developed, targeting CDK6 or CDK8 should be considered in combination with paclitaxel to induce cancer cell death. In addition to the CDKs, the sensitization screen also identified unique sets of genes, including the feline-sarcoma non-receptor tyrosine kinase, FER. FER levels were reduced with siRNAs specific to FER for 48 hours (Figure 4), and followed by treatment of cells with drugs for an additional 24 hours. The dose-dependent increase in apoptosis was observed with paclitaxel (Figure 7D), etoposide (Figure 7E), and cisplatin (Figure 7F). As we have shown, the introduction of siRNAs directed against survival kinases contributes to apoptosis and in combination with low doses of paclitaxel enhances apoptosis. In contrast to HeLa cells, the BT474 breast carcinoma cell line utilizes additional survival pathways to evade apoptosis (Pasleau et al., Oncogene 8, 849-54 (1993); Lane et al., MoI. Cell. Biol. 20, 3210-3223 (2000)). Although BT474 cells exhibit a greater resistance to cell death upon knockdown of many survival kinases (Figure 8 A-C), we asked if in combination with Taxol these resistant cells would become sensitized toward apoptosis. To test this, a combination of siRNAs directed against survival kinases plus low doses of paclitaxel were tested in the BT474 cell line and cell death measured. Low nanomolar doses of paclitaxel synergized with FER (Figure 8A), JIK (Figure 8B), and PLK2 (Figure 8C) to induce apoptosis in the BT474 cells.

Synergistic apoptosis was also observed with PTEN-induced putative ldnase 1 (PINKl) siRNA in HeLa cells (Figure 8D). Activation of caspase 9 and PARP cleavage only occurred in cells transfected with PINKl siRNA and treated with paclitaxel

(Figure 8G). Under these conditions, PINKl mRNA was reduced by approximately 90% (Figure 8F). Importantly, PINKl siRNA tilted the balance to apoptosis in the resistant BT474 cells (Figure 8E). The identification of PINKl in this siRNA sensitization screen uncovers the pivotal role this gene plays in controlling cell survival and apoptosis of cancer cells. Recently, inherited mutations at chromosomal locations lp35-36 have been identified in familial Parkinson's disease, a common neurodegenerative disease due to the loss of neurons (Bonifati et al., Science 299, 256-9 (2003); Valente et al., Science 304, 1158-60 (2004)). The two mutated genes that mapped to this region were PINKl and PARK7/DJ-1, a gene responsible for early onset autosomal recessive parkinsonism and a putative oncogene. Interestingly, we previously discovered PARK7/DJ-1 overexpression in non- small cell lung carcinoma and downregulation enhances apoptosis (MacKeigan et al., Cancer Res 63, 6928-34 (2003)). Taken together, these results emphasize the power of rational combination treatments using conventional chemotherapy in conjunction with targeting novel survival proteins. Discussion

The observations described above have enabled the first large-scale classification of kinase and phosphatase gene families based on their role in apoptosis regulation, a process that is required for normal organism function. This study also identifies a large group of phosphatases and their regulatory subunits whose loss of function results in dramatic chemoresistance and therefore may indicate a previously unrecognized role for these proteins as tumor suppressors. Finally, our data demonstrate that down regulation of novel survival kinases, sensitizes the apoptotic machinery to low concentrations of chemotherapeutic agents. This combination treatment potentiates cell death to levels that greatly exceed that observed with chemotherapeutic agent alone. These survival kinases are potentially important targets for the future development of small molecule inhibitors.

Based on our results, we suggest that approximately 13% of the kinome is essential for promoting cell survival. As expected, known survival kinases such as SGK, AKT2, PKCδ and PKD2, members of the AGC family of kinases, were identified (Datta et al., Genes Dev 13, 2905-27 (1999); Storz, P. & Toker, A., Embo J22, 109-20 (2003)). We also functionally identified several calcium-regulated kinases such as CaMKlG, CaMKIINa, CaMKIIB and CaMKIID as survival kinases which is consistent with the observation that a CamKII inhibitor promotes apoptosis in certain cell types (Yang et al., J Biol. Chem. 278,

7043-7050 (2003)). Interestingly, four of the five type II TGFβR family members (TGFBR2, ACVR2, ACVR2B and ACVRLl) potently control cell survival in HeLa cells which underscores the emerging role for TGFβ signaling in transformation and oncogenesis. Furthermore, since over-production of TGFβ or activation of TGFβR potently cooperates with the ErbB/neu oncogene in models of mammary carcinoma (Seton-Rogers et al., Proc Natl Acad Sd U S A 101, 1257-62 (2004); Muraoka et al., MoI Cell Biol 23, 8691-703 (2003); Siegel et al., Proc Natl Acad Sd USA 100, 8430-5 (2003)), identifying novel type II TGFβR inhibitors may be important for their future use in combination therapy with trastuzumab, which inhibits ErbB/neu function. Contrary to the number of survival kinases, a remarkably large proportion of phosphatases (42%) are responsible for promoting cell survival. This previously unrecognized observation opposes the traditional view that phosphatases merely act to terminate or dampen active signaling. Instead, a great number of diverse phosphatases and associated regulatory proteins are potent mediators of cell survival and therefore may be novel drug targets. Interestingly, loss of PTEN function, a known tumor suppressor, surprisingly resulted in potent cell death. Although this observation appears to contradict the role of PTEN as a negative regulator of cell survival, it is consistent with the lethal phenotype seen mpten ^ Drosophila and mice (Di Cristofano et al., Nat Genet 19, 348-55 (1998); Goberdhan et al., Genes Dev 13, 3244-58 (1999); Stambolic et al., Cell 95, 29-39 (1998); Suzuld et al., Curr Biol 8, 1169-78 (1998)). It is possible that because of the importance of signal strength in cell fate determination (Murphy et al., MoI Cell Biol 24, 144-53 (2004)), in certain cases the cell death resulting from phosphatase loss of function described above could be a checkpoint response to aberrant signaling (Alvarez et al., Nature 413, 744-7 (2001)). Phosphatases that sensitize or promote cell death are of extreme importance because of their possible role as tumor suppressors. When these tumor suppressor proteins are down regulated or mutated, cell survival increases. In this study, 27 phosphatases (12% of total phosphatases) were shown to act in this way. Two examples of this class of phosphatases are the receptor-like tyrosine phosphatases PTPRJ and PTPRG, both of which are known tumor suppressors. PTPRJ is normally believed to inactivate the PDGF receptor and is mutated in colon cancer (Wang et al., Science 304, 1164-6 (2004)) whereas PTPRG is localized to the renal cell carcinoma chromosomal translocation site 3pl4.2 (Tsukamoto et al., Cancer Res 52, 3506-9 (1992); LaForgia et al., Proc Natl Acad Sd USA 88, 5036-40

(1991)). In the present study, three additional receptor-like PTPs, PTPRD, PTPRS and PTPRT also function to promote cell death and may represent novel tumor suppressors. Down-regulation of many of these "death" phosphatases resulted in dramatic cellular resistance to apoptosis-inducing stimuli, which is consistent with up-regulated survival signals in these cells. The most potent protection was observed following suppression of MK-STYX, PP3CB, ACP6, PPP4R1L, PTPRS or PTPRD expression. Intriguingly, many of the genes encoding these phosphatases are either mutated in specific cancer cell lines or have chromosomal locations that map to mutational hot spots. For example, the expression of PPPl R7 is decreased in cervical cancer cell lines and mutations are found in a variety of tumor-derived cell lines (Narayan et al., Oncogene 22, 3489-99 (2003)). MK-STYX is located at chromosome 7pl 1.23, a region mutated in colon cancer indicating a possible role for MK-STYX in this disease. MK-STYX is similar to MKP-I which inactivates MAPKs (Brondello et al., Science 286, 2514-7 (1999)). MK-STYX, however, is predicted to be a dead phosphatase (Wishart, M. J. & Dixon, J. E., Trends Biochem Sd 23, 301-6 (1998)). Our observations suggest that MK-STYX antagonizes cell survival by sequestering pro- survival signaling components analogous to the "substrate-trapping" effects of catalytically inactive phosphatases.

Up-regulation of survival pathways that override critical checkpoint responses to mutations normally associated with cell cycle arrest or apoptosis are a hallmark of many cancers. Survival signaling, therefore, is a major factor responsible for cellular resistance to conventional cancer therapies. Importantly, down-regulation of survival signaling sensitizes the cell to concentrations of chemotherapeutic agents that alone do not induce significant cell death. Such an approach could therefore result in less toxicity and side effects currently associated with cancer therapies (Makin, G. & Dive, C, Breast Cancer Res 3, 150-3 (2001)). Suppression of several known and novel survival kinases, such as FER, JIK, PLK2, or mTOR, significantly enhanced cell death in the presence of low concentrations of chemotherapeutic stimuli highlighting these kinases as potential drug targets. Suppression of PINKl also sensitizes HeLa and BT474 cells to paclitaxel which results in a dramatic increase in cell death. Although PINKl does not fall into a particular kinase sub-family (Manning et al, Science 298, 1912-34 (2002)), it has a known role in maintaining mitochondrial membrane potential (Valente et al., Science 304, 1158-60 (2004)). Loss of PINKl function has been documented in a small group of individuals with

Parkinson's disease (Valente et al, Science 304, 1158-60 (2004)) supporting its role as a regulator of mitochondrial integrity and cell survival.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 2 ^nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); MuIHs et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Antibodies: A Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)), Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986).

Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the word wide web at tigr.org and/or the National Center for Biotechnology Information

(NCBI) on the world wide web at ncbi.nlm.nih.gov.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Table 1. Cell Survival Kinases > 3-Fold Cell Survival Kinase

Accession # Symbol Gene Name

NM_001259 CDK6 cyclin-dependent kinase 6

NMJB1464 RPS6KL1 ribosomal protein S6 kinase-like 1

NM_0050l2 RORl receptor tyrosine kinase-like orphan receptor 1

NM_016231 NLK nemo like kinase

NM_017900 AKIP aurora- A kinase interacting protein

NMJ 82948 PRKACB protein kinase, cAMP-dependent, catalytic, beta

NM_174922 ADCK5 aarF domain containing kinase 5

NM_p00051 ATM ataxia telangiectasia mutated (includes comp. groups A, C and D)

NM_174858 AK5 adenylate kinase 5

NM_001292 CLK3 CDC-like kinase 3

NMJ 82982 GRK4 G protein-coupled receptor kinase 4

NM_006254 PRKCD protein kinase C, delta

NM_020439 CAMKlG calcium/calmodulin-dependent protein kinase IG

NM_002356 MARCKS myristoylated alanine-rich protein kinase C substrate

NM_018584 CaMKIINalpha calcium/calmodulin-dependent protein kinase II

NM_016281 JIK STE20-like kinase

NM_000292 PHKA2 phosphorylase kinase, alpha 2 (liver)

NM_007194 CHEK2 CHK2 checkpoint homolog (S. pombe)

NM_054111 IHPK3 inositol hexaphosphate kinase 3

NM_001211 BUBlB BUBl budding uninhibited by benzimidazoles 1 homolog beta

NM_052947 HAK heart alpha-kinase

NM_006212 PFKFB2 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2

NM_021970 MAP2K1IP1 mitogen-activated protein kinase kinase 1 interacting protein 1

NM 001556 IKBKB inhibitor of kappa light polypep. enhancer in B-cells, kinase beta

> 2-Fold Cell Survival Kinase

Accession # Symbol Gene Name

NM_000077 CDKN2A cyclin-dependent kinase inliib. 2A (melanoma, pi 6, inhibits CDK4)

NMJ45695 DGKB diacylglycerol kinase, beta 9OkDa

NM_020804 PACSINl protein kinase C and casein kinase substrate in neurons 1

NMJ 81358 HIPKl homeodomain interacting protein kinase 1

XM_039796 TNIK TRAF2 and NCK interacting kinase

NM_022445 TPKl thiamin pyrophosphokinase 1

NM_052841 STK22C serine/threonine kinase 22C (spermiogenesis associated)

NMJ316457 PRKD2 protein kinase D2

NMJ ⁾ 01616 ACVR2 activin A receptor, type II

NM_003242 TGFBR2 transforming growth factor, beta receptor II (70/8OkDa)

NM_001220 CAMK2B calcium/calmodulin-dependent protein kinase (CaM kinase) II beta

NM_001260 CDK8 cyclin-dependent kinase 8

NM_005922 MAP3K4 mitogen-activated protein kinase kinase kinase 4

NMJ 52720 NEK3 NIMA (never in mitosis gene a)-related kinase 3

NM_002227 JAKl Janus kinase 1 (a protein tyrosine kinase)

NM_005781 ACKl activated Cdc42-associated kinase 1

NM_003607 CDC42BPA CDC42 binding protein kinase alpha (DMPK-like)

NM_018571 ALS2CR2 amyotrophic lateral sclerosis 2 chromosome region, candidate 2

NM_001184 ATR ataxia telangiectasia and Rad3 related

NM_001626 AKT2 v-akt murine thymoma viral oncogene homolog 2

NM_000289 PFKM phosphofructokinase, muscle

NM_006257 PRKCQ protein kinase C, theta

NM_005027 PIK3R2 phosphoinositide-3-kinase, regulatory subunit, polypep 2 (p85 beta)

NM 005356 LCK lymphocyte-specific protein tyrosine kinase

NM_005248 FGR Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog

NM_004560 ROR2 receptor tyrosine kinase-like orphan receptor 2

NM_018339 FLJl 1149 riboflavin kinase

NM_018425 PI4KII phosphatidylinositol 4-kinase type II

NM_022158 FN3K fructosamine-3-kinase

NM_178432 CCRK cell cycle related kinase

NM_001787 CDC2L1 cell division cycle 2-like 1 (PITSLRE proteins)

BX640919 EIF2AK4 eukaryotic translation initiation factor 2 alpha kinase 4

NM_014572 LATS2 LATS, large tumor suppressor, homolog 2 (Drosophila)

NM_016508 CDKL3 cyclin-dependent kinase-like 3

NM_001106 ACVR2B activin A receptor, type HB

NM_001345 DGKA diacylglycerol kinase, alpha 8OkDa

NM_003582 DYRK3 dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3

NM_004842 AKAP7 A kinase (PRKA) anchor protein 7

NM_018979 PRKWNKl protein kinase, lysine deficient 1

NM_001123 ADK adenosine kinase

NM_000269 NMEl non-metastatic cells 1 , protein (NM23 A) expressed in

NM_006738 AKAP13 A kinase (PRKA) anchor protein 13

NM_006622 PLK2 polo-like kinase 2 (Drosophila)

NMJ)Ol 221 CAMK2D calcium/calmodulin-dependent protein kinase (CaM kinase) II delta

NM_139021 ERK8 extracellular signal-regulated kinase 8

NM_000221 KHK ketohexokinase (fructokinase)

NM_001277 CHK choline kinase

NMJJ04857 AKAP5 A kinase (PRKA) anchor protein 5

NM_005246 FER fer (fps/fes related) tyrosine kinase (phosphoprotein NCP94)

NM_005881 BCKDK branched chain alpha-ketoacid dehydrogenase kinase

NM_020526 EPHA8 EphA8

XMJW2066 MAP3K1 mitogen-activated protein kinase kinase kinase 1

NM_014371 AKAP8L A kinase (PRKA) anchor protein 8-like

NM_004714 DYRKlB dual-specificity tyrosine-(Y)-phosρhorylation regulated kinase IB

NM_021972 SPHKl sphingosine kinase 1

NM_003137 SRPKl SFRS protein kinase 1

NM_000020 ACVRLl activin A receptor type II-like 1

NM_003688 CASK calcium/calmodulin-dependent serine protein kinase (MAGUK)

NM 178170 NEK8 NIMA (never in mitosis gene a)- related kinase 8

Table 2. Kinases Required for Drug-induced Apoptosis (Candidate Tumor Suppressor Proteins)

Accession # Symbol Description

NM_003684 MKNKl MAP kinase-interacting serine/threonine kinase 1

NM_003718 CDC2L5 cell division cycle 2-like 5 (cholinesterase-related cell division controller)

NM_005108 XYLB xylulokinase homolog (H. influenzae)

NM_004938 DAPKl death-associated protein kinase 1

NM_020990 CKMTl creatine kinase, mitochondrial 1 (ubiquitous)

XM_051221 SKIP SPHKl (sphingosine kinase type 1) interacting protein

XM_027237 MAP3K9 mitogen-activated protein kinase kinase kinase 9

NM_006285 TESKl testis-specific kinase 1

NM_080836 STK35 serine/threonine kinase 35

NM_003258 TKl thymidine kinase 1, soluble

NM_004448 ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2,

NM_004327 BCR breakpoint cluster region

NM_006251 PRKAAl protein kinase, AMP-activated, alpha 1 catalytic subunit

NM_000476 AKl adenylate kinase 1

NM_015093 MAP3K7IP2 mitogen-activated protein kinase kinase kinase 7 interacting protein 2

NMJ 81504 PIK3R1 phosρhoinositide-3 -kinase, regulatory subunit, polypeptide 1 (p85 alpha)

XM_370946 LOC388226 similar to protein kinase/ribonuclease IREl beta

NMJ)01396 DYRKlA dual-specificity tyrosine-(Y)-phosphorylation regulated kinase IA

NM_004567 PFKFB4 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4

NMJ39158 ALS2CR7 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 7

NM_014586 HUNK hormonally upregulated Neu-associated kinase

NM_001320 CSNK2B casein kinase 2, beta polypeptide

NM_145040 PRKCDBP protein kinase C, delta binding protein

XM_290793 KSR kinase suppressor of ras

NM_152883 PTK7 PTK7 protein tyrosine kinase 7

NM_173176 PTK2B PTK2B protein tyrosine kinase 2 beta

NM_182687 PKMYTl membrane-associated tyrosine- and threonine-specifϊc cdc2-inhibitory kinase

NM_007229 PACSIN2 protein kinase C and casein kinase substrate in neurons 2

NM_004783 TAOl thousand and one amino acid protein kinase

NM_001929 DGUOK deoxyguanosine kinase

NM_006259 PRKG2 protein kinase, cGMP-dependent, type II

NM_002344 LTK leukocyte tyrosine kinase

NM_004712 HGS hepatocyte growth factor-regulated tyrosine kinase substrate

NM_014791 MELK maternal embryonic leucine zipper kinase

NM_005592 MUSK muscle, skeletal, receptor tyrosine kinase

NM_022344 NJMU-Rl protein kinase Njmu-Rl

NM_004217 AURKB aurora kinase B

NM_032037 SSTK serine/threonine protein kinase SSTK

NM_173354 SNFlLK SNFl -like kinase

NMJU4226 RAGE renal tumor antigen

NM 014365 HSPB8 heat shock 27kDa protein 8

Table 3. Cell Survival Phosphatases > 3-Fold Cell Survival Phosphatases

Accession # Symbol Gene Name

NM _{, .} 014906 PPMlE protein phosphatase IE (PP2C domain containing)

NM _. .016532 SKIP skeletal muscle and kidney enriched inositol phosphatase

AKl 26624 PPFIA4 PTP, receptor type, f polypep. (PTPRF), interacting protein (liprin), alpha 4

NM _, .001789 CDC25A cell division cycle 25A

NM _{, .} 080876 DUSP 19 dual specificity phosphatase 19

NM _{, .} 005539 INPP5A inositol polyphosphate-5-phosphatase, 4OkDa

NM _{, .} 004577 PSPH phosphoserine phosphatase

NM _{, .} 004419 DUSP5 dual specificity phosphatase 5

XM _{, ,} 044727 MTMR7 myotubularin related protein 7

NM[ _. 002721 PPP6C protein phosphatase 6, catalytic subunit

NM_ _. 005021 ENPP3 ectonucleotide pyrophosphatase/phosphodiesterase 3

NM ^" _. 000945 PPP3R1 protein phosphatase 3 (formerly 2B), regulatory B, alpha (calcineurin B)

NM_ _019061 PBP3AP phosphatidylinositol-3-phosphate associated protein

NM _{, .} 006241 PPP 1R2 protein phosphatase 1, regulatory (inhibitor) subunit 2

NM_ _. 014172 PHPTl phosphohistidine phosphatase 1

NM] .032781 PTPN5 protein tyrosine phosphatase, non-receptor type 5 (striatum-enriched)

NM_ _. 005192 CDKN3 cyclin-dependent kinase inhibitor 3 (CDK2-assoc. DUSP)

NM_ _. 004685 MTMR6 myotubularin related protein 6

NM. _. 007240 DUSP 12 dual specificity phosphatase 12

NM_ _. 005134 PPP4R1 protein phosphatase 4, regulatory subunit 1

NM _. .002706 PPMlB protein phosphatase IB (formerly 2C), magnesium-dependent, beta isoform

NM. _. 015568 PPP1R16B protein phosphatase 1, regulatory (inhibitor) subunit 16B

NM _. .002713 PPP 1R8 protein phosphatase 1 , regulatory (inhibitor) subunit 8

NM. _. 017726 PPP1R14D protein phosphatase 1, regulatory (inhibitor) subunit 14D

NM. .001790 CDC25C cell division cycle 25C

NM. _. 001099 ACPP acid phosphatase, prostate

NM. _. 002833 PTPN9 protein tyrosine phosphatase, non-receptor type 9

NM _{. .} 015134 M-RIP myosin phosphatase-Rho interacting protein

NM _. .002708 PPPlCA protein phosphatase 1, catalytic subunit, alpha isoform

NM. _. 002832 PTPN7 protein tyrosine phosphatase, non-receptor type 7

NM. _. 020185 DUSP22 dual specificity phosphatase 22

NM. _. 139245 PPMlL protein phosphatase 1 (formerly 2C)-like

NM. _. 014422 PIB5PA phosphatidylinositol (4,5) bisphosphate 5-phosphatase, A (PIB5PA), mRNA

NM. _. 002851 PTPRZl protein tyrosine phosphatase, receptor-type, Z polypeptide 1

NM. _. 001566 INPP4A inositol polyphosphate-4-phosphatase, type I, 107kDa

NM_ _. 002840 PTPRF protein tyrosine phosphatase, receptor type, F

NM_ _. 013315 TPTE transmembrane phosphatase with tensin homology

XM_ _. 209363 PTPNS1L3 protein tyrosine phosphatase, non-receptor type substrate 1-like 3

NM. _. 181699 PPP2R1B Protein phosphatase 2 (formerly 2A), regulatory A (PR 65), beta isoform

NM. _. 000478 ALPL alkaline phosphatase, liver/bone/kidney

NM. _. 144641 FLJ32332 likely ortholog of mouse protein phosphatase 2C eta

NM 021874 CDC25B cell division cycle 25B

> 2-Fold Cell Survival Phosphatases

Accession # Symbol Gene Name

NM_005608 PTPRCAP protein tyrosine phosphatase, receptor type, C-associated protein

NM_002707 PPMlG protein phosphatase IG (formerly 2C), Mg-dependent, gamma isoform

NM_015466 PTPN23 protein tyrosine phosphatase, non-receptor type 23

NM_004576 PPP2R2B protein phosphatase 2 (formerly 2A), regulatory B (PR 52), beta isoform

NM_017677 MTMR8 myotubularin related protein 8

NM 002844 PTPRK protein tyrosine phosphatase, receptor type, K

NM_002846 PTPRN protein tyrosine phosphatase, receptor type, N

NM_178588 PPP2R5C protein phosphatase 2, regulatory subunit B (B56), gamma isoform

NM_002710 PPPlCC protein phosphatase 1, catalytic subunit, gamma isoform

NM_030667 PTPRO protein tyrosine phosphatase, receptor type, O

NM_002481 PPP IRl 2B protein phosphatase 1, regulatory (inhibitor) subunit 12B

NM_002845 PTPRM protein tyrosine phosphatase, receptor type, M

NM_004300 ACPI acid phosphatase 1 , soluble

NM_002715 PPP2CA protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform

NM_005730 CTDSP2 CTD (C-terminal domain, RNA pol. II polypep. A) small phosphatase 2

NM_015967 PTPN22 protein tyrosine phosphatase, non-receptor type 22 (lymphoid)

XM_087137 LOC151242 protein phosphatase 1 regulatory subunit IA

NM_003660 PPFIA3 PTP, receptor f polypep. (PTPRF), interacting protein (liprin), alpha 3

XMJ74491 PPP1R9A protein phosphatase 1, regulatory (inhibitor) subunit 9 A

NM_003479 PTP4A2 protein tyrosine phosphatase type IVA, member 2

NM_007254 PNKP polynucleotide kinase 3 '-phosphatase

NM_003828 MTMRl myotubularin related protein 1

NM_176895 PPAP2A phosphatidic acid phosphatase type 2A

NM_002847 PTPRN2 protein tyrosine phosphatase, receptor type, N polypeptide 2

NM_004897 MINPPl multiple inositol polyphosphate histidine phosphatase, 1

NM_002972 SBFl SET binding factor 1

NM_003625 PPFIA2 PTP receptor f polypep. (PTPRF), interacting protein (liprin), alpha 2

NM_001947 DUSP7 Homo sapiens dual specificity phosphatase 7 (DUSP7), mRNA

NMJB0791 SGPPl sphingosine-1 -phosphate phosphatase 1

BC058932 INPP5B inositol polyphosphate-5-phosphatase, 75kDa

XM_301056.1 PPP1R3F protein phosphatase 1, regulatory (inhibitor) subunit 3F (PPP 1R3F)

NM_018444 PPM2C protein phosphatase 2C, magnesium-dependent, catalytic subunit

NM_021959 PPPlRI l protein phosphatase 1 , regulatory (inhibitor) subunit 11

NM_006239 PPEF2 protein phosphatase, EF hand calcium-binding domain 2

NM_007207 DUSPlO dual specificity phosphatase 10

NM_030764 SPAPl SH2 domain containing phosphatase anchor protein 1

NM_014225 PPP2R1A protein phosphatase 2 (formerly 2A), regulatory A, PR 65, alpha isoform

NM_000314 PTEN Phosphatase & tensin homolog (mutated in multiple advanced cancers 1)

NMJ81897 PPP2R3A protein phosphatase 2 (formerly 2A), regulatory subunit B", alpha

NM_006242 PPP1R3D protein phosphatase 1, regulatory subunit 3D

NM_001611 ACP5 acid phosphatase 5, tartrate resistant

NM_002194 INPPl inositol polyphosρhate-1-phosphatase

NM_002841 PTPRG protein tyrosine phosphatase, receptor type, G

NM_007026 DUSP 14 dual specificity phosphatase 14

NM_016364 DUSP 13 dual specificity phosphatase 13

NM_002836 PTPRA protein tyrosine phosphatase, receptor type, A

NM_022126 LHPP phospholysine phosphohistidine inorganic pyrophosphate phosphatase

NM_130785 TPTE2 transmembrane phosphoinositide 3-phosphatase and tensin homolog 2

NM_006243 PPP2R5A protein phosphatase 2, regulatory subunit B (B56), alpha isoform

NM_003800 RNGTT RNA guanylyltransferase and 5 '-phosphatase

NM 001946 DUSP6 dual specificity phosphatase 6

Table 4. Cell Death Phosphatases (Candidate Tumor Suppressor Proteins)

Accession # Symbol Gene Name

NM_ .016086 MK-STYX map kinase phosphatase-like protein MK-STYX

NM. _. 021132 PPP3CB PP3 (formerly 2B), catalytic beta isoform (calcineurin A beta)

NM_ _002712 PPP 1R7 protein phosphatase 1, regulatory subunit 7

NM. _. 000151 G6PC glucose-6-ρhosphatase, catalytic (glycogen storage disease type I)

NM _{. .} 002709 PPPlCB protein phosphatase 1, catalytic subunit, beta isoform

NM. _. 016361 ACP6 lysophosphatidic acid phosphatase

NM. _. 017607 PPP1R12C protein phosphatase 1, regulatory (inhibitor) subunit 12C

NM. _. 177414 PPAP2B phosphatidic acid phosphatase type 2B

NM _{. .} 014369 PTPNl 8 protein tyrosine phosphatase, non-receptor type 18 (brain-derived)

NM. _. 178003 PPP2R4 protein phosphatase 2A, regulatory subunit B' (PR 53)

NM. _. 004156 PPP2CB protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform

XM. _. 086650 PPP4R1L protein phosphatase 4, regulatory subunit 1 -like

NM. _. 002711 PPP1R3A protein phosphatase 1, regulatory (inhibitor) subunit 3 A

NM. _. 006209 ENPP2 ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin)

NM _. _021090 MTMR3 myotubularin related protein 3

X53279 ALPPL2 alkaline phosphatase, placental-like 2

NM. _. 002850 PTPRS protein tyrosine phosphatase, receptor type, S

NM _. J33170 PTPRT protein tyrosine phosphatase, receptor type, T

NM _. _002831 PTPN6 protein tyrosine phosphatase, non-receptor type 6

NM _{. .} 021176 G6PC2 glucose-6-phosphatase, catalytic, 2

NM_ _. 002829 PTPN3 protein tyrosine phosphatase, non-receptor type 3

NM. _182642 CTDSPl CTD (C-term domain, RNA pol. II, polypep. A) small phosphatase 1

NM _{. .} 003463 PTP4A1 protein tyrosine phosphatase type IVA, member 1

NM. _005541 INPP5D inositol polyphosphate-5-phosphatase, 145kDa

NM. _. 004417 DUSPl dual specificity phosphatase 1

NM. 002843 PTPRJ protein tyrosine phosphatase, receptor type, J

NM 002839 PTPRD protein tyrosine phosphatase, receptor type, D

Table 5. Functional Categories of Cell Survival Kinases. Cell Cycle Control

Accession # Symbol Gene Name

NM_001259 CDK6 cyclin-dependent kinase 6

NM_017900 AKIP aurora-A kinase interacting protein

NM_000051 ATM ataxia telangiectasia mutated (includes comp. groups A, C and D)

NMJ)01292 CLK3 CDC-like kinase 3

NMJ)07194 CHEK2 CHK2 checkpoint homolog (S. pombe)

NMJ)01211 BUBlB BUB 1 budding uninhibited by benzimidazoles 1 homolog beta

NM_000077 CDKN2A cyclin-dependent kinase inhib. 2A (melanoma, pi 6, inhibits CDK4)

NMJ)01260 CDK8 cyclin-dependent kinase 8

NM_152720 NEK3 NIMA (never in mitosis gene a)-related kinase 3

NMJ)01184 ATR ataxia telangiectasia and Rad3 related

NM_178432 CCRK cell cycle related kinase

BX640919 EIF2AK4 eukaryotic translation initiation factor 2 alpha kinase 4

NMJ)01787 CDC2L1 cell division cycle 2-like 1 (PITSLRE proteins)

NMJ) 16508 CDKL3 cyclin-dependent kinase-like 3

NMJ)06622 PLK2 polo-like kinase 2 (Drosophila)

NM 178170 NEK8 NIMA (never in mitosis gene a)- related kinase 8

Calcium Signaling

Accession # Symbol Gene Name

NM_020439 CAMKl G calcium/calmodulin-dependent protein kinase IG NM_018584 CaMKIINalpha calcium/calmodulin-dependent protein kinase II NM_145695 DGKB diacylglycerol kinase, beta 9OkDa NMJ)01220 CAMK2B calcium/calmodulin-dependent protein kinase (CaM kinase) II beta NMJ)01221 CAMK2D calcium/calmodulin-dependent protein kinase (CaM kinase) II delta NM 003688 CASK calcium/calmodulin-dependent serine protein kinase (MAGUK)

AGC Kinases

Accession # Symbol Gene Name

NMJ 82948 PRKACB protein kinase, cAMP-dependent, catalytic, beta

NM_182982 GRK4 G protein-coupled receptor kinase 4

NM_006254 PRKCD protein kinase C, delta

NM_016457 PRKD2 protein kinase D2

NM_005627 SGK seram/glucocorticoid regulated kinase

NMJ)01626 AKT2 v-akt murine thymoma viral oncogene homolog 2

NMJJ06257 PRKCQ protein kinase C, theta

NM_004842 AKAP7 A kinase (PRKA) anchor protein 7

NM_006738 AKAP 13 A kinase (PRKA) anchor protein 13

NM_004857 AKAP5 A kinase (PRKA) anchor protein 5

NM 014371 AKAP8L A kinase (PRKA) anchor protein 8-like

Lipid Signaling

Accession # Symbol Gene Name

NM_054111 IHPK3 inositol hexaphosphate kinase 3

NMJ45695 DGKB diacylglycerol kinase, beta 9OkDa

NM_005781 ACKl activated Cdc42-associated kinase 1

NM_003607 CDC42BPA CDC42 binding protein kinase alpha (DMPK-like)

NM_005027 PIK3R2 phosphoinositide-3 -kinase, regulatory subunit, poly. 2 (p85 beta)

NMJ)01277 CHK choline kinase

NM_182661 CERK ceramide kinase

NM_021972 SPHKl sphingosine kinase 1

NM_018425 PI4KII phosphatidylinositol 4-kinase type II

NM 001345 DGKA diacylglycerol kinase, alpha 8OkDa

MAPK Signaling

Accession # Symbol Gene Name

NM_016231 NLK nemo-like kinase

NMJ) 16281 JIK STE20-like kinase

NM_021970 MAP2K1IP1 mitogen-activated protein kinase kinase 1 interacting protein 1

NM_005922 MAP3K4 mitogen-activated protein kinase kinase kinase 4

XM_042066 MAP3K1 mitogen-activated protein kinase kinase kinase 1

NM 139021 ERK8 extracellular signal-regulated kinase 8

Tyrosine Kinase Signaling

Accession # Symbol Gene Name

NMJJ02227 JAKl Janus kinase 1 (a protein tyrosine kinase)

NM_005356 LCK lymphocyte-specific protein tyrosine kinase

NM_005248 FGR Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog

NMJ) 14572 LATS2 LATS, large tumor suppressor, homolog 2 (Drosophila)

NM_003582 DYRK3 dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3

NMJ)05246 FER fer (fps/fes related) tyrosine kinase (phosphoprotein NCP94)

NM 004714 DYRKlB dual-specificity tyrosine-(Y)-ρhosphorylation regulated kinase IB

TGFβ Signaling

Accession # Symbol Gene Name

NMJ)Ol 616 ACVR2 activin A receptor, type II

NMJJ03242 TGFBR2 transforming growth factor, beta receptor II (70/8OkDa)

NMJ ⁾ 01106 ACVR2B activin A receptor, type IIB

NM 000020 ACVRLl activin A receptor type II-like 1

Metabolic Regulation

Accession # Symbol Gene Name

NM_174858 AK5 adenylate kinase 5

NM_000292 PHKA2 phosphorylase kinase, alpha 2 (liver)

NM_052947 HAK heart alpha-kinase

NM_006212 PFKFB2 6-phosphorructo-2-kinase/fructose-2,6-biphosphatase 2

NM_022445 TPKl thiamin pyrophosphokinase 1

NM_000289 PFKM phosphofructokinase, muscle

NM_018339 FLJl 1149 riboflavin kinase

NM_022158 FN3K fhictosamine-3-kinase

NMJ)01123 ADK adenosine kinase

NM_000221 KHK ketohexokinase (fructokinase)

NM 005881 BCKDK branched chain alpha-ketoacid dehydrogenase kinase

Novel

Accession # Symbol Gene Name

NMJ)31464 RPS6KL1 ribosomal protein S6 kinase-Iike 1 NM_005012 RORl receptor tyrosine kinase-like orphan receptor 1 NM_004560 ROR2 receptor tyrosine kinase-like orphan receptor 2

Table 6. Functional Categories of Cell Survival Phosphatases Lipid Signaling

Accession # Symbol Gene Name

NM _, _016532 SKIP skeletal muscle and kidney enriched inositol phosphatase

NM_ _. 005539 INPP5A inositol polyphosphate-5-phosρhatase, 4OkDa

XM ^~ _044727 MTMR7 myotubularin related protein 7

NM _, _019061 PIP3AP phosphatidylinositol-3-phosphate associated protein

NM] _004685 MTMR6 myotubularin related protein 6

NM_ _, 014422 PIB5PA phosphatidylinositol (4,5) bisphosphate 5-phosphatase, A (PIB5PA)

NM_ _. 001566 INPP4A inositol polyphosphate-4-phosphatase, type I, 107kDa

NM] _, 013315 TPTE transmembrane phosphatase with tensin homology

NM] _. 017677 MTMR8 myotubularin related protein 8

NM[ _. 003828 MTMRl myotubularin related protein 1

NM_ _, 176895 PPAP2A phosphatidic acid phosphatase type 2A

NM_ _, 004897 MINPPl multiple inositol polyphosphate histidine phosphatase, 1

NM[ J)02972 SBFl SET binding factor 1

NM _{, ,} 030791 SGPPl sphingosine-1 -phosphate phosphatase 1

BC058932 INPP5B inositol polyphosphate-5-phosphatase, 75kDa.

NM _{, ,} 000314 PTEN phosphatase and tensin homolog (mutated in multiple advanced cancers 1)

NM _{, .} 002194 INPPl inositol polyphosphate- 1-phosphatase

NM ^" 130785 TPTE2 transmembrane phosphoinositide 3-phosphatase and tensin homolog 2

Dual Specificity Phosphatases

Accession # Symbol Gene Name

NM_080876 DUSP 19 dual specificity phosphatase 19

NM_004419 DUSP5 dual specificity phosphatase 5

NM_007240 DUSP 12 dual specificity phosphatase 12

NM_020185 DUSP22 dual specificity phosphatase 22

NM_001947 DUSP7 Homo sapiens dual specificity phosphatase 7 (DUSP7), mRNA

NM_007207 DUSPlO dual specificity phosphatase 10

NM_007026 DUSP 14 dual specificity phosphatase 14

NMJH6364 DUSP13 dual specificity phosphatase 13

NM_001946 DUSP6 dual specificity phosphatase 6

Protein Phosphatases

Accession # Symbol Gene Name

NMJ) 14906 PPMlE protein phosphatase IE (PP2C domain containing)

NMJ)02721 PPP6C protein phosphatase 6, catalytic subunit

NMJ)05134 PPP4R1 protein phosphatase 4, regulatory subunit 1

NMJ)02706 PPMlB protein phosphatase IB (formerly 2C), Mg-dependent, beta isoform

NMJ)15568 PPP IRl 6B protein phosphatase 1, regulatory (inhibitor) subunit 16B

NMJ)02713 PPP 1R8 protein phosphatase 1, regulatory (inhibitor) subunit 8

NM_017726 PPP1R14D protein phosphatase 1, regulatory (inhibitor) subunit 14D

NMJ)02708 PPPlCA protein phosphatase 1, catalytic subunit, alpha isoform

NM_139245 PPMlL protein phosphatase 1 (formerly 2C)-like

NM_181699 PPP2R1B protein phosphatase 2 (formerly 2A), regulatory A (PR 65), beta isoform

NM_144641 FLJ32332 likely ortholog of mouse protein phosphatase 2C eta

NMJ)02707 PPMlG protein phosphatase IG (formerly 2C), magnesium-dependent, gamma

NMJ)04576 PPP2R2B protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta

NM_178588 PPP2R5C protein phosphatase 2, regulatory subunit B (B56), gamma isoforms

NMJ302710 PPPlCC protein phosphatase 1 , catalytic subunit, gamma isoforms

NMJ)02481 PPP1R12B protein phosphatase 1, regulatory (inhibitor) subunit 12B

NM 002715 PPP2CA protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoforms

XM _. _087137 LOC151242 protein phosphatase 1 regulatory subunit IA

XM _374491 PPP1R9A protein phosphatase 1, regulatory (inhibitor) subunit 9A

XM. J01056.1 PPP1R3F protein phosphatase 1, regulatory (inhibitor) subunit 3F (PPP1R3F)

NM. _018444 PPM2C protein phosphatase 2C, magnesium-dependent, catalytic subunit

NM _021959 PPPlRI l protein phosphatase 1, regulatory (inhibitor) subunit 11

NM _. _006239 PPEF2 protein phosphatase, EF hand calcium-binding domain 2

NM. _014225 PPP2R1A protein phosphatase 2 (formerly 2A), regulatory A (PR 65), alpha

NM J81897 PPP2R3A protein phosphatase 2 (formerly 2A), regulatory subunit B", alpha

NM _006242 PPP1R3D protein phosphatase 1, regulatory subunit 3D

NM 006243 PPP2R5A protein phosphatase 2, regulatory subunit B (B56), alpha isoform

Protein tyrosine phosphatases

Accession # Symbol Gene Name

AK126624 PPFIA4 PTP, receptor type, f (PTPRF), interacting protein (liprin), alpha 4

NM_032781 PTPN5 protein tyrosine phosphatase, non-receptor type 5 (striatum-enriched)

NM_002833 PTPN9 protein tyrosine phosphatase, non-receptor type 9

NM_002832 PTPN7 protein tyrosine phosphatase, non-receptor type 7

NM_002851 PTPRZl protein tyrosine phosphatase, receptor-type, Z polypeptide 1

NM_005608 PTPRCAP protein tyrosine phosphatase, receptor type, C-associated protein

NM_015466 PTPN23 protein tyrosine phosphatase, non-receptor type 23

NM_002844 PTPRK protein tyrosine phosphatase, receptor type, K

NM_002846 PTPRN protein tyrosine phosphatase, receptor type, N

NM_030667 PTPRO protein tyrosine phosphatase, receptor type, O

NMJ302845 PTPRM protein tyrosine phosphatase, receptor type, M

NM_015967 PTPN22 protein tyrosine phosphatase, non-receptor type 22 (lymphoid)

NM_003660 PPFIA3 PTP, receptor type, f (PTPRF), interacting protein (liprin), alpha 3

NM_003479 PTP4A2 protein tyrosine phosphatase type IVA, member 2

NM_002847 PTPRN2 protein tyrosine phosphatase, receptor type, N polypeptide 2

NM_003625 PPFIA2 PTP, receptor type, f (PTPRF), interacting protein (liprin), alpha 2

NM_002841 PTPRG protein tyrosine phosphatase, receptor type, G

NM 002836 PTPRA protein tyrosine phosphatase, receptor type, A

HMV- ϊ 00.25

HMV-100.25

61

HMV- 100.25

HMV- 100.25

HMV- 100.25

Table 8. Cell Survival Kinase DNA Tar et 2 and siRNA Tar et 2

HMV-100.25

HMV- 100.25

HMV- 100.25

HMV-100.25

HMV- 100.25

HMV- 100.25

>

X

HMV-100.25

HMV- 100.25

HMV- 100.25

HMV- 100.25

HMV- 100.25

HMV- 100.25