WO2005056825A1 - Screening for modulators of mekk2 and mekk3 - Google Patents

Abstract

The present invention provides a method for screening for modulators of MEKK2 and MEKK3. In particular, the present invention provides methods of screening for modulators that recognize a phosphorylation site on MEKK2 at serine 519 or MEKK3 at serine 526. In other embodiments, the present invention concerns modulators for inhibiting dimerization of MEKK2, MEKK3, or both. In further embodiments the present invention provides prognostic and diagnostic methods for a disease state due to MEKK2 or MEKK3 activation.

Description

SCREENING FOR MODULATORS OF MEKK2 AND MEKKS

BACKGROUND OF THE INVENTION

The government owns rights in the present invention pursuant to grant numbers AI44016 and HL070225 from the National Institutes of Health. This application claims priority to and incorporates by reference herein in their entirety U.S. Provisional Patent Application No. 60/527,240, filed December 5, 2003, and U.S. Provisional Patent Application. No. 60/620,758, filed October 21, 2004 1. Field of the Invention The present invention relates generally to the fields of molecular biology and pathology. More particularly, it concerns methods of screening for modulators of MEKK2 and MEKK3 activation. In preferred embodiments, it concerns modulators that recognize phosphorylation of MEKK2 at serine 519 or MEKK3 at serine 526. In other preferred embodiments, the present invention regards modulators that affect dimerization of MEKK2, MEKK3, or both, 2. Description of the Related Art Mitogen-activated protein kinase (MAPK) pathways provide an important connection between external stimuli that activate a wide variety of cell-signaling systems and the nucleus. At the core of each MAPK cascade is a three-kinase module in which the most downstream member, the MAPK, is activated by a MAPK kinase (MAPKK or MEK), which is in turn activated by a MAPKK kinase (MAPKKK or MEKK) (Su and Karin, 1994, English et al., 1999; Huang, 2004). The MEKK family of MAP3Ks contains a C-termmal catalytic domain and an N-teι^*miιιal regulatory domain. It is known in the art that MEKK2 and ME K3 are closely related in their primary sequences, sharing 94% identity between their catalytic domains and 77% overall. The catalytic domains of MEKK2 and MEKK3 are less closely related to other MEKKs, showing approximately 50% identity to ME K1 and MEKK4 catalytic domains. It has previously been demonstrated that MEKK2 and MEKKS are crucial activators of the JJCK/NF-KB, JNK, p38 and Erk MAPK cascades (Deacon and Blank 1999; Cheng et al. 2000; Garringtoti et al, 2000). The role of MEKK2 in regulating INK activation has been demonstrated using MEKK2 -deficient ES cell-derived mast cells and T cells (Garrington et al, 2000, Guo et al, 2002). The involvement of MEKK3 as an IKK activator has been demonstrated using MEKK3 -deficient dominant negative mouse embryo fibroblast cells. MEKK3 has also been shown to play a role in blocking cell proliferation and cell cycle progression (Deacon and Blank, 1999; EUinger-Ziwegelbauer et al., 1999). Other studies using genetically modified mouse models revealed pivotal roles of

MEKK2 and MEKK3 cardiovascular development, angiogenesis, lymphocyte development/activation, and regulation of innate immune response through TNFR family and IL-1R/TLR signaling (Garrington et al, 2000; Yang et al, 2000; Chayama et al, 2001; Su et al. 2001; Yang et al, 2001; Guo et al., 2002). Gelfand and Johnson (WO 97/45736; U.S. Patent No. 5,910,417; U.S. Patent No.

6,495,331; US2003/0129752) provide methods and compositions related to regulation of cytokine production, such as in a hematopoietic cell, by regulating an MEKK JNKK- contingent signal transduction pathway, particularly wherein the regulation relates to inhibition of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2. Johnson (WO 99/47686; US2002/0146798; U.S. 5,981,265; U.S. 6,074,861; U.S. 6,312,934; U.S. 6,333,170) concerns isolated nucleic acid molecules encoding MEKK proteins, MEKK proteins, and products and methods for regulating signal transduction, such as by regulating MEKK activity. Whalen (US2003/0064496 and US2004/0019918) details MEKK2 compositions, including nucleic acids and proteins and their use, such as for identifying a MEKK2 modulator. WO 03/023362 describes agents that modulate JNK signalsome-mediated signal transduction and methods of ameliorating arthritis through inhibition of JNK signalsome activity. Such modulation of JNK signalsome-mediated signal transduction in specific embodiments regards inhibition of MEKK2 with other components of the protein complex. WO 94/24159 regards MEKK proteins having catalytic domains and being capable of phosphorylating MEK proteins, and methods of use thereof. These molecules therefore provide ideal targets for identifying therapeutic agents involved in various diseased states such as cancer, cardiovascular diseases, and autoimmune diseases. SUMMARY OF THE INVENTION The present invention provides a method of screening for candidate modulators of MEKK2 and MEKK3 activity comprising: (a) providing a MEKK2 or MEKK3 containing-sample; (b) contacting the MEKK2 and MEKK3 containing-sample with a candidate substance; and (c) determining phosphorylation of MEKK2 at serine 519 or MEKK3 at serine 526, wherein a change in MEKK2 or MEKK3 phosphorylation in the presence of the candidate substance, as compared with the phosphorylation of MEKK2 or MEKK3 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK2 and/or MEKK3 activity. The candidate modulator of the present invention may be a small organic molecule, a small inorganic molecule, a peptide or protein, a nucleic acid molecule, a DNA molecule, or an RNA molecule, hi further embodiments of the invention, the candidate modulator may be an inhibitor or activator of MEKK2. In still a further embodiment of the invention the candidate modulator may be an inhibitor or activator of MEKK3. hi another embodiment of the invention, the MEKK2 containing-sample may be an organ sample, a tissue sample, or a cell sample. In yet another embodiment of the invention, the MEKK3 containing-sample may be an organ sample, a tissue sample, or a cell sample. In some embodiments, the present invention contemplates determining MEKK2 phosphorylation comprising assaying by Western blotting, DNA microarray, multiplexed bead array, flow cytometry, mass spectrometry, 2D gel electrophoresis, immunoprecipitation, or antibody array. In further embodiments, the present invention contemplates determining MEKK2 phosphorylation comprising assaying by gel mobility shift assay or a radiolabeled phosphate assay. In yet another embodiment, the present invention contemplates determining MEKK3 phosphorylation comprising assaying by Western blotting, DNA microarray, multiplexed bead array, flow cytometry, mass spectrometry, 2D gel electrophoresis, immunoprecipitation, or antibody array, h further embodiments, the present invention contemplates determining MEKK3 phosphorylation comprising assaying by gel mobility shift assay or a radiolabeled phosphate assay. In another particular embodiment of the present invention, there is provided a method of predicting or diagnosing a disease state due to MEKK2 and MEKK3 activation comprising: (a) obtaining a cell sample from the subject; and (b) assessing MEKK2 phosphorylation at serine 519 or MEKK3 phosphorylation at serine 526 in the sample. The subject may be a mammal such as a human. It is contemplated in the present invention that the cell sample may be a tissue sample or an organ sample. hi another embodiment the present invention contemplates assessing MEKK2 phosphorylation comprising assaying by Western blotting, DNA microarray, multiplexed bead array, flow cytometry, mass spectrometry, 2D gel electrophoresis, immunoprecipitation, or antibody array. In a further embodiment of the invention, assessing MEKK2 phosphorylation comprises assaying by gel mobility shift assay or a radiolabeled phosphate assay. hi a further embodiment, the present invention contemplates assessing MEKK3 phosphorylation comprising assaying by Western blotting, DNA microarray, multiplexed bead array, flow cytometry, mass spectrometry, 2D gel electrophoresis, immunoprecipitation, or antibody array, hi still a further embodiment of the invention, assessing MEKK3 phosphorylation comprises assaying by gel mobility shift assay or a radiolabeled phosphate assay. In yet another particular embodiment, the present invention provides a method of treating a subject comprising administering to the subject a therapeutically effective amount of an MEKK2 and/or MEKK3 modulator. It is contemplated that the subject may have a cancer, an inflammatory disease, an autoimmune disease, a cardiovascular disease or condition, or a disease or condition due to a genetic disorder. The cancer may be a bladder cancer, a breast cancer, a lung cancer, a colon cancer, a prostate cancer, a liver cancer, a pancreatic cancer, a stomach cancer, a testicular cancer, a brain cancer, an ovarian cancer, a lymphatic cancer, a skin cancer, a brain cancer, a bone cancer, a soft tissue cancer, but is not limited to such cancers. Administering to a subject a therapeutically effective amount of an MEKK2 and/or MEKK3 modulator may be by intravenous, intradermal, intramuscular, intraarterial, intralesional, percutaneous, subcutaneous, by aerosol routes, or a combination thereof. However, these methods of administering are not meant to be limiting and any method known to one of ordinary skill in the art may be applied. The MEKK2 and/or MEKK3 modulator administered to a subject may be a protein or a nucleic acid expression construct, hi further embodiments, the MEKK2 and/or MEKK3 modulator may be an antisense construct, or a small organic or inorganic molecule, or an organo-pharmaceutical. i still yet another particular embodiment of the present invention, there is provided an antibody that recognizes phosphorylated MEKK2 and MEKK3. The MEKK2 antibody recognizes the phosphorylation site at serine 519 whereas the MEKK3 antibody recognizes the phosphorylation site at serine 526 (this site is equivalent to serine 519 in MEKK2). still yet another particular embodiment of the present invention, there is

'provided a kit comprising an antibody that recognizes MEKK2 phosphorylation at serine

519 and an antibody that recognizes MEKK3 phosphorylation at serine 526 and may further include reagents therefor. The kit contemplated in the present invention may include both the MEKK2 and the MEKK3 antibodies, hi other embodiments, the MEKK2 antibody and reagents therefor may be provided in a separate kit than the MEKK3 antibody and reagents therefor. In an additional embodiment of the present invention, there is a modulator of MEKK2 that affects the dimerization of MEKK2. In an additional embodiment of the present invention, there is a modulator of MEKK3 that affects the dimerization of MEKK3. The modulator may be an inhibitor or it may be an activator, embodiments wherein the modulator is an inhibitor of MEKK2 or MEKK3 dimerization, the inhibitor may comprise at least part of the MEKK2 or MEKK3 dimerization domain. The inhibitor may be further defined as a peptide inhibitor, such as, for example, one that comprises SEQ ID NO:5. h other embodiments, the inhibitor is further defined as a polypeptide of SEQ ID NO:6. a specific embodiment, the modulator is comprised in a pharmaceutically suitable excipient. I-n a further embodiment of the present invention, there is a method of treating a subject comprising administering to the subject a therapeutically effective amount of a modulator that affects the dimerization of MEKK2 and/or the dimerization of MEKK3. The subject may be afflicted with a medical condition, such as cancer, an inflammatory disease, an autoimmune disease, a disease or condition due to a genetic disorder, or a cardiovascular disease or condition. Administration of the modulator may be intravenously, intradermally, intramuscularly, intraarterially, intralesionally, percutaneously, subcutaneously, by an aerosol, or a combination thereof. The modulator may be encoded by a polynucleotide expressed in cells of the subject, hi further specific embodiments, the polynucleotide is comprised on a vector, such as a viral vector or a non-viral vector. In a specific embodiment, the viral vector is an adenoviral vector, a retroviral vector, or an adeno-associated viral vector. In another embodiment of the present invention, there is a method of screening for candidate modulators of MEKK2 activity comprising: (a) providing a MEKK2- containing sample; (b) contacting the MEKK2-containing sample with a candidate substance; and (c) determining the effect of the candidate substance on dimerization of MEKK2, wherein a change in MEKK2 dimerization in the presence of the candidate substance, as compared with the dimerization of MEKK2 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK2 dimerization. The method may be further defined as comprising GST pulldown assay, two-hybrid assay, gel shift assay, or a combination thereof. In another embodiment of the present invention, there is a method of screening for candidate modulators of MEKK3 activity comprising: (a) providing a MEKK3- containing sample; (b) contacting the MEKK3 -containing sample with a candidate substance; and (c) determining the effect of the candidate substance on dimerization of MEKK3, wherein a change in MEKK3 dimerization in the presence of the candidate substance, as compared with the dimerization of MEKK3 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK3 dimerization. The method may be further defined as comprising GST pulldown assay, two-hybrid assay, gel shift assay, or a combination thereof. In specific embodiments, the candidate modulator is a small organic molecule or a small inorganic molecule. In other embodiments, the candidate modulator is a peptide or protein, although it may be a nucleic acid molecule, such as a DNA molecule, an RNA molecule, or a mixture thereof. In particular embodiments, the candidate modulator is an inhibitor of MEKK2, although it may be an activator of MEKK2. hi other embodiments, the candidate modulator is an inhibitor of MEKK3, although it may be an activator of MEKK3. The modulator that affects dimerization of MEKK2 is one that targets at least part of the dimerization domain; the catalytic domain; a region of MEKK2 located between the kinase subdomains I to Ul; a region of MEKK2 comprising at least amino acids 342- 619 (SEQ J-D NO: 10); and/or a region of MEKK2 comprising at least amino acids 342- 424 (SEQ JJD NO:5). In another specific embodiment, there is provided an isolated polypeptide comprising SEQ ID NO:5, such as, for example, one being comprised in a pharmaceutically acceptable excipient. i an additional specific embodiment, there is provided an isolated polypeptide comprising SEQ ID NO: 10, such as, for example, one being comprised in a pharmaceutically acceptable excipient. A skilled artisan recognizes that MEKK3-related peptides, polypeptides, and proteins may be utilized in the invention, such as, for example, in embodiments analogous to those described herein for the exemplary MEKK2 embodiment. In particular, there is a region of MEKK3 that is homologous to an analogous region of MEKK2. This homology may be further defined as comprising one or more domains having similar functions, such as dimerization functions, phosphorylation sites, or both. hi particular, the region of 342-424 in MEKK2 (SEQ ID NO:5) is homologous to the region of 378-460 in MEKK3 (SEQ ID NO: 11). Comparisons of these regions indicate that they are 89% identical, and are 92% positive, As used herein, the term "identical" refers to invariant amino acids upon comparison. As used herein, the term "positive" refers to identical amino acid sequences plus the sequence of amino acids having similar physico-chemical properties, which may be referred to as being conservative amino acids. The term "conservative" as used herein refers to a particular amino acid in a peptide or polypeptide that is a different amino acid in comparison to another amino acid but is of a similar chemical nature. For example, a nonpolar amino acid may be conservatively substituted with another nonpolar amino acid. In specific embodiments, a hydrophobic amino acid may be substituted with another hydrophobic amino acid. The region of 342-619 of MEKK2 (SEQ ID NO:10) is homologous to the region of 378-656 in MEKK3 (SEQ ID NO: 12). Comparisons of these regions indicate that they are 87% identical and 94% positive. In another specific embodiment of the present invention, there is provided an isolated polypeptide comprising SEQ ID NO:l 1 or SEQ J-D NO: 12, such as, for example, one being comprised in a pharmaceutically acceptable excipient. As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Expression and purification of active and kinase dead MEKK2 proteins in COS-1 cells. Maldi-MS identification of active phosphorylation site in MEKK2. Top panel -immunoblot analysis of MEKK2(CT) and MEKK2(CTKM) expression in COS-1 cells with anti-HA antibody. Middle and bottom panels - MEKK2(CT) and MEKK2(CTKM) proteins from the above expressed COS-1 cells were affinity purified and subjected to Maldi-MS analysis. Middle panel shows the peptide elution pattern and bottom panel shpws the amino acid sequencing data of the peptide containing Ser519. MEKK2(CT) and MEKK2(CTKM). FIGS. 2A-2C. Mutagenesis analysis of Ser519 mutation. S519 mutation severely impaired MEKK2's ability to activate its downstream MAPKs JNK and ER5. FIG. 2 A - Flag-JNK was co-transfected with control vector or vectors for MEKK2CT or MEKK2CT519A as indicated. In vitro kinase assay was performed as described (Cheng et al, 2000). FIG. 2B - MEKK2CT or MEKK2CT519A were co-transfected with flagERK5 or FlagERK5(mu) and ERK5 activity was assayed by its phosphorylation. FIG. 2C - MEKK2(CT) and MEKK2(CT519A) expressed in COS-1 cells were immunoprecipitated and assayed for their activity by auto-phosphorylation in the presence of γ- P-ATP. FIG. 3. Loss of MEKK2 activity following Ser519 mutation in both wild-type full length MEKK2 and active MEKK2 (CT) protein. Full length MEKK2 or MEKK2(519A) were transfected alone or with flagJNKl into COS-1 cells. The transfected proteins were isolated by immunoprecipitation and their activities assayed by in vitro kinase assay as described in FIG. 2 (top panel). Botom panel shows the expression level of JNK by immunoblotting. FIGS. 4A-4B. Characterization of peptide antibody against phospho-Ser519. Mutagenesis analysis of S519 phosphorylation in MEKK2 and MEKK3. FIG. 4A - MEKK2(CT) and its derive mutants were expressed in COS-1 cells and analyzed by anti- HA antibody and by phosphor-specific antibody PI as indicated. FIG. 4B - Full length MEKK2, or MEKK3, or MEKK2 mutants were expressed in COS-1 cells and analyzed by anti-HA and PI antibody as described in FIG. 4A. FIGS. 5A-5B. Characterization of MEKK2 and MEKK3 specific antibodies. Characterization of MEKK2 and MEKK3 specific peptide antibodies. FIG. 5A - Cell lysates prepared from wild type, MEKK3KO and MEKK2&MEKK3 double-KO mouse embryonic fibroblasts (MEFs) were subjected to immunoprecipitation with pre-immune serum, MEKK2 specific (8384) and MEKK3 specific (1415) anti-sera as indicated. The immune complex were separated by a SDS-PAGE gel and followed by immunoblot analysis by 8384. FIG. 5B - Same as FIG. 5A except 1415 (anti-MEKK3) and 8384 were used for immunobloting. MEKK2 and MEKK3 were indicated. FIGS. 6A-6B. PI antibody detects endogenous active MEKK2 in stimulated Jurkat T cells and LPS stimulated mouse embryonic fibroblasts. 1 antibody (anti- phosphor-MEKK) detects endogenous MEKK2 stimulated by LPS and TCR CD3. FIG. 6A - Wild type MEFs were either un-treated or stimulated with LPS (100 ug/ml) for different time points before being lysated. The lysates were first immunoprecipitated (JP) with 8384 then blotted with 8384 or PI as indicated. Pre-hn, control IP with non-specific serum. IgG, p-MEKK2 and MEKK2 bands are indicated. FIG. 6B - Jurkat T cells stimulated with TPA, anti-CD28 or anti-CD3 were subjected for JP-WB as described in FIG. 6A except that 1128 (anti-MEKK2CT polyclonal antibody) was used for JP. p- MEKK2 and MEKK2 bands are indicated. FIGS. 7A-7C. PI antibody detects endogenous active MEKK2 and MEKK3 from small cell lysates. Detection of endogenous active MEKK2 and MEKK3 with PI antibody. FIG. 7 A - Macrophage cell line Raw246.7 were untreated or treated with 50 ng/ml of anisomycine, or 100J/m2 of UVC or polyLC before being lysed. Cell lysates were subjected to IP with anti-MEKK2 antibody 8384 followed by immunobloting with pi antibody. FIGS. 7B-7C - Raw246.7 cells were untreated or stimulated with IL- l(10ng/ml) or LPS (lOOng/ml) before being lysed for immunobloting with PI antibody. Phosphor-MEKK2 and phosphor-MEKK3 were indicated. FIGS. 8A-8B. PI antibody detects endogenous active MEKK2 and MEKK3 activated by stress and cytokines. Immunoblotting of stress and cytokine stimulated cell lysates. FIG. 8A - RAW246.7 cells were stimulated with UNC (120-240J/M²), sorbitol (200mM), anisomycine (50ng/ml), and nocdazol (0.5 mg/ml) for the time indicated. The cells were then lysed and analyzed by immunoblotting with PI antibody. FIG. 8B - RAW246.7 ells were stimulated with PGΝ (a bacteria product that bind to toll-like receptor 1/2), IL-1, and LPS and then analyzed by PI antibody. Anti-β-actin immunoblotting was used as a loading control. FIG. 9. PI antibody used as a means to detect MEKK2 and MEKK3 inhibitors. Expression vectors SRαHA-MEKK2 (express an active MEKK2) and SR HA-ΝB that expresses a novel inhibitor of MEKK2 were transfected into COS-1 cells and the active status of the expressed MEKK2 were determined by immunoblotting with PI antibody (top panel). Percentage of inhibition was indicated with the increased expressing of the inhibitor. p-MEKK2, phospho-MEKK2. Bottom panel shows the expression levels of MEKK2 and the inhibitor. FIGS. 10A-10E. MEKK2 Is a Phosphor-protein and Forms Dimers through Its Catalytic Domain. FIG. 10A- MEKK2 is a phosphor-protein. HA-tagged MEKK2(1- 619), MEKK2(l-619)(KM)(top panel), MEKK2(342-619), and MEKK2(342-619)(KM) (bottom panel) were expressed in COS-1 cells, as indicated, and examined by immunoblotting (IB) with an anti-HA antibody. For calf intestine phosphatase (CIP) treatment, cell lysates were prepared in the absence of phosphatase inhibitors and incubated with 1 unit/ml CIP at 37°C for 30 min before IB analysis. NS, nonspecific bands. FIG. lOB-Dephosphorylation inactivates MEKK2. HA-tagged MEKK2(342-619) and MEKK2(342-619)KM were expressed as described in FIG. 10A and subjected to immunoprecipitation with an anti-HA antibody. The MEKK2(342-619) immunocomplex was divided into two parts with either no treatment or CIP treatment as described above before being used for an in vitro kinase assay using JNKK2(KM) as a substrate. Phosphorylated MEKK2 (pMEKK2) and JNKK2 (pMEKK2) are indicated. FIG. 10C- MEKK2 forms dimers through the C-terminal catalytic domain. HA-tagged MEKK2(1- 341) and MEKK2(342-619) were co-transfected with or without GST-MEKK2 in COS-1 cells as indicated for a GST pull-down assay (right panel). The total cell lysates were analyzed by IB with an anti-HA antibody (left panel). NS, nonspecific bands. FIG. 10D- Mapping the dimerization motif in MEKK2. GST-MEKK2(342-619) was co-transfected into COS-1 cells with an empty vector or expression vectors for HA-tagged MEKK2(1- 619), MEKK2(1-341), MEKK2(342-619), and MEKK2(342-424) for a GST pull-down assay as described in FIG. 10C (right panel). Total cell lysates were analyzed by IB with an anti-HA antibody (left panel). FIG. lOE-Expression of the MEKK2 dimerization motif disrupts MEKK2 dimer formation. GSTMEKK2( 342-619) and HA-tagged MEKK2(342-619) were co-transfected into COS-1 cells alone or with an increased amount of MEKK2(342-424) for a GST pull-down assay as described above. Total cell lysates were analyzed by IB (bottom panels). FIGS. 11A-11C. MEKK2 Dimer Formation Is Essential for Its Activation. FIG. 11 A Nonphosphorylated MEKK2 forms dimers preferentially. GST-MEKK2(342-619) was co-transfected into COS-1 cells with expression vectors for HA-tagged MEKK2(342- 619) and MEKK2(342-619)KM as indicated for a GST pull-down assay (top panel). Treatment with CIP was performed as described in FIG. 10. The total cell lysates were analyzed by IB (bottom panel). FIG. HB-Dimer formation is required for MEKK2 phosphorylation. GST-MEKK2(342-619) was co-transfected into COS-1 cells with control vector or expression vectors for HA-tagged MEKK2(342-619) or MEKK2(342-619)KM, as indicated. The cell lysates were subjected to immunoprecipitation with an anti-HA antibody for an in vitro kinase assay (top panel). KA, kinase assay. The total cell lysates were analyzed by IB with an anti- MEKK2 antibody (bottom panel). FIG. HC-Disruption of MEKK2 dimers inhibits JNK1 activation by MEKK2. HA-tagged MEKK2(342-619) and Flag-tagged JNK1 were co- transfected into COS-1 cells with an increased amount of MEKK2(342-424). JNK1 was immunoprecipitated for an in vitro kinase assay. The total cell lysates were analyzed by IB (middle and bottom panels). KA, kinase assay. FIGS. 12A-12G. Identification and Cloning of Human MEKK2 Interacting Protein Mipl. FIG. 12A-Purification and mass spectrometric analysis of Mipl. Cells (293T) were transfected with an empty vector or GST-MEKK2(342-619). The total cell lysates were prepared for precipitation with glutathione beads, and the complex was thoroughly washed, resolved by a SDS-polyacrylamide gel and visualized by silver staining. The protein bands that were specifically precipitated by GST-MEKK2(342-619) but were not in the control were excised from the gel and analyzed by mass spectrometry. Endogenous GST and GST-MEKK2(342-619) were indicated. FIG. 12B-The HPLC/MS/MS spectra of one representative Mipl tryptic peptide. The predicted amino acid sequence of Mipl cDNA is shown in the insert. Other peptides identified by MS/MS are also shown (underlined in bold face). FIG. 12C-Sequence comparison of human Miplα,β,γ (accession number AY633624, AY633625, and AY633626 respectively), JC310, and chick Sinl. Identical residues are highlighted. FIG. 12D-Northern blot analysis of mipl expression in human tissues. A Pstl (0.6 kb) fragment of 19 mipl cDNA was labeled with 32P and used to probe an mRNA blot prepared from human tissues as indicated. FIG. 12E-Mipl binds to transfected MEKK2. HA-tagged MEKK2 and MEKK2(342-619) were expressed alone or with GST-Mipl in COS-1 cells for a GST pull-down assay (right panel). The total lysates were analyzed by IB (left panel). FIG. 12F-Mipl binds to MEKK2 but not MEKK1. HA-tagged MEKK1 or MEKK2 were transfected alone or with GST-Mipl into COS-1 cells for a GST pull-down assay as described in FIG. 10. FIG. 12G-MEKK2 interacts with endogenous Mipl protein detected by an Mipl -specific antibody. An empty vector or HA-tagged Mipl expression vector was transfected into COS-1 cells. The cell lysates were subjected to immunoprecipitation with an anti-HA antibody followed by IB analysis with an anti- Mipl peptide antibody K87 (left panel). The lysates prepared from 293T cells were subjected to immunoprecipitation with pre-immune serum (Pre) or K87 followed by IB analysis with K87 (middle panel). Cells (293T) were transfected with an empty vector or a GST-MEKK2(342-619) expression vector (right panel). The cell lysates were precipitated with glutathione beads followed by IB analysis with K87 antibody. FIGS. 13A-13D. Mipl Inhibits MEKK2-Mediated JNKl Activation and AP-1 Reporter Gene Activation. FIG. 13A-Flag-tagged JNKlwas co-transfected into COS-1 cells with an empty vector, GSTMEKK1, or GST-MEKK2 in the absence or presence of HA-tagged Mipl, as indicated. JNKl was immunoprecipitated for an in vitro kinase assay. Relative fold induction of JNK activity was determined by a phospholmage (BioRad FX) and normalized to the JNKl expression level. KA, kinase assay. FIG. 13B- Mipl blocks MEKK2 phosphorylation and kinase activity toward JNKK2. HA-tagged MEKK2 expressed with an empty vector or Mipl in COS-1 cells was immunoprecipitated with anti-HA antibody for an in vitro kinase assay by using JNKK2(KR) as a substrate. The expression levels of MEKK2 and Mipl were determined by IB (middle and bottom panels). FIG. 13C-Mipl does not block JNKK2-mediated JNK activation. Flag-tagged JNKl was co- 20 transfected with an empty vector or HA-tagged JNKK2(DD) (an active form of JNKK2) in the absence or presence of GST-Mipl as indicated. JNKl activity was determined by an in vitro kinase assay as described in panel A. Expression level of Flag- JNKl and GST-Mipl, was determined by IB. FIG. 13D- Mipl inhibits the AP-1 reporter gene expression induced by MEKK2 but not MEKK1. Gal4-Luc reporter plasmid and Gal4-cJun plasmid were co-transfected with either empty vector, MEKK1, or MEKK2 expression vectors in the absence or presence of Mipl plasmid as indicated. An actin-β-gal plasmid was cotiansfected in every transfection as a control for transfection efficiency. The reporter gene expression was determined 36 hr later and normalized to the β-gal activity. The results shown are the average of three independent experiments. FIGS. 14A-14D Mapping the Mipl-MEKK2 Interaction Motif. FIG. 14A-The recombinant GST and GST fused with various regions of Mipl were purified from bacteria (left panel) for a GST pull-down assay by using COS-1 -expressed HA-tagged MEKK2(342-619) (right panel). FIG. 14B-Mipl binds to the MEKK2 dimerization motif. HA-tagged MEKK2(342-619) or MEKK2(342-424) were co-transfected into COS-1 cells with an empty vector or GSTMipl. The cell lysates were prepared 36 hr later for a GST pull-down assay, as described. The expression levels of MEKK2(342-619) and MEKK2(342-424) in cell lysates were determined by IB (left panel). FIG. 14C-Mipl disrupts MEKK2 dimers. GST-MEKK2 was co-transfected with HA-tagged MEKK2(342-619) or MEKK2(342-619)KM into COS-1 cells with or without Mipl. The cell lysates were prepared 36 hr later for a GST pull-down assay as described above. The expression levels of MEKK2(342- 19), MEKK2(342-619)KM, and Mipl were determined by IB (bottom two panels). FIG. 14D-Identifϊcation of potential coiled-coil structures in MEKK2 and Mipl. The COILS program (available on the World Wide Web) was used to calculate the probabilities of formation of coiled-coil motifs in MEKK2 and Mipl with a window of 14 residues. The bars indicate the sequences in MEKK2 and Mipl mapped by a GST pull-down assay that are required for MEKK2 dimer formation and MEKK2- 21 Mipl interaction. FIGS. 15A-15E Mipl Is a Negative Regulator of MEKK2 Signaling Pathway. FIG. 15A-Mipl interacts with nonphosphorylated MEKK2 preferentially. GST-Mipl was cotiansfected into COS-1 cells with expression vectors for HA-tagged MEKK2(342- 619) and MEKK2(342-619)KM for a GST pull-down assay as indicated (top panel). Treatment by CIP was performed as described in FIG. 10. The level of MEKK2 and Mipl proteins in the lysates was determined by IB (bottom two panels). FIG. 15B-Mipl- associated MEKK2 is inactive. HA-tagged MEKK2(342-619) was co-transfected with an empty vector or GST-Mipl into COS-1 cells. The cells were lysed 36 hr later and subjected to precipitation with glutathione beads or with an anti-HA antibody. The amount of HA-tagged MEKK2(342-619) precipitated by immunoprecipitation and by glutathione beads was determined by immunoblotting (middle panel). Equal amounts of HA-tagged MEKK2(342-619) from each precipitation were used for an in vitro kinase assay (top panel). KA, kinase assay. FIG. 15C-The knockdown of Mipl expression by siRNA induces JNK activity. Left graph, GFPMipl was co-transfected into 293T cells with control siRNA (NS-siRNA) or mipl- siRNA (mipl-siRNA). Cells were harvested 72 hr later and analyzed by flow cytometric analysis with a FACS-caliber for GFP- positive cells. Right panels, Flag-tagged JNKl and HA-tagged MEKK2 were cotiansfected into 293T cells with control siRNA (NSsiRNA) or mipl -specific siRNA (mipl-siRNA). JNKl activity was determined by an in vitro kinase assay as described in FIG. 11 (top panel). The JNKl expression level and the endogenous Mipl level were determined by IB as indicated (middle and bottom panels). FIG. 15D-The knockdown of Mipl expression activates AP-1 reporter gene expression. AP-1 -Luc reporter plasmid and HA-tagged MEKK2 were cotiansfected into 293T cells with an empty vector, mipl- siRNA, or control siRNA. Luciferase activity was determined 48 hr later and normalized to renila luciferase activity by using Promega's dual luciferase system. The results shown are the average of three independent experiments. FIG. 15E-EGF stimulation dissociates endogenous Mipl-MEKK2 complex. Ten million 293T cells were either untreated, or stimulated with EGF (25 ng/ml) for the time points indicated before being lysed for immunoprecipitation (JP) with anti-MEKK2 antibody 8384. The immunocomplex were thoroughly washed and separated by a SDS-PAGE gel for immunoblotting (IB) with anti- Mip 1 antibody K87 (top panel) or anti-MEKK2 antibody 8384 (bottom panel). FIG. 16- Diagram of an Exemplary Model of MEKK2 Regulation and Activation.

DESCRIPTION OF PREFERRED EMBODIMENTS

I. The Present Invention MAP3Ks, particularly MEKK2 and MEKK3, are involved in a variety of disease states. However, agents that target these molecules, particularly the active molecule, are lacking in the art. The present invention seeks to screen for modulators of MEKK2 and MEKK3 activation that are therapeutic agents for predicting, diagnosing and/or treating a MEKK2- or MEKK3-related disease. The inventors of the present invention have identified a unique site in MEKK2 and MEKK3 whose phosphorylation is essential for their activation. Antibodies that specifically recognize the phosphorylated and nonphosphorylated MEKK2 and MEKK3 have been generated and can distinguish phosphorylated versus unphosphorylated MEKK2 at serine 519 or MEKK3 at serine 526. Thus, this novel site provides a means of identifying therapeutic agents that target MEKK2 and MEKK3 through activation of these molecules. Therefore, in particular embodiments, methods for screening for inhibitors and activators of MEKK2 and MEKK3 both at the molecular and cellular levels are provided. More specifically, methods of screening for modulators that alter the phosphorylation at serine 519 of MEKK2 or phosphorylation at serine 526 of MEKK3 are provided. The present invention provides screening methods for identifying potential targets that intervene with the biological processes involving MEKK2 and MEKK3 using cell-based and/or solid-phase techniques. The present invention also provides prognostic and diagnostic methods to screen for MEKK2 or MEKK3 activity in a cell, tissue or organ sample. Additionally, the present invention provides methods of treating a subject having a disease state due to MEKK2 or MEKK3. Such disease states include, but are not limited to, cancer, cardiovascular diseases, autoimmune diseases or genetic disorders. In particular embodiments, the present invention concerns antibodies that recognize active MEKK2 and MEKK3 molecules. Monoclonal and polyclonal antibodies against active MEKK2 or MEKK3, phosphorylated at serine 519 and serine 526 respectively, can be identified and generated based on the methods of the present invention. The present invention also provides modulators of MEKK2 dimerization, such as inhibitors of MEKK2 dimerization, which may be identified by screening methods described herein, for example. In particular, the present invention provides both an exemplary peptide inhibitor (SEQ ID NO:5) and an exemplary polyprotein (SEQ LO NO:6; Mipl) for inhibition of MEKK2 dimerization. The present invention also provides exemplary peptide inhibitors for inhibition of activity, function, or interaction with other molecules for MEKK3. Exemplary embodiments include SEQ ID NO: 11 and SEQ LD NO:12. II. Nucleic Acids The present invention provides nucleic acid sequences encoding MEKK2 or

MEKK3, and methods of screening for modulators of such nucleic acid sequences. Thus, the present invention employs the nucleic acid sequence of MEKK2 (Accession No. NM_006609; SEQ ID NO:l) and MEKK3 (Accession No. NM 002401; SEQ ID NO:3) and expression constructs encoding such nucleic acid sequences. Nucleic acids according to the present invention may encode an entire MEKK2 or MEKK3 gene, a domain of MEKK2 or MEKK3, or any other fragment of MEKK2 or MEKK3 as set forth herein. The nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid comprises complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intion or an intron derived from another gene; such engineered molecules are sometime referred to as "mini-genes." At a minimum, these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis. The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non- coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy. The cDNAs for MEKK2 and MEKK3 encode for proteins with predicated molecular weight of 69.5 and 70 kDa, respectively. It also is contemplated that a given MEKK2 or MEKK3 polynucleotide may be represented by natural or synthetic variants that have slightly different nucleic acid sequences but, nonetheless, encode the same or homologous protein (Table 1). As used in this application, the term "a polynucleotide encoding a polypeptide" refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid. In exemplary embodiments, the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO:l or SEQ ID NO:3. The term "comprises SEQ ID NO:l or SEQ TD NO:3" means that the nucleic acid sequence substantially corresponds to a portion of SEQ TD NO:l or SEQ ID NO:3. The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine (Table 1), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.

TABLE 1

Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%>, more usually about 70%o, most usually about 80%, preferably at least about 90% and most preferably about 95%> of nucleotides that are identical to the nucleotides of SEQ ID NO:l or SEQ ID NO:3 are contemplated. Sequences that are essentially the same as those set forth in SEQ ID NO:l or SEQ ID NO:3 also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:l under standard conditions. The DNA segments of the present invention include those encoding biologically functional equivalent MEKK2 or MEKK3 proteins, peptides and fragments thereof, as described elsewhere herein. Such sequences may arise as a consequence of codon redundancy and/or amino acid functional equivalency that are known to those of skill in the art. For example, polynucleotides encoding MEKK2 or MEKK3 polypeptides analogous to the exemplary MEKK2 or MEKK3 protein of SEQ ID NO:2 or SEQ ID NO:4 are likewise contemplated herein. As discussed further below, and as known to those of skill in the art, various amino acid substitutions, deletions and/or additions may be made to a known amino acid sequence without adversely affecting the function and/or usefulness thereof. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below. Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequences set forth herein, for example in SEQ ID NO:l or SEQ ID NO:3. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the terms "complementary sequences" and "essentially complementary sequences" means nucleic acid sequences that are substantially complementary to, as may be assessed by the same nucleotide comparison set forth above, or are able to hybridize to a nucleic acid segment of one or more sequences set forth herein, for example SEQ ID NO:l or SEQ TD NO:3, under relatively stringent conditions such as those described herein. Such sequences may encode an entire MEKK2 or MEKK3 protein or peptide or functional or non-functional fragments thereof. The hybridizing segments may be short oligonucleotides. Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 750, 1000, 1250, 1500, 2000, 2500, 3000 or 4000 bases and longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions. Suitable hybridization conditions will be well known to those of skill in the art.

In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of the probe and the target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCI at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results. In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mm KCI, 3 mM MgCl₂, 10 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 μM MgCl₂, at temperatures ranging from approximately 40°C to about 72°C. Formamide and SDS also may be used to alter the hybridization conditions. One method of using probes and primers of the present invention is in the search for molecules related to MEKK2 and/or MEKK3 proteins and peptides, including for example, MEKK2 and/or MEKK3 proteins from other species. Normally, the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules. By varying the stringency of hybridization, and the region of the probe, different degrees of homology may be discovered.

III. Proteinaceous Compositions The present invention concerns evaluating the expression and/or activity of a MEKK2 or MEKK3 polypeptide. The present invention also provides MEKK2 and MEKK3 protein/polypeptide sequences. For example, SEQ ID NO:2 and SEQ ID NO:4 provides a full-length amino acid sequence for MEKK2 and MEKK3 respectively. As used herein, a "proteinaceous molecule," "proteinaceous composition," "proteinaceous compound," "proteinaceous chain" or "proteinaceous material" generally refers, but is not limited to, a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids. All the "proteinaceous" terms described above may be used interchangeably herein. In certain embodiments, the size of the at least one proteinaceous molecule may be at least, at most or may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 582, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or greater amino molecule residues, and any range derivable therein. It is specifically contemplated that such lengths of contiguous amino acids from SEQ TD NO:2 or SEQ TD NO:4 are part of the invention. As used herein, an "amino molecule" refers to any amino acid, amino acid derivative or amino acid mimic as would be known to one of ordinary skill in the art. hi certain embodiments, the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties. The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 1 shows the codons that encode particular amino acids. Amino acid sequence variants of a MEKK2 or MEKK3 polypeptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Amino acid sequence variants contemplated in the present invention may also include variant MEKK2 or MEKK3 molecules that lack the phosphorylation site at serine 519 of MEKK2 or serine 526 of MEKK3. Amino acid sequence variants contemplated in the present invention may also include MEKK2 and MEKK3 variants molecules that have constitutively activated sites. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein, hi making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophihcity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophihcity of a protein, as governed by the hydrophihcity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophihcity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure (Johnson et al, 1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used, in conjunction with the principles outline above, to engineer second generation molecules having many of the natural properties of MEKK2 or MEKK3, but with altered and even improved characteristics. Proteinaceous compositions may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials. The nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases found on the internet at the National Institutes of Health website. The coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art. Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term "purified protein or peptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur. Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%>, about 80%, about 90%, about 95% or more of the proteins in the composition.

A. Purification of MEKK2 or MEKK3 Polypeptides It may be desirable to purify MEKK2 and MEKK3 or variants thereof. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using cTiromatographic and electiophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion- exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC. Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term "purified protein or peptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur. Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%> or more of the proteins in the composition. Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number." The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity. Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide. There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein. It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al, 1977). It will therefore be appreciated that under differing electiophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample. Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight. Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.). A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteiris. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fructose will bind to lectins from lotus. The matrix should be a substance that itself does not adsorb nαolecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below. B. Synthetic Peptides The present invention also includes smaller MEKK2 and MEKK3 related peptides for use in various embodiments of the present invention. Because of their relatively small size, the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young (1984); Tarn et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.

C. Antigen Compositions The present invention also provides for the use of MEKK2 and MEKK3 proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. In particular, the present invention provides MEKK2 phosphorylated at serine 519 or MEKK3 phosphorylated at serine 526. It is envisioned that MEKK2 and MEKK3 or portions thereof, will be coupled, bonded, bound, conjugated or chemically- linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable. Preferred agents are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA). IV. Prognostic and Diagnostic Applications In accordance with the present invention, it will also be useful to predict or diagnose a disease state, in a subject, due to MEKK2 or MEKK3 activation. Assays for examining protein levels, mRNA levels, and phosphorylation of a protein may be applied for examining a clinical sample for activity of MEKK2 or MEKK3. Such assays are well known to one of ordinary skill in the art. Thus, in particular embodiments, the present invention contemplates assessing the level of expression of MEKK2 or MEKK3 in a MEKK2 or MEKK3 containing-sample. Assays for MEKK2 or MEKK3 mRNA levels, mRNA stability or turnover, as well as protein expression and phosphorylation levels may be employed in the present invention. It is further contemplated that any post- translational processing of MEKK2 or MEKK3 may also be assessed. In one particular embodiment, dimerization of MEKK2 and/or MEKK3 is determined, hi preferred embodiments, an antibody that specifically recognizes phosphorylation of MEKK2 at serine 519 or MEKK3 at serine 526 may be used. In other embodiments, there is an antibody that specifically recognizes at least part of a dimerization domain of MEKKL2, MEKK3, or both.

A. Northern Blotting Techniques It is contemplated that the present invention may employ Northern blotting techniques in assessing the expression of MEKK2 or MEKK3 in a cell such as cancer cell or cardiac cell but is not limited to such cells. The tecliniques involved in Northern blotting are commonly used in molecular biology and are well known to one of skilled in the art. These techniques can be found in many standard books on molecular protocols (e.g., Sambrook et al, 2001). This technique allows for the detection of RNA, i. e., hybridization with a labeled probe. Briefly, RNA is separated by gel electiophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non- covalent binding. Subsequently, the membrane is incubated with, e.g., a chromophore- coηjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices. U.S. Patent 5,279,721, incorporated by reference herein, discloses an apparatus and method for the automated electiophoresis and transfer of nucleic acids. The apparatus permits electiophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.

B. Immunohistochemistry It is also contemplated that the present invention may employ quantitative immunohistochemistry in assessing the expression of MEKK2 or MEKK3 in a cell, tissue or organ sample. Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen

"pulverized" tumor at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections containing an average of about 500 remarkably intact cell, tissue or organ sample. Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 h fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial pennanent sections. Other immunohistochemistry techniques that may be employed in the present invention include tissue microarray immunohistochemistry. This method enables the simultaneous examination of multiple tissues sections concurrently as compared to the more conventional technique of one section at a time. This technique is used for high throughput molecular profiling of tumor specimen (Kononen et al, 1998).

C. Western Blotting The present invention may also employ the use of Western blotting

(immunoblotting) to analyze MEKK2 or MEKK3 activity or expression in a cell such as cancer cell, or cardiac cell, but is not limited to such. This technique is well known to those of skill in the art, see U.S. Patent 4,452,901 incorporated herein by reference and Sambrook et al. (2001). In brief, this technique generally comprises separating proteins in a sample such as a cell or tissue sample by SDS-PAGE gel electrophoresis. In SDS- PAGE proteins are separated on the basis of molecular weight, then are transferred to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), followed by incubation of the proteins on the solid support with antibodies that specifically bind to the proteins, for example, an antibody that specifically recognizes phosphorylation of MEKK2 serine 519 or MEKK3 on serine 526.

D. ELISA The present invention may also employ the use of immunoassays such as an enzyme linked immunosorbent assay (ELISA) in assessing the activity or expression of MEKK2 or MEKK3 in a cell such as cancer cell, or cardiac cell, but is not limited to such. An ELISA generally involves the steps of coating, incubating and binding, washing to remove species that are non-specifically bound, and detecting the bound immune complexes. This technique is well known in the art, for example see U.S. Patent 4,367,110 and Harlow and Lane, 1988. In an ELISA assay, a MEKK2 or MEKK3 protein sample may be immobilized onto a selected surface, preferably a surface exliibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powder. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. After binding of the antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from 2 to 4 or more hours to allow effective binding, at temperatures preferably on the order of 25°C to 37°C (or overnight at 4°C). Following incubation, the antisera-contacted surface is washed so as to remove non- immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. To provide a detecting means, the second antibody preferably has an associated enzyme that generates a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti- human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS- containing solution such as PBS-Tween). After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3- ethyl-benzthiazoline-6-sulfonic acid (ABTS) and H O , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. The use of labels for immunoassays are described in U.S. Patents 5,310,687, 5,238,808 and 5,221,605. Other immunodetection methods that may be contemplated in the present invention include radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay. These methods are well known to those of ordinary skill and have been described in Doolittle et al. (1999); Gulbis et al (1993); De Jager et al. (1993); and Nakamura et al. (1987), each incorporated herein by reference. V. Screening for Modulators A. Screening for Modulators of MEKK2 or MEKK3 The present invention further comprises methods for identifying modulators of MEKK2 or MEKK3 activity. MEKK2 or MEKK3 may be used as a target in screening for compounds that inhibit, decrease, down-regulate or activate its activity in cancer or cardiac cells, but is not limited to such cells. These assays may comprise random screening of large libraries of candidate substances; alternatively, these assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to modulate the function of MEKK2 or MEKK3, or the inhibition or activation of MEKK2 or MEKK3. To identify a MEKK2 or MEKK3 modulator, one generally will determine MEKK2 or MEKK3 activity in the presence and absence of the candidate substance, wherein a modulator is defined as any substance that regulates MEKK2 or MEKK3 activity. For example, a method may generally comprise: a) providing a MEKK2 or MEKK3 containing-sample; b) contacting the MEKK2 or MEKK3 containing-sample with a candidate substance; and c) determining phosphorylation of MEKK2 serine 5 19 or MEKK3 at serine 526, wherein a change in MEKK2 or MEKK3 phosphorylation in the presence of the candidate substance, as compared with the phosphorylation of MEKK2 or MEKK3 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK and/or MEKK3 activity. Thus, methods of screening for molecules that bind to and modulate phosphorylation of MEKK2 at serine 519 or MEKK3 at serine 526 are contemplated in the present invention. Such methods may employ assays well known to one of ordinary skill in the art, for example, see U.S. Patents 5,695, 944, 5.496,703 and 5,672,470. The present invention also contemplates the use of colorimetric assays using an ELISA method, as discussed above, or may employ FACS analysis in screening for molecules that bind to phosphorylated MEKK2 or MEKK3 at serine 519 and 526 respectively. Other methods of screening for candidate modulators of MEKK2 activity are also provided, such as those comprising: (a) providing a MEKK2-containing sample; (b) contacting the MEKK2-containing sample with a candidate substance; and (c) determining the effect of the candidate substance on dimerization of MEKK2, wherein a change in MEKK2 dimerization in the presence of the candidate substance, as compared with the dimerization of MEKK2 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK2 dimerization.

Assays may be conducted in cell free systems, in isolated cells, or in organisms including transgenic animals. It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.

B. MEKK2 or MEKK3 Modulators As used herein the term "candidate substance" or "candidate compound" refers to any molecule that may regulate the activity of MEKK2 or MEKK3. A MEKK2 or MEKK3 modulator, may be a compound that overall effects inhibition or activation of MEKK2 or MEKK3. Any compound or molecule described in the methods and compositions herein may be a modulator of MEKK2 or MEKK3 activity. An inhibitor according to the present invention may be one that exerts an inhibitory effect on the expression or function of MEKK2 and/or MEKK3. By the same token, an activator according to the present invention may be one that exerts a stimulatory effect on the expression or function of MEKK2 and/or MEKK3. The candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to MEKK2 or MEKK3 or that binds MEKK2 or MEKK3. Using lead compounds to help develop improved compounds is known as "rational drug design" and includes not only comparisons with known inhibitors, but predictions relating to the structure of target molecules. A candidate modulator may be any molecule that affects dimerizationof MEKK2 or MEKK3. The modulator may inhibit dimerization or, in alternative embodiments, the modulator may enhance dimerization. In specific embodiments, a modulator is an inhibitor of MEKK2 dimerization, such as a peptide (for example, that of SEQ ID NO:5) or a polypeptide (for example, that of SEQ ID NO:6). hi particular, the modulator that affects dimerization of MEKK2 is one that targets at least part of the dimerization domain, the catalytic domain, a region of MEKK2 located between the kinase subdomains I to Ul, a region of MEKK2 comprising amino acids 342-619 (SEQ ID NO: 10), and/or a region of MEKK2 comprising amino acids 342-424 (SEQ ID NO:5). In particular, the modulator that affects dimerization of MEKK3 is one that targets at least part of the dimerization domain, the catalytic domain, a region of MEKK3 comprising amino acids 378-656 (SEQ ID NO: 12), and/or a region of MEKK3 comprising amino acids 378-460 (SEQ ID NO: 11). Candidate modulators of the present invention will likely function to inhibit, decrease or down-regulate, activate or stimulate the activity of MEKK2 or MEKK3 in a cancer cell or a cardiac cell but is not limited to such. These candidate compounds may be antisense molecules, ribozymes, interfering RNAs, antibodies (including single chain antibodies), or organopharmaceuticals, but are not limited to such. 1. Antisense Constructs A modulator of MEKK2 or MEKK3 activity, as contemplated in the present invention may be an antisense molecule. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementary rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing. Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA leads to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense constructs, or DNA encoding such antisense constructs, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject. Such molecules may include MEKK2 or MEKK3 peptides derived from the relevant regions of MEKK2 or MEKK3 of the present invention. Thus, antisense molecules are one potential class of a MEKK2 or a MEKK3 inhibitor. Antisense constructs may be designed to bind to the promoter and/or other control regions, exons, intions or even exon-intion boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intion/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intion sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected. As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions. It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intion is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence. 2. Ribozymes Another modulator of MEKK2 or MEKK3 activity, as contemplated in the present invention, includes ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, ribozymes have been found useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et al, 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction. Thus, ribozymes are another potential class of MEKK2 and/or MEKK3 inhibitor. Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989). For example, U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et α/., 1990). 3. RNA Interference (RNAi) RNA interference (also referred to as "RNA-mediated interference" or RNAi) is a mechanism by which gene expression can be reduced or eliminated. Double stranded RNA (dsRNA) has been observed to mediate the reduction, which is a multi-step process. dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and tiansposon activity (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin and Avery, 1999; Montgomery et al, 1998; Sharp, 1999; Sharp and Zamore, 2000; Tabara et al, 1999; Hurvagner et al, 2001; Tuschl, 2001; Waterhouse et al, 2001; Zamore, 2001). Activation of these mechanisms target mature, dsRNA-complementary mRNA for destruction. Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C. elegans, Trypanasoma and Drosophila (Grishok et al, 2000; Sharp, 1999; Sharp and Zamore 2000). RNAi offers major experimental advantages for the study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene. In RNAi the dsRNA is typically directed to an exon, although some exceptions to this have been shown (see Plasterk and Ketting, 2000). Also, a homology threshold (probably about 80-85% over 200 bases) is required. Most tested sequences are 500 base pairs or greater, though sequences of 30 nucleotides or fewer evade the antiviral response in mammalian cells (Baglioni et al, 1983; Williams, 1997). The targeted mRNA is lost after RNAi. The effect of RNAi is non-stoichiometric, and thus incredibly potent. In fact, it has been estimated that only a few copies of dsRNA are required to knock down >95% of targeted gene expression in a cell (Fire et al, 1998). Due to a potent antiviral response pathway in mammalian cells that induces global changes in gene expression when the cells are challenged with long (>30 nucleotides) dsRNA molecules, RNAi was used in non-mammalian cells. This limitation in the art was overcome by the discovery of a method to bypass the antiviral response and induce gene specific silencing in mammalian cells (Elbashir et al, 2001). Several 21 nucleotide (nt) dsRNAs with 2 nt 3' overhangs were transfected into mammalian cells without inducing the antiviral response. These small dsRNAs, referred to as small interfering RNAs (siRNAs) proved capable of inducing the specific suppression of target genes. In addition, it was demonstrated that siRNAs could reduce the expression of several endogenous genes in human cells. The use of siRNAs to modulate gene expression in mammalian cells has since been demonstrated (Caplen et al, 2001; Hutvagner et al, 2001). Although the precise mechanism of RNAi is still unknown, the involvement of permanent gene modification or the disruption of transcription have been experimentally eliminated. It is now generally accepted that RNAi acts post-tianscriptionally, targeting RNA transcripts for degradation. It appears that both nuclear and cytoplasmic RNA can be targeted. (Bosher and Labouesse et al. , 2000). RNAi may be used to identify genes that are essential for a particular biological pathway, identify disease-causing genes, study structure function relationships, and implement therapeutics and diagnostics. As with other types of gene inhibitory compounds, such as antisense and triplex forming oligonucleotides, tracking these potential drugs in vivo and in vitro is important for drug development, pharmacokmetics, biodistribution, macro and microimaging metabolism and for gaining a basic understanding of how these compounds behave and function.

C. Rational Drug Design The present invention also provides methods for developing drugs that modulate

MEKK2 or MEKK3 activity or expression that may be used to treat a disease state due to MEKK2 or MEKK3 activation. Rational drug design may be used to produce structural analogs of MEKK2 that recognize a phosphorylation site at serine 519 or MEKK3 that recognize a phosphorylation site at serine 526. They may also be used to produce structural analogs of at least part of a MEKK2 dimerization domain, at least part of a MEKK3 dimerization domain, or both. By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for the MEKK2 or MEKK3 targets of the invention or a fragment thereof, such as a domain comprising one or more phosphorylation sites or at least part of a dimerization domain.. This could be accomplished by X-ray crystallography, computer modeling or by a combination of both approaches. It also is possible to use antibodies to ascertain the structure of a target compound modulator. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen. On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force" the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries (e.g., peptide libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds. Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators. Other suitable compounds include antisense molecules, ribozymes, and antibodies

(including single chain antibodies), each of which would be specific for the target molecule. Such compounds are described in greater detail elsewhere in this document.

For example, an antisense molecule that bound to a translational or transcriptional start site, or splice junctions, would be ideal candidate inhibitors. In addition, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the structure of the modulators. Such compounds, which may include peptidomimetics of peptide molecules, may be used in the same manner as the modulators described herein. A modulator according to the present invention may be one which exerts its inhibitory or activating effect upstream, downstream or directly on MEKK2 or MEKK3 or other related members of the MAP kinase pathway. Regardless of the type of modulator identified by the present screening methods, the effect by such a compound results in the regulation of MEKK2 or MEKK3 activity or expression as compared to that observed in the absence of the added candidate substance. The term "drug" as used herein is intended to refer to a chemical entity, whether in the solid, liquid, or gaseous phase which is capable of providing a desired therapeutic effect when administered to a subject. The term "drug" should be read to include synthetic compounds, natural products and macromolecular entities such as polypeptides, polynucleotides, or lipids and also small entities such as neurotiansmitters, ligands, hormones or elemental compounds. The term "drug" is meant to refer to that compound whether it is in a crude mixture or purified and isolated.

D. In Vitro Assays A quick, inexpensive and easy assay to run is a binding assay. Binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge- charge interactions. This can be performed in solution or on a solid phase and can be utilized as a first round screen to rapidly eliminate certain compounds before moving into more sophisticated screening assays. In one embodiment of this kind, the screening of compounds that bind to a MEKK2 and/or MEKK3 molecule or fragment thereof is provided. The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determination of binding, hi another embodiment, the assay may measure the inhibition of binding of a target to a natural or artificial substrate or binding partner (such as MEKK2 and/or MEKK3). Competitive binding assays can be performed in which one of the agents (MEKK2 and/or MEKK3 for example) is labeled. Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with the binding moiety's function. One may measure the amount of free label versus bound label to determine binding or inhibition of binding. A technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with, for example, MEKK2 and/or MEKK3 and washed. Bound polypeptide is detected by various methods. Purified target, such as MEKK2 and/or MEKK3, can be coated directly onto plates for use in the aforementioned drug screening techniques. Non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase. Also, fusion proteins containing a reactive region (preferably a terminal region) may be used to link an active region (e.g., the C-terminus of MEKK2 and/or MEKK3) to a solid phase.

E. In Cyto Assays Various cells that express at least part of MEKK2 and/or MEKK3 can be utilized for screening of candidate substances. For example, cells containing at least part of MEKK2 and/or MEKK3 with an engineered indicator can be used to study various functional attributes of candidate compounds. In such assays, the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell. Depending on the assay, culture may be required. As discussed above, the cell may then be examined by virtue of a number of different physiologic assays (growth, size, calcium effects). Alternatively, molecular analysis may be performed in which the activity of MEKK2 and/or MEKK3 and related pathways may be explored. This involves assays such as those for protein expression, enzyme function, substrate utilization, mRNA expression (including differential display of whole cell or polyA RNA) and others.

F. In Vivo Assays The present invention also contemplates the use of various animal models for analyzing the expression or activity of MEKK2 or MEKK3 modulators. Treatment of these animals with test compounds will involve the administiation of the compound, in an appropriate form, to the animal. Administration will be by any route the could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, or even topical. Alternatively, administiation may be by intratracheal instillation, bronchial instillation, intiadermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Specifically contemplated are systemic intravenous injection, regional administration via blood or lymph supply.

VI. Generating Antibodies Reactive with MEKK2 and MEKK3 In a particular embodiment, the present invention contemplates an antibody that is immunoreactive with a MEKK2 molecule phosphorylated at serine 519 or MEKK3 molecule phosphorylated at serine 526. Such an antibody can be a polyclonal or a monoclonal antibody, hi a preferred embodiment, the antibody is a polyclonal antibody. Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988). The genes for MEKK2 or MEKK3 polyclonal antibodies can be utilized as inhibitors of MEKK2 and/or MEKK3 activation. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a non- human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies. Antibodies, both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood. It is proposed that the antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to MEKK2 or MEKK3-related antigen epitopes. Additionally, it is proposed that antibodies specific to the particular MEKK2 or MEKK3 of different species may be utilized in other useful applications In general, both polyclonal and monoclonal antibodies against serine phosphorylated MEKK2 or MEKK3 may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding other MEKK2 molecules phosphorylated at serine 519 or MEKK3 molecules phosphorylated at serine 526. They may also be used in inhibition studies to analyze the effects of MEKK2 or MEKK3-related peptides in cells or animals. MEKK2 or MEKK3 antibodies will also be useful in immunolocalization studies to analyze the distribution of MEKK2 or MEKK3 during various cellular events, for example, to determine the cellular or tissue-specific distribution of MEKK2 or MEKK3 polypeptides at different points in the cell cycle. A particularly useful application of such antibodies is in purifying native or recombinant MEKK2 or MEKK3, for example, using an antibody affinity column. The operation of such immunological techniques are well known to those of skill in the art. Another use for a MEKK2 antibody is to target at least part of a MEKK2 to prevent dimerization, such as, for example, targeting a dimerization domain, the catalytic domain, a region of MEKK2 located between the kinase subdomains I to III, a region of MEKK2 comprising amino acids 342-619, and/or a region of MEKK2 comprising amino acids 342-424. Another use for a MEKK3 antibody is to target at least part of a MEKK3 to prevent dimerization, such as, for example, targeting a dimerization domain, the catalytic domain, a region of MEKK3 comprising amino acids 378-656, and/or a region of

MEKK2 comprising amino acids 378-460. In respective embodiments, MEKK3 antibodies are used similarly. Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988; incoφorated herein by reference). More specific examples of antibody preparation are provided in the examples herein. As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine. It is also well known in the art, that the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intiadermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs. MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified MCJP protein, polypeptide or peptide or cell expressing high levels of MCIP. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

VII. Drug Formulations and Routes for Administration to Patients Clinical applications of MEKK2 or MEKK3 modulators to treat a subject having a disease state due to MEKK2 or MEKK3 are contemplated in the present invention. Thus, pharmaceutical compositions, for eample, expression vectors, virus stocks and drugs, may be prepared in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. Where delivery of an expression construct is desired, one will generally employ appropriate salts and buffers to render the vectors stable and allow for uptake by target cells. Aqueous compositions of the present invention may comprise an effective amount of a MEKK2 or MEKK3 modulator, or an expression construct encoding therefor, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administiation to humans. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incoφorated into the compositions, provided they do not inactivate the compositions. The compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administiation may be by orthotopic, intiadermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra. The MEKK2 or MEKK3 modulators or expression constructs encoding therefor, may also be administered parenterally or intraperitoneally. By way of illustration, solutions of these compositions as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy syringability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin. Sterile injectable solutions may be prepared by incoφorating the MEKK2 or MEKK3 modulators or expression constructs thereof in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof. For oral administiation the MEKK2 or MEKK3 modulator or expression constructs thereof generally may be incoφorated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incoφorating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incoφorated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. The compositions of the present invention generally may be formulated in a neutial or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administiation. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administiation will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

VIII. Combination Therapy with MEKK2 or MEKK3 Modulators In another embodiment, it is envisioned to use MEKK2 and MEKK3 modulators in combination with other therapeutic modalities. Thus, in addition to the therapies described herein, one may also provide to the patient more "standard" pharmaceutical cancer or cardiac therapies but is not limited to such therapies. Examples of standard cancer therapies include, without limitation, anticancer therapies such as chemotherapy, radiotherapy, gene therapy or surgery. Examples of standard cardiac therapies include, without limitation, so-called "beta blockers", anti-hypertensives, cardiotonics, anti- thrombotics, vasodilators, hormone antagonists, endothelin antagonists, calcium channel blockers, phosphodiesterase inhibitors, angiotensin type 2 antagonists and cytokine blockers/inhibitors. In further embodiments, it is contemplated that standard therapies for treating autoimmune diseases may also be employed in conjunction with the MEKK2 or MEKK3 modulators of the present invention. The diseases or therapies discussed herein are provided as examples and therefore are not meant to be limiting. Combinations may be achieved by contacting a MEKK2 or MEKK3 containing- sample such as a cell, tissue or organ with a single composition or pharmacological formulation that includes both agents, or by contacting the sample with two distinct compositions or formulations, at the same time, wherein one composition includes the modulator or expression construct comprising a nucleic acid sequence encoding a modulator and the other includes the cancer, cardiac or other agent. Alternatively, the MEKK2 or MEKK3 modulator may precede or follow administration of the cancer or cardiac or other agent by intervals ranging from minutes to weeks. In embodiments where the other agent and modulator or expression construct encoding therefor are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and modulator or expression construct encoding therefor would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would typically contact the cell with both modalities witliin about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred, hi some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. It also is conceivable that more than one administiation of either a MEKK2 or

MEKK3 modulator, or the cancer, cardiac or other agent will be desired. Various combinations may be employed; for example, where the MEKK2 or MEKK3 modulator is "A" and the cancer, cardiac or other agent is "B" as follows: A/B/A B/A B B/B/A A/A/B A B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A A/B/B A B/A/B A B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the MEKK2 or MEKK3 modulator to a patient will follow general protocols for the administiation of the cancer, cardiac or other therapy, taking into account the toxicity, if any, of the MEKK2 or MEKK3 modulator. It is expected that the treatment cycles would be repeated as necessary.

A. Anticancer Therapy An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. Anti-cancer agents include biological agents (biotherapy), chemotherapy agents, and radiotherapy agents. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

1. Chemotherapy Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitiosurea, dactinomycin, daunorubicin, doxorabicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, Temazolomide (an aqueous form of DTIC), or any analog or derivative variant of the foregoing. The combination of chemotherapy with biological therapy is known as biochemotherapy. The present invention contemplates any chemotherapeutic agent that may be employed or known in the art for treating or preventing cancers.

2. Radiotherapy Other factors that cause DNA damage and have been used extensively include what are commonly known as -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

3. Immunotherapy hnmuiiotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. Immunotherapy could also be used as part of a combined therapy. The general approach for combined therapy is discussed below. In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55. An alternative aspect of immunotherapy is to anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor such as mda-7 has been shown to enhance anti-tumor effects (Ju et al, 2000). Examples of immunotherapies currently under investigation or in use are immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitiochlorobenzene and aromatic compounds) (U.S. Patent 5,801,005; U.S. Patent 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998), cytokine therapy (e.g., interferons, and; IL-1, GM- CSF and TNF) (Bukowski et al, 1998; Davidson et al, 1998; Hellstiand et al, 1998) gene therapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al, 1998; Austin- Ward and Villaseca, 1998; U.S. Patent 5,830,880 and U.S. Patent 5,846,945) and monoclonal antibodies (e.g., anti-ganglioside GM2, anti-HER-2, anti-pl85) (Pietras et al, 1998; Hanibuchi et al, 1998; U.S. Patent 5,824,311). Herceptin (tiastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It possesses anti-tumor activity and has been approved for use in the treatment of malignant tumors (Dillman, 1999). Combination therapy of cancer with herceptin and chemotherapy has been shown to be more effective than the individual therapies. i) Adoptive Immunotherapy In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al, 1988; 1989). To achieve this, one would administer to an animal, or human patient, an immunologically effective amount of activated lymphocytes in combination with an adjuvant-incoφorated antigenic peptide composition as described herein. The activated lymphocytes will most preferably be the patient's own cells that were earlier isolated from a blood or tumor sample and activated (or "expanded") in vitro. This form of immunotherapy has produced several cases of regression of melanoma and renal carcinoma, but the percentage of responders were few compared to those who did not respond. ii) Passive Immunotherapy A number of different approaches for passive immunotherapy of cancer exist. They may be broadly categorized into the following: injection of antibodies alone; injection of antibodies coupled to toxins or chemotherapeutic agents; injection of antibodies coupled to radioactive isotopes; injection of anti-idiotype antibodies; and finally, purging of tumor cells in bone marrow. Preferably, human monoclonal antibodies are employed in passive immunotherapy, as they produce few or no side effects in the patient. However, their application is somewhat limited by their scarcity and have so far only been administered intialesionally. Human monoclonal antibodies to ganglioside antigens have been administered intialesionally to patients suffering from cutaneous recurrent melanoma ( ie and Morton, 1986). Regression was observed in six out of ten patients, following, daily or weekly, intialesional injections. In another study, moderate success was achieved from intialesional injections of two human monoclonal antibodies (Irie et al, 1989). It may be favorable to administer more than one monoclonal antibody directed against two different antigens or even antibodies with multiple antigen specificity. Treatment protocols also may include administration of lymphokines or other immune enhancers as described by Bajorin et al. (1988). The development of human monoclonal antibodies is described in further detail elsewhere in the specification. iii) Active Immunotherapy In active immunotherapy, an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or "vaccine" is administered, generally with a distinct bacterial adjuvant (Ravindranath and Mitchell et al, 1990; Mitchell et al, 1993). hi melanoma immunotherapy, those patients who elicit high IgM response often survive better than those who elicit no or low IgM antibodies (Morton et al, 1992). IgM antibodies are often transient antibodies and the exception to the rule appears to be anti- ganglioside or anticarbohydrate antibodies.

4. Genes In yet another embodiment, the anticancer therapy is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the MEKK2 or MEKK3 modulator is administered. Delivery of an expression vector encoding a MEKK2 or MEKK3 modulator in conjunction with a second expression vector encoding one of the following gene products will have a combined anti- hypeφroliferative effect on target tissues. Alternatively, a single vector encoding both genes may be used. A variety of proteins are encompassed within the invention, some of which are described below. Various genes that may be targeted for gene therapy of some form in combination with the present invention are will known to one of ordinary skill in the art and may comprise any gene involve in cancers, cardiovascular disease or autoimmune diseases/conditions. i) Inducers of Cellular Proliferation The proteins that induce cellular proliferation further fall into various categories dependent on function. The commonality of all of these proteins is their ability to regulate cellular proliferation. For example, a form of PDGF, the sis oncogene, is a secreted growth factor. Oncogenes rarely arise from genes encoding growth factors, and at the present, sis is the only known naturally-occurring oncogenic growth factor. In one embodiment of the present invention, it is contemplated that anti-sense mRNA directed to a particular inducer of cellular proliferation is used to prevent expression of the inducer of cellular proliferation. The proteins FMS, ErbA, ErbB and neu are growth factor receptors. Mutations to these receptors result in loss of regulatable function. For example, a point mutation affecting the tiansmembrane domain of the Neu receptor protein results in the neu oncogene. The erbA oncogene is derived from the intracellular receptor for thyroid hormone. The modified oncogenic ErbA receptor is believed to compete with the endogenous thyroid hormone receptor, causing uncontrolled growth. The largest class of oncogenes includes the signal transducing proteins (e.g., Src, Abl and Ras). The protein Src is a cytoplasmic protein-tyrosine kinase, and its transformation from proto-oncogene to oncogene in some cases, results via mutations at tyrosine residue 527. In contrast, transformation of GTPase protein ras from proto- oncogene to oncogene, in one example, results from a valine to glycine mutation at amino acid 12 in the sequence, reducing ras GTPase activity. The proteins Jun, Fos and Myc are proteins that directly exert their effects on nuclear functions as transcription factors. ii) Inhibitors of Cellular Proliferation The tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. The tumor suppressors p53, pl6 and C-CAM are described below. hi addition to p53, which has been described above, another inhibitor of cellular proliferation is pi 6. The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4), regulates progression through the Gl. The activity of this enzyme may be to phosphorylate Rb at late Gl. The activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the pl6INK4 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al, 1993; Serrano et al, 1995). Since the pl6INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hypeφhosphorylation of the Rb protein, pi 6 also is known to regulate the function of CDK6. pl6INK4 belongs to a newly described class of CDK-inhibitory proteins that also includes pl6B, pl9, p21WAFl, and p27KIPl. The pl6INK4 gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pl6INK4 gene are frequent in human tumor cell lines. This evidence suggests that the pl6INK4 gene is a tumor suppressor gene. This inteφretation has been challenged, however, by the observation that the frequency of the pl 6INK4 gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al, 1994; Cheng et al, 1994; Hussussian et al, 1994; Kamb et al, 1994; Kamb et al, 1994; Mori et al, 1994; Okamoto et al, 1994; Nobori et al, 1995; Orlow et al, 1994; Arap et al, 1995). Restoration of wild-type pl6INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995). Other genes that may be employed according to the present invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, MMACl / PTEN, DBCCR-1, FCC, rsk-3, p27, p27/pl6 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, ra erb, fins, trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC. iii) Regulators of Programmed Cell Death Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al, 1972). The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al, 1985; Cleary and Sklar, 1985; Cleary et al, 1986; Tsujimoto et al, 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists. Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 (e.g., BclXL, BclW, BclS, Mcl-1, Al, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

5. Surgery Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destioyed. Tumor resection refers to physical removal of at least part of a tumor, hi addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue. Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 ^"weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

B. Cardiac Therapy Non-limiting examples of a cardiac agent that may be used in combination with the MEKK2 or MEKK3 modulator of the present invention may include an antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmic agent, an antihypertensive agent, a vasopressor, a treatment agent for congestive heart failure, an antianginal agent, an antibacterial agent or a combination thereof, but is not limited to such. 1. Antihyperlipoproteinemics In certain embodiments, administration of an agent that lowers the concentration of one of more blood lipids and/or lipoproteins, known herein as an "antihyperlipoproteinemic," may be combined with a MEKK2 or MEKK3 modulator of the present invention, particularly in treatment of atherosclerosis and thickenings or blockages of vascular tissues, hi certain aspects, an antihyperlipoproteinemic agent may comprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acid sequesterant, a HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a miscellaneous agent or a combination thereof. Antihyperlipoproteinemic agents may include: Aryloxyalkanoic Acid/Fibric Acid Derivatives. Non-limiting examples of aryloxyalkanoic/fibric acid derivatives include beclobrate, enzafibrate, binifibrate, ciprofibrate, clinofibrate, clofibrate (atiomide-S), clofibric acid, etofibrate, fenofibrate, gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrate and theofibrate. Resins/Bile Acid Sequesterants. Non-limiting examples of resins/bile acid sequesterants include cholestyramine (cholybar, questran), colestipol (colestid) and polidexide. HMG CoA Reductase Inhibitors. Non-limiting examples of FfMG CoA reductase inhibitors include lovastatin (mevacor), pravastatin (pravochol) or simvastatin (zocor). Nicotinic Acid Derivatives. Non-limiting examples of nicotinic acid derivatives include nicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid. Thryroid Hormones and Analogs. Non-limiting examples of thyroid hormones and analogs thereof include etoroxate, thyropropic acid and thyroxine. Miscellaneous Antihyperlipoproteinemics. Non-limiting examples of miscellaneous antihyperlipoproteinemics include acifran, azacosterol, benfluorex, - benzalbutyramide, camitine, chondroitin sulfate, clomestione, detaxtran, dextran sulfate sodium, 5, 8, 11, 14, 17-eicosapentaenoic acid, eritadenine, furazabol, meglutol, melinamide, mytatrienediol, ornithine, -oryzanol, pantethine, pentaerythritol tetraacetate, -phenylbutyramide, pirozadil, probucol (lorelco), -sitosterol, sultosilic acid-piperazine salt, tiadenol, triparanol and xenbucin. 2. Antiarteriosclerotics Antiarteriosclerotic agents may also be used in combination with the MEKK2 or MEKK3 modulators of the present invention. A non-limiting example of an antiarteriosclerotic include pyridinol carbamate.

3. Antithrombotic/Fibrinolytic Agents In certain embodiments, administration of an agent that aids in the removal or prevention of blood clots may be combined with administration of a modulator, particularly in tieatment of athersclerosis and vasculature (e.g., arterial) blockages. Non- limiting examples of antithrombotic and/or fibrinolytic agents include anticoagulants, anticoagulant antagonists, antiplatelet agents, thrombolytic agents, thrombolytic agent antagonists or combinations thereof. hi certain aspects, antithrombotic agents that can be administered orally, such as, for example, aspirin and wafarin (coumadin), are preferred. Antithrombotic agents may include agents such as anticoagulants. A non-limiting example of an anticoagulant include acenocoumarol, ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide, tioclomarol and warfarin. Antithrombotic agents may also include antiplatelet agents such as aspirin, a dextran, dipyridamole (persantin), heparin, sulfinpyranone (anturane) and ticlopidine (ticlid), but are not limited to such agents. Antithrombotic agents may further include thrombolytic agents such as tissue plaminogen activator (activase), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase (streptase), anistreplase/APSAC (eminase), but are not limited to such agents. 4. Blood Coagulants In certain embodiments wherein a patient is suffering from a hemmorage or an increased likelyhood of hemmoraging, an agent that may enhance blood coagulation may be used. Non-limiting examples of a blood coagulation promoting agent include

, thrombolytic agent antagonists and anticoagulant antagonists. Non-limiting examples of anticoagulant antagonists include protamine and vitamine Kl . Non-limiting examples of thrombolytic agent antagonists include amiocaproic acid (amicar) and tranexamic acid (amstat). Non-limiting examples of antithrombotics include anagrelide, argatroban, cilstazol, daltioban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

5. Antiarrhythmic Agents Non-limiting examples of antiarrhythmic agents include Class I antiarrythmic agents (sodium channel blockers), Class II antiarrythmic agents (beta-adrenergic blockers), Class II antiarrythmic agents (repolarization prolonging drugs), Class TV antiarrhythmic agents (calcium channel blockers) and miscellaneous antiarrythmic agents. Non-limiting examples of sodium channel blockers include Class IA, Class IB and Class IC antiarrhythmic agents. Non-limiting examples of Class IA antiarrhythmic agents include disppyramide (noφace), procainamide (pronestyl) and quinidine (quinidex). Non-limiting examples of Class IB antiarrhythmic agents include lidocaine (xylocaine), tocainide (tonocard) and mexiletine (mexitil). Non-limiting examples of Class IC antiarrhythmic agents include encainide (enkaid) and flecainide (tambocor). Non-limiting examples of a beta blocker, otherwise known as a -adrenergic blocker, a -adrenergic antagonist or a Class II antiarrhythmic agent, include acebutolol (sectial), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitiolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol, propanolol (inderal), sotalol (betapace), sulfinalol, talinolol, tertatolol, timolol, toliprolol and xibinolol. hi certain aspects, the beta blocker comprises an aryloxypropanolamine derivative. Non-limiting examples of aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitiolol, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol. Non-limiting examples of an agent that prolong repolarization, also known as a Class Ul antiarrhythmic agent, include amiodarone (cordarone) and sotalol (betapace). Non-limiting examples of a calcium channel blocker, otherwise known as a Class IN antiarrythmic agent, include an arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline, verapamil), a dihydropyridine derivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitiendipine) a piperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a micellaneous calcium channel blocker such as bencyclane, etafenone, magnesium, mibefradil or perhexiline. hi certain embodiments a calcium channel blocker comprises a long-acting dihydropyridine (nifedipine-type) calcium antagonist. Νon-limiting examples of miscellaneous antiarrhymic agents include adenosine (adenocard), digoxin (lanoxin), acecainide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, disopyranide, hydroquinidine, indecainide, ipatiopium bromide, lidocaine, lorajmine, lorcainide, meobentine, moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate and viquidil.

6. Antihypertensive Agents The MEKK2 or MEKK3 modulators of the preset invention may also be used in combination with cardiac therapies such as sympatholytic agents, alpha/beta blockers, alpha blockers, anti-angiotensin II agents, beta blockers, calcium channel blockers, vasodilators and miscellaneous antihypertensives. Νon-limiting examples of an alpha blocker, also known as an -adrenergic blocker or an -adrenergic antagonist, include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin and yohimbine. hi certain embodiments, an alpha blocker may comprise a quinazoline derivative. Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin and trimazosin. In certain embodiments, an antihypertensive agent is both an alpha and beta adrenergic antagonist. Non-limiting examples of an alpha/beta blocker comprise labetalol (normodyne, tiandate). Non-limiting examples of anti-angiotension II agents include include angiotensin converting enzyme inhibitors and angiotension II receptor antagonists. Non-limiting examples of angiotension converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (vasotec), captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril, perindopril, quinapril and ramipril.. Non-limiting examples of an angiotensin II receptor blocker, also known as an angiotension II receptor antagonist, an ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS), include angiocandesartan, eprosartan, irbesartan, losartan and valsartan. Non-limiting examples of a sympatholytic include a centrally acting sympatholytic or a peripherially acting sympatholytic. Non-limiting examples of a centrally acting sympatholytic, also known as an central nervous system (CNS) sympatholytic, include clonidine (catapres), guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet). Non-limiting examples of a peripherally acting sympatholytic include a ganglion blocking agent, an adrenergic neuron blocking agent, a β-adrenergic blocking agent or a alphal -adrenergic blocking agent. Non-limiting examples of a ganglion blocking agent include mecamylamine (inversine) and trimethaphan (arfonad). Non-limiting of an adrenergic neuron blocking agent include guanethidine (ismelin) and reseφine (seφasil). Non-limiting examples of a β-adrenergic blocker include acenitolol (sectial), atenolol (tenormin), betaxolol (kerlone), carteolol (cartiol), labetalol (normodyne, tiandate), metoprolol (lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken), propranolol (inderal) and timolol (blocadren). Non-limiting examples of alphal -adrenergic blocker include prazosin (minipress), doxazocin (cardura) and terazosin (hytrin). ha certain embodiments a cardiovasculator therapeutic agent may comprise a vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or a peripheral vasodilator), hi certain preferred embodiments, a vasodilator comprises a coronary vasodilator. Non-limiting examples of a coronary vasodilator include amotriphene, bendazol, benfurodil hemisuccinate, benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole, droprenilamine, efloxate, erythrityl tetianitiane, etafenone, fendiline, floredil, ganglefene, herestrol bis(-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin, lidoflanine, mannitol hexanitiane, medibazine, nicorglycerin, pentaerythritol tetranitiate, pentrinitiol, perhexiline, pimefylline, trapidil, tricromyl, trimetazidine, trolnitiate phosphate and visnadine. In certain aspects, a vasodilator may comprise a chronic therapy vasodilator or a hypertensive emergency vasodilator. Non-limiting examples of a chronic therapy vasodilator include hydralazine (apresoline) and minoxidil (loniten). Non-limiting examples of a hypertensive emergency vasodilator include nitioprusside (nipride), diazoxide (hyperstat TV), hydralazine (apresoline), minoxidil (loniten) and verapamil. Non-limiting examples of miscellaneous antihypertensives include ajmaline, - aminobutyric acid, bufeniode, cicletainine, ciclosidomine, a cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, a protoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodium nitiorusside, ticrynafen, trimethaphan camsylate, tyrosinase and urapidil. In certain aspects, an antihypertensive may comprise an arylethanolamine derivative, a benzothiadiazine derivative, a N-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative, a guanidine derivative, a hydrazines/phthalazine, an imidazole derivative, a quanternary ammonium compound, a reseφine derivative or a suflonamide derivative. Arylethanolamine Derivatives. Νon-limiting examples of arylethanolamine derivatives include amosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol and sulfinalol. Benzothiadiazine Derivatives. Νon-limiting examples of benzothiadiazine derivatives include althizide, bendroflumethiazide, benzthiazide, benzyUiydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachlormethiazide and trichlormethiazide. N-carboxyalkyl(peptide/lactam) Derivatives. Non-limiting examples of N- carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapril and ramipril. Dihydropyridine Derivatives. Νon-limiting examples of dihydropyridine derivatives include amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitiendipine. Guanidine Derivatives. Νon-limiting examples of guanidine derivatives include bethanidine, debrisoquin, guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan. Hydrazines/Phthalazines. Νon-limiting examples of hydrazines/phthalazines include budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine and todralazine. Imidazole Derivatives. Νon-limiting examples of imidazole derivatives include clonidine, lofexidine, phentolamine, tiamenidine and tolonidine. Quanternary Ammonium Compounds. Νon-limiting examples of quantemary ammonium compounds include azamethonium bromide, chlorisondamine chloride, hexamethonium, pentacynium bis(methylsulfate), pentamethonium bromide, pentolinium tartiate, phenactropinium chloride and trimethidinium methosulfate. Reserpine Derivatives. Νon-limiting examples of reseφine derivatives include bietaseφine, deseφidine, rescinnamine, reseφine and syrosingopine. Suflonamide Derivatives. Νon-limiting examples of sulfonamide derivatives include ambuside, clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide.

7. Vasopressors Vasopressors generally are used to increase blood pressure during shock, which may occur during a surgical procedure may also be combined with the MEKK2 or

MEKK3 modulators of the present invention. Νon-limiting examples of a vasopressor, also known as an antihypotensive, include amezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine. 8. Treatment Agents for Congestive Heart Failure The MEKK2 or MEKK3 modulators of the present invention may also be combined with agents for the treatment of congestive heart failure. Such agents may include anti-angiotension II agents, afterload-preload reduction tieatment, diuretics and inotropic agents. In certain embodiments, an animal patient that cannot tolerate an angiotension antagonist may be tieated with a combination therapy. Such therapy may combine adminstiation of hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitrate). Non-limiting examples of a diuretic include a thiazide or benzothiadiazine derivative (e.g., althiazide, bendroflumethazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetiachloromethiazide, trichlormethiazide), an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurous chloride, mersalyl), a pteridine (e.g., furterene, triamterene), purines (e.g., acefylline, 7-moφholinomethyltheophylline, pamobrom, protheobromine, theobromine), steroids including aldosterone antagonists (e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative (e.g. , acetazolamide, ambuside, azosemide, bumetanide, butazolamide, chloraminophenamide, clofenamide, clopamide, clorexolone, diphenylmethane-4,4'-disulfonamide, disulfamide, ethoxzolamide, furosemide, indapamide, mefruside, methazolamide, piretanide, quinethazone, torasemide, tripamide, xipamide), a uracil (e.g., aminometiadine, amisometradine), a potassium sparing antagonist (e.g., amiloride, triamterene)or a miscellaneous diuretic such as aminozine, arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine, isosorbide, mannitol, metochalcone, muzolimine, perhexiline, ticmafen and urea. Non-limiting examples of a positive inotiopic agent, also known as a cardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline, amrinone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin, stiphanthin, sulmazole, theobromine and xamoterol. In particular aspects, an intiopic agent is a cardiac glycoside, a beta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limiting examples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin (crystodigin). Non-limiting examples of a - adrenergic agonist include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, cloφrenaline, denopamine, dioxethedrine, dobutamine (dobutiex), dopamine (intiopin), dopexamine, ephedrine, etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol and xamoterol. Non-limiting examples of a phosphodiesterase inhibitor include amrinone (inocor). 9. Antianginal Agents The MEKK2 or MEKK3 modulators of the present invention may also be combined with antianginal agents. Such agents may comprise organonitiates, calcium channel blockers, beta blockers and combinations thereof. Non-limiting examples of organonitiates, also known as nitrovasodilators, include nitroglycerin (nitro-bid, nitiostat), isosorbide dinitiate (isordil, sorbitrate) and amyl nitrate (aspirol, vaporole).

10. Surgical Therapy for Cardiac Diseases hi certain aspects, the secondary therapeutic agent for treating a cardiac disease may comprise a surgery of some type, which includes, for example, preventative, diagnostic or staging, curative and palliative surgery. Surgery, and in particular a curative surgery, may be used in conjunction with other therapies, such as the present invention and one or more other agents. Such surgical therapeutic agents for vascular and cardiovascular diseases and disorders are well known to those of skill in the art, and may comprise, but are not limited to, performing surgery on an organism, providing a cardiovascular mechanical prostheses, angioplasty, coronary artery reperfusion, catheter ablation, providing an implantable cardioverter defibrillator to the subject, mechanical circulatory support or a combination thereof. Non-limiting examples of a mechanical circulatory support that may be used in the present invention comprise an intra-aortic balloon counteφulsation, left ventricular assist device or combination thereof.

IX. Dimerization of MEKK2, MEKK3, or Both In specific embodiments of the present invention, MEKK2 comprises dimerization activity. In other embodiments, MEKK3 comprises dimerization activity, although MEKK2 dimer will be referred to herein as the exemplary embodiment. In one aspect of the invention, MEKK2 is a homodimer, whereas in alternative aspects MEKK2 is a heterodimer. The dimerization activity may be provided in one or more regions of MEKK2, although in a specific embodiment it is provided in the C-terminal region, such as specifically in the region from about amino acids 342-619, and such as more specifically in the region from about 342 to about 424. The dimerization activity may be provided in one or more regions of MEKK3, although in a specific embodiment it is provided in the C-terminal region, such as specifically in the region from about amino acids 378-656, and such as more specifically in the region from about 378 to about 460. The dimerization activity of MEKK2 or a fragment or derivative thereof may be characterized by any suitable method, hi a particular embodiment, dimerization is identified by GST pulldown assays, gel-shift assays, and/or two-hybrid assays, for example. In additional aspects of the invention, the dimerization activity of MEKK2 is exploited to identify one or more candidate modulators that interfere with the dimerization. For example, a screen for candidate modulators of MEKK2 activity may be employed wherein a MEKK2-containing sample is provided and contacted with a candidate substance; and the effect of the candidate substance on dimerization of MEKK2 is determined. In specific embodiments of the assay, a change in MEKK2 dimerization in the presence of the candidate substance, as compared with the dimerization of MEKK2 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK2 dimerization. Particular examples for identifying candidate modulators of MEKK2 dimerization is to utilize two-hybrid assay, GST-pulldown assays, gel shift assays, or a combination thereof. X. Examples The following examples are included to further illustrate various aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques and/or compositions discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 Experimental Procedures for Examples 2-10

Cell Culture. COS-1 cells, Jurkat cells, Raw277.1 and 293T were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin. Generation of MEKK2 and MEKK3-deficient MEFs. The Mekk≠ MEFs were established from E9.5 embryos dissected from Mekk3^+I~ mice as described previously (Yang, 2000; Yang, 2001). Mekk2^~l~ and Mekk2^+/~ MEFs were established from El 1 embryos, MekkT^IMekk ^1', Mekk2^''^'Mekk3^+/- and Mekk2^+l'Mekk3^+l~ MEFs were isolated from E9.5 embryos from Mekk2^+l'Mekk3^+l" using the same protocol as described for Mekk3^''^~ MEFs (Yang, 2001). All MEFs were maintained in DMEM with 15% FBS as described previously (Yang, 2001). The MEFs were transfected with FuGene 6 (Boeheringer Mannheim, Indianapolis, IN) according to the manufacturer's protocol. Plasmids. The HA-tagged MEKK3 expression vector SRα3HA-MEKK3 was described previously (Yang, 2000), Lys (391)-Met Mekk3 mutant was generated by polymerase chain reaction-directed mutagenesis, and the mutant cDNA was sub cloned into the SR 3HA vector as described previously (Cheng et al, 2000). HA-tagged JNKl, ERK5, were cloned into pSRα3 vector as previously described (Minden et al, 1994; Su et al, 1994; Lin et al, 1995; Yang et al, 1998). HA-tagged MEKK2(CT) expression vector was constructed by introducing an Ncol site at codon 343 by the PCR-based method, followed by subcloning into the expression vector pSRα3HA at the Ncol site, in which MEKK2 (CT) fused in frame with the HA tag (Cheng et al, 2000). GST-tagged MEKK2(FL), MEKK2(CT), MEKKl(CT), and JNKK2 mammalian expression vectors were cloned into pEGB vector (Cheng et al, 2000). Bacterial recombinant protein expression vectors for GST-c-Jun (Su et al, 1994). Anti-HA antibody 12CA5 was prepared from 12CA5 hybridoma (Wilson et al, 1984) and further purified using a protein-A sepharose column. MEKK2-specific antibody 1128 was prepared by immunizing rabbits with a GST-fusion protein fused in frame with a fragment of human MEKK2 peptide (amino acids 343-428).

Immunoprecipitation and In Vitro Kinase Assay Cell Assay. Cell lysates were prepared 40 hr after transfection, as described (Su et al, 1994; Xia et al, 1998) and incubated with appropriate antibodies for 4 hr at 4°C in a rotator, followed by addition of protein-A sepharose beads for another 45 min. The beads were washed four times with lysis buffer (20 mM Tris, pH 7.5, 0.5% NP-40, 250 mM NaCI, 3 mM EDTA, 3 mM EGTA, and 100 mM Na₃VO₄), and twice with kinase reaction buffer (20 mM HEPES, pH 7.6, 1 mM MgCl₂, and 10% glycerol). The immunoprecipitates were then subjected to kinase assays in 30 μl of kinase buffer with appropriate substrates in the presence of 0.5 μl γ- P-ATP and 20 μM cold ATP. After 20 min at 30°C, the reactions were terminated by SDS-PAGE loading buffer and boiling for 5 min. The proteins were separated by SDS-PAGE, and ³²P incoφoration was determined by X-ray film. Co-Precipitation Assay. COS-1 cells transfected with HA-, Flag-, or GST- tagged expression vectors were lysed 40 hr after transfection using low-salt lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCI, 1.5 mM MgCl₂, 1 mM EDTA, 1% Triton-X 100, and 10% glycerol). Nucleus and cell debris was removed from the lysates by centrifugation in a microfuge for 20 min at 4°C. Expressed proteins were precipitated from the clarified lysates with GSH-sepharose beads at 4°C for 4-hr incubation in a rotator. The beads were washed four times with low-salt lysis buffer, and the precipitates were eluted with sample buffer and resolved by SDS-PAGE. After electrophoresis and transfer, the proteins were analyzed by western blotting with appropriate antibodies.

EXAMPLE 2 Expression and Purification of Active and Kinase Dead MEKK2 Proteins in COS-1 Cells

Expression vectors for HA-MEKK2CT and HA-MEKK2CT (KM) were transfected into COS-1 cells as described previously (Cheng et al, 2000). Cell lysates were prepared 42 hours later. The proteins were purified by immnunoprecipitation and visualized by western blotting as shown in FIG. 1 (top panel). The expressed proteins were excised from a SDS-PAGE gel and in-gel digested with trypsin and the peptides recovered for MS analysis. One phospho-peptide was identified and the phosphorylation site was determined as serine 519. No phosphor-peptide was found in HA-MEKK2CT (KM). Data is shown in FIG. 1 (middle and bottom panels).

EXAMPLE 3 Mutagenesis Analysis of Ser519 Mutation

To confirm the function of Ser519, mutants with Ser519-Ala, Thr521, 523-Ala, Ser519Thr521, 523-Ala [HA-MEKK2 (CT-S519A), HA-MEKK2 (CT-521/523A), HA-

MEKK2 (CT-519/521/523 A) were constructed and their activity towards the MEKK2 downstream targets, JNK and ERK5, determined as shown in FIGS. 2A - 2B. Auto- phosphorylation. an indicator of self-activity, was also determined in these mutants by standard in vitro kinase assay (FIG. 2C). These mutants were further analyzed by Maldi MS as described in FIG. 1 and the loss of Ser519 phosphorylation in Ser519-Ala mutant protein confirmed. An equal amount of Thr521 and Thr523 phosphorylation was also observed in Ser519 mutant.

EXAMPLE 4 Loss of MEKK2 Activity Following Ser519 Mutation

Loss of MEKK2 activity following Ser519 mutation in both wild-type full-length MEKK2 and active MEKK2 (CT) protein was next detennined. To confirm that the function of Ser519 is the same in the full-length protein and in the truncated protein, constitutively active MEKK2(CT), and HA-MEKK2 (S519A) were generated and the activity determined using the methods previously described. The result is the same as in FIG. 3.

EXAMPLE 5 Characterization of Peptide Antibody Against Phospho-Ser519

To further confirm that the active MEKK2 is phosphorylated at Ser519, a polyclonal antibody was generated (Bethyl Laboratories, Inc., Montgomery, TX) using a short peptide that encompasses the Ser519 region where the Ser519 is phosphorylated as an antigen. Antibody PI was analyzed with the mutant and wild-type MEKK2 constructs and was also characterized with endogenous MEKK2 in combination with anti-MEKK2 and anti-MEKK3 specific antibodies as described herein. The results are shown in FIGS. 4A-4B.

EXAMPLE 6 Characterization of MEKK2 and MEKK3 Specific Antibodies Three peptide antibodies against unique MEKK2 and MEKK3 (8384 for MEKK2, and 1415 for MEKK3), and a shared region in MEKK2 and MEKK3 (33/1) were generated as described above. Their specificities were determined by western blot, immunoprecipitation and IP -WB using transfected proteins and cell lysates prepared from wild-type cells, MEKK2 knockout cells, MEKK3 knockout cells and MEKK2 and MEKK3 double knockout cells. The results are shown in FIGS. 5A-5B.

EXAMPLE 7 PI antibody Detects Endogenous Active MEKK2

PI antibody detects endogenous active MEKK2 in stimulated Jurkat T cells and LPS stimulated mouse embryonic fibroblasts. To confirm that the active site identified by Maldi-MS is biological relevant, the activation of MEKK2 in Jurkat T cells by TCR- stimulation and in MEFs by LPS stimulation was determined. As shown in FIGS. 6 A and 6B, the anti-phospho-MEKK2 antibody detected a specific band only when the cells were stimulated.

EXAMPLE 8 PI Antibody Detects Endogenous Active MEKK2 and MEKK3

PI antibody detects endogenous active MEKK2 and MEKK3 in small cell lysates. The results shown in FIGS. 6A-6B and FIG. 7A demonstrate that MEKK2 and MEKK3 could be first captured by total anti- MEKK2 and MEKK3 antibodies followed by analysis of the amount of active MEKK2 and MEKK3 molecules in the total kinases with PI antibody. This provides the base for solid phase screening of inhibitors and activators of MEKK2 and MEKK3. hi addition, the PI antibody can be used directly to measure the active MEKK2 and MEKK3 in total cell lysates or in tissues (mouse brain, heart, thymus etc.). As shown in FIGS. 7B and 7C, non-treated or various stimulated fibroblasts were lysed and analyzed by western blot with PI antibody directly. Active MEKK2 and MEKK3 are indicated.

EXAMPLE 9 PI Antibody Detects Endogenous Active MEKK2 and MEKK3 Activated by Stress and Cytokines

PI antibody detects endogenous active MEKK2 in stimulated macrophage line RAW246.7. Because the MEKK2 and MEKK3 may be mediating various upstream signals to the downstieam MAPK cascades, their induction with pi antibody was examined, hi FIG. 8A, RAW246.7 cells were stimulated with UVC (120-240J/M²), sorbitol (200mM), anisomycine (50ng/ml), and nocdazol (0.5 mg/ml) for the time indicated. The cells were then lysed and analyzed by immunoblotting with PI antibody. FIG. 8B, RAW246.7 cells were stimulated with PGN (a bacteria product that bind to tolllike receptor 1/2), IL-1, and LPS and then analyzed by PI antibody. Anti-β-actin immunoblotting was used as a loading control.

EXAMPLE 10 PI Antibody Used As a means To Detect MEKK2 and MEKK3 inhibitors To confirm that PI antibody can be utilized to detect inhibitors of MEKK2 and

MEKK3, SRaHA-MEKK2 was transfected (this will express an active MEKK2) with SRaHA-NB, that will express a known inhibitor of MEKK2 (inhibitor not shown) and then determined the active status of the expressed MEKK2 by immunoblotting with PI antibody. FIG. 9 shows that with the increased expressing of the inhibitor, MEKK2 activity as indicated by its phosphorylation was dramatically decreased. p-MEKK2, phospho-MEKK2.

EXAMPLE 11 MEKK2 activation requires phosphorylation Previously, the inventors and others found that transient transfection of MEKK2 in various cell lines led to the strong activation of its downstieam targets, including the JNK and p38 MAPKs (Blank et al., 1996; Cheng et al, 2000; Su et al., 2001). These results indicated that MEKK2 was capable of self-activation without any upstream agonist stimulation. The inventors therefore characterized how endogenous MEKK2 is activated (or self-activated) and how MEKK2 maintains an inactive status before stimulation. Although in a specific embodiment MEKK2 is under negative regulation, no direct evidence is available so far to formally prove this hypothesis. Full-length MEKK2 and a constitutively active MEKK2 mutant, MEKK2 (342- 619), when transiently expressed in COS-1 cells, displayed multiple protein bands on an SDS-PAGE gel (FIG. 10A). However, when treated with a protein phosphatase, the bands with slower mobility in the full-length MEKK2 were significantly reduced and MEKK2 (342-619) became a single band suggesting that MEKK2 was a phosphor- protein (FIG. 10A). Transfection of the kinase-inactive forms of MEKK2, MEKK2 (KM), or MEKK2 (342-619)KM, only the faster moving bands were observed suggesting that the phosphorylation of MEKK2 is dependent on the catalytic activity of MEKK2 (FIG. 10A). hi addition, as shown in FIG. 10B, neither MEKK2(342-619)KM, nor the protein phosphatase-treated MEKK2(342-619), was able to phosphorylate JNKK2, the major substrate for MEKK2, which the inventors have shown previously (Cheng et al., 2000). Consistently, neither MEKK2(342-619)KM nor the CIP treated MEKK2(342-619) was phosphorylated. In contrast, expression of MEKK2(342-619) led to efficient JNKK2 phosphorylation and also self-phosphorylation (FIG. 10B). This indicates, in specific embodiments, that MEKK2 phosphorylation is required for its activation.

EXAMPLE 12 MEKK2 forms dimers through its catalytic domain The present and subsequent examples concern the exemplary MEKK2 dimerization embodiment, but a skilled artisan recognizes that corresponding studies with dimerization of MEKK3 may be employed based on the disclosure provided herein.

Concerning how MEKK2 activated itself when overexpressed, in one specific embodiment the mechanism utilized formation of self-dimers, since dimer formation mediated by ligands or adaptor molecules has been shown to activate many receptor tyrosine kinases and many Ser/Thr kinases (Farrar et al, 6 1996; Goetz et al, 2003; Heldin, 1995; Khokhlatchev et al., 1998). To characterize this embodiment, a GST-fused MEKK2 (GST-MEKK2) was cotiansfected with either an HA-tagged MEKK2(l-342) or MEKK2(342-619) into COS-1 cells. A GST pull-down assay was performed to determine MEKK2 self-interaction. As shown in FIG. 10C, the carboxyl-terminal catalytic domain (amino acid 342-619), but not the amino-terminal domain of MEKK2 (amino acid 1- 342), was co-precipitated with the full-length MEKK2. This result indicated that MEKK2 might form dimers through its catalytic domain. To confirm this, GST-MEKK2(342- 619) was used to carry out the GST pull-down assay. Consistently, the catalytic domain of MEKK2 formed dimers with the full-length and the COOH-terminal but not the N- terminal regions of MEKK2 (FIG. 10D).

It was further determined that the motif required for MEKK2 dimerization is located between the kinase subdomains I to III. As shown in FIG. 10D, MEKK2(342- 424) interacted with the MEKK2 catalytic domain as effectively as it did with the whole catalytic domain of MEKK2. To further confirm this, this motif was expressed to determine whether it would disrupt MEKK2 dimer formation. Indeed, as shown in FIG. 10E, with increased expression of MEKK2(342-424), MEKK2 dimer formation was significantly decreased. As expected, the interaction between MEKK2(342-619) and MEKK2(342-424) was concomitantly increased (FIG. 10E). EXAMPLE 13 Dimerization of MEKK2 is essential for its activation .Interestingly, although there were multiple protein species of MEKK2(342-619) in the cell lysates due to phosphorylation as described above, the band with the fastest mobility was preferentially precipitated in the GST pull-down assay (FIG. 10C). To further confirm this, the same pull-down assay was utilized to compare the co- precipitation of untreated, or protein phosphatase treated MEKK2, or MEKK2(342- 619)(KM), with GST-MEKK2.

As shown in FIG. 11 A, only the nonphosphorylated species of MEKK2 were co- precipitated as dimers. This result indicated that only nonphosphorylated MEKK2 forms dimers and MEKK2 dimerization occurred prior to its phosphorylation and was required for its activation. If this is the case, it would be expected that a kinase-inactive MEKK2 in the dimer would prevent MEKK2 self-activation. To test this, a GST-MEKK2 fusion protein was expressed with either MEKK2(342-619) or MEKK2(342-619)(KM). The MEKK2 dimers were precipitated with an anti-HA antibody. After being thoroughly washed, the immunocomplex was subjected to in vitro kinase assay in the presence of ³²P-ATP. As shown in FIG. 11B, the full-length MEKK2 precipitated by MEKK2(342- 619) was capable of self-phosphorylation, suggesting that it was activated. Consistently, the MEKK2(342-619) was also phosphorylated in this complex. However, neither the full-length MEKK2 was co-precipitated by the kinase-inactive MEKK2(342-619)(KM) nor was the MEKK2(343-619)KM phosphorylated. This result indicated that the activation of MEKK2 required dimerization between two wild type catalytic domains. To further determine whether MEKK2 dimerization is functionally important, the MEKK2 dimerization motif MEKK2(342-424) that was shown to disrupt MEKK2 dimers (Figure IE) was overexpressed, and it was determined whether it would inhibit MEKK2- mediated JNK activation. As shown in FIG. 11C, JNK activation was significantly inhibited by MEKK2(342-424) expression, demonstrating that MEKK2 dimer formation is required for MEKK2 signaling.

EXAMPLE 14 Cloning of Mipl, an MEKK2 interacting protein The above results indicated that MEKK2 dimerization is essential for its activation and function. These data also at least partially explain why the overexpression of MEKK2 led to MEKK2 self-activation, because in transient transfection there is an increased chance for MEKK2 to form dimers thus leading to its activation. If MEKK2 dimer formation is crucial for MEKK2 activation, it would be beneficial to characterize how this dimerization is regulated under normal conditions to prevent unwanted MEKK2 activation, hi one specific embodiment, MEKK2 is regulated by inhibitors that prevent its dimerization and, hence, activation. To investigate this possibility, GST-MEKK2(342- 619) was expressed as a bait to isolate the MEKK2 interacting proteins (Mips), using the same GST pull-down condition as described above. As shown in FIG. 12 A, many unique protein bands (putative Mips) were precipitated specifically by GST-MEKK2(342-619) but not GST alone. Because of potential problems with the degradation products, only those Mips larger than GST- MEKK2(342-619) were isolated for peptide sequencing by mass spectrometry. Comparing the sequence data with the National Center for Biotechnology Information's GenBank database revealed that the Mips included hsp70, hsp90, kinesin-like protein- 1, PARPl, and several novel proteins (FIG. 12 A; data not shown). One of the Mips, which is referred to herein as Mipl, with an apparent molecular weight of 65 kDa, was particularly interesting because it shares sequence homology with a conserved gene called JC310, encoding a protein that was characterized as a Ras inhibitory factor (Colicelli et al., 1991) (a full-length cDNA for JC310 was recently deposited to NCBI GenBank during the course of this study). Although the function of the JC310 clone has not been studied in mammalian cells, it was shown that a yeast gene encoding a protein called Sty-1 interacting protein (Sin) 1 that is involved in the yeast MAPK pathway shared a considerable homology with the JC310 clone (Wilkinson et al., 1999). In addition, the function of yeast (y)Sinl could be compensated by a chicken homolog gene isolated from chicken hindbrain with unknown function in mammals (Wilkinson et al., 1999). These results strongly indicate that Mipl is likely a critical MAPK kinase regulator in mammalian cells. To obtain the full-length Mipl cDNA, the human EST-cDNA database was searched and several EST clones were obtained. One clone (IMAGE clone ID 303135) containing a 1.9-kb insert was completely sequenced, which revealed an open-reading frame of 1458 bp encoding a polypeptide of 486 amino acids. The predicted polypeptide sequence is shown in FIG. 12B, and the peptide sequences corresponding to the HPLC/MS/MS data of Mipl are underlined, and the MS sequence is high-lighted. There is an in-frame stop codon upstream of the first methionine indicating that this EST-clone is the full-length cDNA encoding human Mipl. Comparing the sequence of human Mipl with those of chicken Sinl and JC310 revealed a stretch of 36 amino acids that was missing from the human Mipl, indicating that additional forms of human Mipl may exist. To obtain the different forms, primers flanking this region were designed, and RT-PCR was carried out using human Jurkat cDNA as a template. Three different isoforms of human Mipl were identified, which are referred to as α, β and γ (FIG. 12C). Searching through the human genomic DNA database revealed that these isoforms are derived from the alternative splicing of different exons on chromosome 9q34.12, locus J-D79109. The current study was performed with Mip 1 α.

Northern blot analysis showed that Mipl is ubiquitously expressed; its highest expression levels are observed in the heart and skeletal muscle (FIG. 12D). To confirm that the human cDKTA indeed encodes the Mipl that interacts with MEKK2, Mipl expression vectors with HA- and GST-tags were constructed, and GST pull-down and co- immunoprecipitation (Co-JP) assays were carried out. As shown in FIG. 12E, Mipl interacted with both the catalytic domain (the bait) and the full-length MEKK2. This interaction appeared specific since Mipl did not interact with MEKK1, another member of the MEKK/STE11 gene family in the same assay (FIG. 12F). Furthermore, we prepared a Mipl -specific peptide antibody (K87), which detects the transiently transfected Mipl (FIG. 12G, left panel) and an endogenous protein with an apparent molecular weight of 65 kDa (FIG. 12G, middle panel). Most importantly, K87 detects a protein that was pulled down by GST-MEKK2(342-619), confirming that the cDNA is indeed the mipl gene (FIG. 12G, right panel). EXAMPLE 15

Mipl is a negative regulator of MEKK2 signaling

To characterize the role of Mipl in MEKK2 signaling, its effect on MEKK2- induced JNK activation was examined. As shown in FIG. 13 A, the expression of Mipl by itself had no effect on JNK basal activity. However, its. expression significantly inhibited MEKK2-induced JNKl activation in a dose-dependent manner. Consistent with no interaction with MEKK1, Mipl did not affect MEKK1 -induced JNKl activation, although both MEKK2 and MEKK1 are potent JNK activators. Since study from Saccharomyces (S). pombe showed that the Mipl orthology Sinl regulated the yeast Sty- 1 MAPK function(Wilkinson et al., 1999), in specific embodiments the inhibition shown above is due to a direct blockade of JNK activity rather than to the inhibition of MEKK2 activity.

To address this issue, MEKK2 activity was examined in the presence and absence of Mipl. As shown in FIG. 13B, Mipl inhibited MEKK2 activity directly (as determined by its autophosphorylation) and its activation toward its substrate JNKK2. Furthermore, Mipl did not inhibit the JNKK2-induced JNKl activation, confirming that Mipl was a direct regulator of MEKK2 in controlling the MEKK2 signaling (FIG. 13C). Consistent with the inhibition of MEKK2-induced JNK activation, Mipl blocked the MEKK2- but not the MEKKl -induced AP-1 reporter gene expression (FIG. 13D). Thus, these results established Mipl as a negative regulator of MEKK2-signaling in the JNK- AP-1 pathway.

EXAMPLE 16

Mipl binds to the same region that is required for MEKK2 dimerization

To understand how Mipl-MEKK2 interaction regulates MEKK2 activation, the domains in both Mipl and MEKK2 that were required for interaction were mapped using the GST pulldown assay. As shown in FIG. 14 A, the N-terminal 184 amino acids of

Mipl were sufficient to confer MEKK2 binding. The carboxyl terminal is not required for and did not interact with MEKK2. Since the carboxyl terminal of MEKK2 is required for dimer formation and is also the region that binds to Mipl, it is possible that Mipl may bind to the MEKK2 dimerization motif to prevent MEKK2 dimerization, an essential step for MEKK2 activation. Indeed, as shown in FIG. 14B, the MEKK2 dimerization motif

MEKK2(342-424) was sufficient to bind to Mipl. This interaction appeared to be as strong as that with the entire catalytic domain, indicating that this is the binding motif of

MEKK2 to Mipl. The above study also indicates that Mipl may negatively regulate MEKK2 activation by preventing MEKK2 from forming dimers.

Thus, Mipl expression was examined for disruption of MEKK2 dimer formation. A GST pull-down assay was performed with either MEKK2(342-619) or MEKK2(342- 619)(KM) in the presence or absence of Mipl. As shown in FIG. 14C, Mipl significantly inhibited MEKK2 dimer formation with either MEKK2(342-619) or MEKK2(342- 619)(KM). Similar results were observed using GSTMEKK2( 342-619) to pull-down full-length MEKK2 protein (data not shown). To reveal the underlying structural features of the interaction motifs of MEKK2 and Mipl, the primary sequence of MEKK2 and Mipl was analyzed using a computer program, COILS. Interestingly, a typical coiled-coil structure residing in the binding motifs of both MEKK2 and Mipl was identified, as shown in FIG. 14D. The coiled-coil motifs are used by a large number of proteins to confer specific protein-protein interaction (Burkhard et al., 2001; Chang et al., 2003; Lupas, 1996). Thus, these data strongly indicate that MEKK2 and Mipl utilize this structure to interact with each other.

EXAMPLE 17

Mipl interacts with nonphosphorylated and inactive MEKK2

As described above, the transiently expressed MEKK2(342-619) was phosphorylated and active, showing multiple bands on an SDS-polyacrylamide gel, whereas the kinase-inactive MEKK2(342-619)(KM) mutant was nonphosphorylated with only the band of fastest mobility (FIG. 10A lower panel). Interestingly, we observed that the MEKK2(342-619) treated with protein phosphatase led to significantly more Mipl interaction than did the untreated sample (FIG. 15 A). In addition, more MEKK2(342- 619)(KM) than MEKK2(342-619) was precipitated by Mipl. Significantly, almost all the pulled-down MEKK2 protein corresponded to the nonphosphorylated, inactive MEKK2, indicating that Mipl preferentially interacts with the inactive and nonphosphorylated MEKK2 (FIG. 15 A). If this is true, it would be expected that the MEKK2 that was associated with Mipl would be inactive and unable to undergo autophosphorylation. To characterize this, GST-Mipl with MEKK2(342-619) were co-expressed. MEKK2(342-619) was either co-precipitated by GST-Mipl or immunoprecipitated by an anti-HA antibody and then subjected to an in vitro kinase assay to determine its activity. As shown in FIG. 15B, although a significant amount of MEKK2 was pulled down by Mipl, those MEKK2 were unable to autophosphorylate, suggesting that those MEKK2 associated with Mipl were inactive. In contrast, the MEKK2 precipitated by an anti- HA antibody was active and autophosphorylated. Thus, these results indicate that Mipl may inhibit MEKK2 signaling by binding to the catalytic domain of MEKK2 to prevent its dimerization and activation.

EXAMPLE 18 The knockdown of Mipl by siRNA in vivo activates the JNK- AP-1 pathway

The above biochemistry study strongly indicated that Mipl was a negative regulator of the MEKK2 signaling pathway, hi particular embodiments, under normal conditions most endogenous MEKK2 was either sequestered into specific intiacellular compartments or associated with Mipl, thus preventing it from forming dimers to activate itself or both. When MEKK2 was ectopically expressed, there may not have been enough Mipl in the cells to prevent MEKK2 dimer formation and activation, thus allowing activation of the downstieam JNK- AP-1 pathway nonspecifically. In this embodiment, one would expect that the knockdown of Mipl would allow MEKK2 to activate the JNK- AP-1 pathway. Thus, endogenous Mipl protein was knocked down with mipl -specific siRNA (FIG. 15C). JNK and AP-1 activation were examined with in vitro kinase assay and AP-1 reporter assay. As shown in FIGS. 15C and 15D, transfection of mipl -specific siRNA but not control siRNA led to augmented JNK activation and AP-1 reporter gene activation. Together, these results showed that Mipl is a negative regulator of MEKK2 in vivo. EXAMPLE 19

EGF stimulation dissociates the MEKK2-Mipl complex

If the MEKK2 activity is negatively regulated through binding to Mipl, activation of MEKK2 during cell stimulation may require Mipl to dissociated from MEKK2. to test this, the association of MEKK2 and Mipl was examined in either unstimulated cells or cells treated with EGF for different time points since EGF has been shown previously as a potent MEKK2 activator (Fanger et al., 1997). Using a MEKK2-specific antibody, the present inventors immunoprecipitated the endogenous MEKK2 from the un- stimulated and stimulated cells and further examined the Mipl that were coprecipitated with MEKK2 by immunoblotting. In the absence of EGF treatment, endogenous MEKK and Mip2 complex was detected (FIG. 15E). However, after 10 min EGF tieatment, where was a significant decrease of the MEKK2-Mipl complex. The MEKK2-Mipl complex appeared to re-form after 60 min of EGF stimulation, indicating that Mipl in specific embodiments is involved in negative feedback regulation of the MEKK2 signaling (FIG. 15E).

EXAMPLE 20

Exemplary Procedures for Examples 11-19

Cell Culture and Transient Transfection

COS-1 and 293T cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Plasmid DNA was transfected with lipofectamine (Invitrogen, La Jolla, CA).

Plasmids, Proteins, and Antibodies

Flag-tagged JNKl, HA-tagged JNKl, JNKK2(DD), MEKK2(l-342), MEKK2(342-619), MEKK2(342-424), MEKK2(1-619), MEKK2(342-619)KM, MEKK2(1-619)KM, and MEKKl, glutathione transferase (GST)-tagged MEKK2 and MEKK2(342-619) mammalian expression plasmids, Gal4-luc, Gal4-c-Jun, and GST-c- Jun were previously described (Cheng et al, 2000; Su et al., 2001; Su et al., 1994; Yang et al., 2000; Yang et al., 1998). HA-tagged MJP1, GSTtagged MJP1 mammalian expression plasmids, GST-fused MIP1(1-184), MJPl(l-457), MIP1(1- 486), MJP1(152- 486), and MJ-Pl(313-486) bacterial expression plasmids were constructed with standard cloning procedures. Anti-HA antibody 12CA5 was prepared from a 12CA5 hybridoma and further purified using a protein-A sepharose column. Anti-Flag antibody M2 was purchased from J-BI-Kodak (New Haven, CT). MEKK2-specific antibody 1128 was described before (Cheng et al., 2000). Mipl -specific antibody K87 was prepared by immunizing rabbits with a peptide, CKNIQWKERSKQSA (SEQ ID NO:7), and further affinity purified.

Protein purification

The whole-cell lysates of 293T cells transfected with either a GST empty vector or GSTMEKK2( 342-619) were prepared by using lysis buffer (50 mM HEPES, pH 7.6, 300 mM NaCI, 1.5 mM MgCl₂, 1 mM EDTA, 1% Triton-X 100, and 10% glycerol). After being pre-cleared with protein-A sepharose beads, the lysates were incubated with GSH-sepharose beads at 4°C for 4 hr on a rotator. The beads were washed six times with lysis buffer, and the precipitates were eluted with sample buffer, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by silver staining. The putative MEKK2 interacting protein bands were excised from the gel and analyzed by mass spectrometry.

Immunoprecipitation, in vitro kinase assay, and GST pull-down assay

Cell lysates preparation, immunoprecipitation, and in vitro kinase assays were performed as described in our previous studies (Cheng et al., 2000; Su et al., 1994). For the GST pull-down assay, COS-1 cells were transfected with HA-, Flag-, or GST- tagged expression vectors, and they were lysed 40 hr after transfection using low-salt lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCI, 1.5 rnM MgCl₂, 1 mM EDTA, 1% Triton-X 100, and 10% glycerol). Nuclear and cellular debris were removed from the lysates by centrifugation for 20 min at 4°C. GST-fusion proteins were precipitated from the clarified lysates with GSH-sepharose at 4°C for a 4-hr incubation in a rotator. The beads were washed four times with low-salt lysis buffer, the precipitates were eluted with a sample buffer and resolved by SDS-PAGE, and the interacting proteins were analyzed by immunoblotting with appropriate antibodies.

RNA interference

Oligo nucleotides (5 ' -accgattcatcctccttcaatgttcaagagacattgaaggaggatgaatctttttc-3 ' ; SEQ J-D NO:8, 3'- taagtaggaggaagttacaagttctctgtaacttcctcctacttagaaaaagagct-5'; SEQ ID NO:9) containing 19-nucleotide sequence matching mipl cDNA (underlined) in reverse orientation were synthesized (Sigma). The mipl and non-specific siRNA oligos (matching lacZ sequence) (a gift from Dr. Qin) were inserted into a pBS-U6 vector (Qin et al., 2003). 293T cells grown in a 6-well plate were tiansfected with siRNA expression plasmids (1 μg) using Hpofectamine (Invitiogen, La Jolla, CA). Knockdown of cotiansfected Mipl-GFP fusion protein was determined by FACS cytometric analysis, and knockdown of the endogenous Mipl expression was analyzed by immunoblotting with K87 antibody.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incoφorated herein by reference.

U.S. Patent 4,196,265

U.S. Patent 4,367,110

U.S. Patent 4,452,901

U.S. Patent 4,554,101 U.S. Patent 5,221,605

U.S. Patent 5,238,808

U.S. Patent 5,279,721

U.S. Patent 5,310,687

U.S. Patent 5,354,855 U.S. Patent 5.496,703

U.S. Patent 5,672,470

U.S. Patent 5,695,944

U.S. Patent 5,739,169

U.S. Patent 5,801,005 U.S. Patent 5,824,311

U.S. Patent 5,830,880

U.S. Patent 5,846,945

Arap et al, Cancer Res., 55(6):1351-1354, 1995. Austin- Ward and Villaseca, Rev. Med. Chil, 126(7):838-45, 1998.

Baglioni and Nilsen, Interferon, 53-42, 1983

Bajorin et «/., J. Clin. Oncol, 6(5):786-792, 1988.

Bakhshi et al, Cell, 41(3):899-906, 1985.

Barany and Merrifield, In: The Peptides, Gross and Meienhofer (Eds.), Academic Press, NY, 1-284, 1979.

Blank et al, J. Biol. Chem. 271:5361-5368, 1996. Bosher and Labouesse, Nat. Cell. Biol, 2:E31-E36, 2000.

Bukowski et al, Clin. Cancer Res., 4(10):2337-2347, 1998.

Burkhard et al, Trends Cell Biol, 11:82-88, 2001.

Caldas et al, Nat. Genet., 8(l):27-32, 1994. Capaldi et al, Biochem. Biophys. Res. Comm., 74(2):425-433, 1977.

Caplen et al, Proc. Natl. Acad. Sci. USA, 98:9742-9747, 2001.

Chang et al, Structure (Camb), 11:637-649, 2003.

Chayama et al, Proc. Natl Acad. Sci. USA, 98(8):4599-604, 2001.

Cheng et al, Cancer Res., 54(21):5547-5551, 1994. Cheng et al. Mol. Cell. Biol. 20:2334-2342, 2000.

Cheng et al, J. Cardiovasc. Pharmacol, 35(l):l-6, 2000.

Choi et al, Cell, 78:499-512, 1994.

Christodoulides et al, Microbiology, 144(Pt ll):3027-3037, 1998.

Cleary and Sklar, Proc. Natl. Acad. Sci. USA, (21): 7439-7443, 1985. Cleary et al, J. Exp. Med., 164(l):315-320, 1986.

Cobb, Prog. Biophys. Mol. Biol, 71:479-500, 1999.

Colicelli et al, Proc. Natl. Acad. Sci. USA, 88:2913-2917, 1991.

Davidson et al, J. Immunother., 21(5):389-398, 1998.

De Jager et al, Semin. Nucl Med., 23(2):165-179, 1993. Deacon and Blank, J. Biol. Chem., 274(23): 16604- 16610, 1999.

Deacon and Blank, J. Biol. Chem., 272:14489-14496, 1997.

Dillman et α/., Antibody Immunocon. Radiopharm., 1:65-77, 1988.

Doolittle and en-Zeev, Methods Mol Biol, 109:215-237, 1999.

Elbashir et al, Genes Dev., 5(2): 188-200, 2001. Elbashir et al, Nature, 411(6836):494-498, 2001.

Elion, Curr. Opin. Microbiol, 3:573-581, 2001.

Elion, J Cell Sci, 114:3967-3978, 2001.

Ellinger-Ziegelbauer et al, JBC, 272:2668-2674, 1997.

English et al, J. Biol. Chem., 274:31588-31592, 1999. Fanger et al, Embo J., 16:4961-4972, 1997.

Farrar et al, Nature, 383:178-181, 1996. Fire et al, Nature, 391(6669):806-811, 1998.

Forster and Symons, Cell, 49(2):211-220, 1987.

Garrington et al, EMBO J., 19(20):5387-5395, 2000.

Garrington and Johnson, Curr. Opin. Cell. Biol, 11:211-218, 1999. Gerlach et al, Nature (London), 328:802-805, 1987.

Gerwins et al, J. Biol. Chem., 272:8288-8295, 1997.

Goding, hi: Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, FL., 60-66, and 71-74, 1986.

Goetz et al. J. Biol. Chem., 278:51184-51189, 2003. Grishok et al. , Science, 287:2494-2497, 2000.

Gulbis and Galand, Hum. Pathol, 24(12):1271-1285, 1993.

Guo et al, Mol. Cell. Biol. 22, 5761-5768, 2002.

Guo et al, Leukemia, 16:2447-2453, 2002.

Hammaker et al, J. Immunol. 172:1612-1618, 2004. Hanibuchi et al, Int. J. Cancer, 78(4):480-485, 1998.

Harlow and Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, 553-612, 1988.

Heldin, Cell, 80:213-223, 1995.

Hellstrand et al, Acta Oncol, 37(4):347-353, 1998. Hui and Hashimoto, Infect. Immun., 66(11):5329-5336, 1998.

Huang et al., Nat. Immunol. 5:98-103, 2004.

Hussussian et α/., N t.Ge/7et., 8(1):15-21, 1994.

Hurvagner et al, Science, 293:834-838, 2001.

Hurvagner et al, Science, 293:834-838, 2001. Ichijo et al, Science, 275:90-94, 1997.

Irie and Morton, Proc. Natl. Acad. Sci. USA, 83(22):8694-8698, 1986. tie et al, Lancet., l(8641):786-787, 1989.

Johnson et al, In: Biotechnology And Pharmacy, Pezzuto et al. (Eds.), Chapman and Hall, ΝY, 1993. Johnson and Lapadat, Science, 298:1911-1912, 2002.

Joyce, Nature, 338:217-244, 1989. Ju et al, J. Neuropathol Exp. Neurol, 59(3):241-50, 2000.

Kamb et al, Nat. Genet, 8(l):23-26, 1994.

Kamb et al, Science, 2674:436-440, 1994.

Kaήn, Ann. NYAcαd. Sci. 851:139-146, 1998. Karin and Ben-Neriah, Annu. Rev. Immunol, 18:621-663, 2000.

Karin and Delhase, Proc. Nαtl Acαd. Sci. USA, 95:9067-9069, 1998.

Kerr et αl, Br. J. Cancer, 26(4)239-257, 1972.

Ketting et al, Cell, 99:133-141, 1999.

Khokhlatchev et al, Cell 93:605-615, 1998. Kim and Cook, Proc. Natl. Acad. Sci. USA, 84(24):8788-8792, 1987.

Kononen et al, Nat. Med., 4(7):844-847, 1998.

Kyte and Doolittle, J Mol. Biol, 57(1): 105-32, 1982.

Lamark et al, J. Biol. Chem., 278:34568-34581, 2003.

Lange-Carter, C. A., et al. (1993). Science 260, 315-319. Lin and Avery, Nature, 402:128-129, 1999.

Urv et al, Science, 268, 286-290, 1995.

Lupas, Trends Biochem. Sci. 21:375-382, 1996.

Merrifield, Science, 232(4748):341-347, 1986.

Michel and Westhof, J. Mol. Biol, 216:585-610, 1990. Minden et al, Science, 266:1719-1723, 1994.

Mitchell et al, Ann. NY Acad. Sci, 690:153-166, 1993.

Mitchell et al, J. Clin. Oncol, 8(5):856-869, 1990.

Montgomery et al, Proc. Natl. Acad. Sci. USA, 95:155-2-15507, 1998.

Mori et al, Cancer Res., 54(13):3396-3397, 1994. Morton et al, Arch. Surg., 127:392-399, 1992.

Nakamura et al, In: Handbook of Experimental Immunology (4^th Ed.), Weir et al, (eds). 1:27, Blackwell Scientific Publ., Oxford, 1987.

Nakamura and Johnson, J. Biol. Chem., 278:36989-36992, 2003.

Nobri et al, Nature (London), 368:753-756, 1995. Noda et al, J. Biol. Chem., 278:43516-43524, 2003.

Okamoto et al, Proc. Natl. Acad. Sci. USA, 91(23):11045-11049, 1994. Orlow et al, Int. J. Oncol, 15(l):17-24, 1994.

PCT Appln. WO 84/03564

Patriotis et al, Proc. Natl. Acad. Sci. USA, 90:2251-2255, 1993.

Pietias et al, Oncogene, 17(17)2235-2249, 1998. Posas et al. Curr. Opin. Microbiol, 1:175-182, 1998.

Plasterk and Ketting, Curr. Opin. Genet. Dev., 10:562-567, 2000.

Qin et al, Proc. Natl. Acad. Sci. USA, 95(24): 14411-14416, 1998.

Qin et al, Proc. Natl. Acad. Sci. USA, 100:183-188, 2003.

Ravindranath and Morton, Intern. Rev. Immunol, 7: 303-329, 1991. Reinhold-Hurek and Shub, Nature, 357:173-176, 1992.

Remington's Pharmaceutical Sciences, 15^th ed., pages 1035-1038 and 1570-1580, Mack Publishing Company, Easton, PA, 1980.

Rosenberg et al, Ann. Surg. 210(4):474-548, 1989.

Rosenberg et al, N Engl J. Med., 319:1676, 1988. Sambrook et al, In: Molecular cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.

Sarver et al, Science, 247:1222-1225, 1990.

Sasaki, et al, J. Exp. Med. 194:757-768, 2001.

Scanlon et al, Proc. Natl. Acad. Sci. USA, 88:10591-10595, 1991. Serrano et al, Nature, 366:704-707, 1993.

Serrano et al, Science, 267(5195)249-252, 1995.

Schaefer et al, Immunity, 11 :411-421, 1999.

Schaeffer et al, Science, 281:1668-1671, 1998.

Schlesinger et al, Front. Biosci., 3:D1181-1186, 1998. Shaφ and Zamore, Science, 287:2431-2433, 2000.

Shaφ, Genes Dev., 13:139-141, 1999.

Stewart and Young, hi: Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co., 1984.

Su and Karin, Curr. Opin. Immunol, 8:402-411, 1996. Su et al, Cell, 77:727-736, 1994.

Su et al, Proteins, 42:279-293, 2001. Su, et al, J. Biol. Chem. 276:14784-14790, 2001.

Tabara et al, Cell, 99:123-132, 1999.

Tam et al., J. Am. Chem. Soc, 105:6442, 1983.

Teramoto et al J. Biol. Chem., 271:27225-27228, 1996. Tibbies et al, Embo J. 15:7026-7035, 1996.

Tsujimoto and Croce, Proc. Natl Acad. Sci. USA, 83(14):5214-5218, 1986.

Tsujimoto et al, Science, 228(4706):1440-1443, 1985.

Tuschl, Chembiochem., 2:239-245, 2001.

Ullrich and Schlessinger, Cell, 61:203-212, 1990. Waterhouse et al, Nature, 411 :834-842, 2001.

Weston et al, Science, 296:2345-2347, 2002.

Williams, Biochem. Soc. Trans., 25(2):509-513, 1997.

Wilkinson et al, Embo J. 18:4210-4221, 1999.

Wilson et al, Cell, 37:767, 1984 Xia et al, Genes Dev 12:3369-3381, 1998.

Yamaguchi et al. Science 270:2008-2011, 1995.

Yang et al, J. Biol. Chem., 275:26892-36898, 2000.

Yang et al, Nat. Immunol, 2:140-142, 2001.

Yang et al, Nuc. Acid. Res., 26 (20) 4771-4777, 1998. Yang et al. Nat. Genet. 24:309-313, 2000.

Yang et al, Nat. Immunol. 2:620-624, 2001.

Yang et al. Gene 212:95-102, 1998.

Zamore Nat. Struct. Biol, 8:746-750, 2001.

Zhang et al, Mol. Vis. 9:584-593, 2003a. Zhang et al, Embo. J. 22:4443-4454, 2003b.

Zhao and Lee, J. Biol. Chem. 274:8355-8358, 1999.

Claims

WHAT IS CLAIMED IS:

1. A method of screening for candidate modulators of MEKK2 and MEKK3 activity comprising:

(a) providing a MEKK2 or MEKK3 containing-sample;

(b) contacting the MEKK2 and MEKK3 containing-sample with a candidate substance; and (c) determining phosphorylation of MEKK2 serine 519 or MEKK3 at serine 526, wherein a change in MEKK2 or MEKK3 phosphorylation in the presence of the candidate substance, as compared with the phosphorylation of MEKK2 or MEKK3 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK2 and/or MEKK3 activity.

2. The method of claim 1, wherein the candidate modulator is a small organic molecule.

3. The method of claim 1, wherein the candidate modulator is a small inorganic molecule.

4. The method of claim 1, wherein the candidate modulator is a peptide or protein.

5. The method of claim 1, wherein the candidate modulator is a nucleic acid molecule.

6. The method of claim 5, wherein the nucleic acid molecule is a DNA molecule.

7. The method of claim 5, wherein the nucleic acid molecule is a RNA molecule.

8. The method of claim 1, wherein the candidate modulator is an inhibitor of MEKK2.

9. The method of claim 1, wherein the candidate modulator is an activator of MEKK2.

10. The method of claim 1, wherein the candidate modulator is an inhibitor of MEKK3.

11. The method of claim 1, wherein the candidate modulator is an activator of MEKK3.

12. The method of claim 1, wherein the MEKK2 containing-sample is a organ sample.

13. The method of claim 1, wherein the MEKK2 containing-sample is a tissue sample.

14. The method of claim 1, wherein the MEKK2 containing-sample is a cell sample.

15. The method of claim 1, wherein the MEKK3 containing-sample is a organ sample.

16. The method of claim 1, wherein the MEKK3 containing-sample is a tissue sample.

17. The method of claim 1, wherein the MEKK3 containing-sample is a cell sample.

18. The method of claim 1, wherein determining MEKK2 phosphorylation comprises assaying by Western blotting, DNA microarray, multiplexed bead array, flow cytometry, mass spectrometry, 2D gel electrophoresis, immunoprecipitation, or antibody array.

19. The method of claim 1, wherein determining MEKK2 phosphorylation comprises assaying by gel mobility shift assay or a radiolabeled phosphate assay.

20. The method of claim 1, wherein determining MEKK3 phosphorylation co prises assaying by Western blotting, DNA microarray, multiplexed bead array, flow cytometry, mass spectrometry, 2D gel electrophoresis, immunoprecipitation, or antibody array.

21. The method of claim 1, wherein deteπnining MEKK3 phosphorylation comprises assaying by gel mobility shift assay or a radiolabeled phosphate assay.

22. A method of predicting or diagnosing a disease state due to MEKK2 and EKK3 activation comprising:

(a) obtaining a cell sample from the subject; and

(b) assessing MEKK2 phosphorylation at serine 519 or EKK3 phosphorylation at serine 526 in said sample

23. The method of claim 22, wherein the subject is a mammal.

24. The method of claim 23, wherein the mammal is a human.

25. The method of claim 22, wherein the cell sample is a tissue sample.

26. The method of claim 22, wherein the cell sample is an organ sample.

27. The method of claim 22, wherein assessing MEKK2 phosphorylation comprises assaying by Western blotting, DNA microarray, multiplexed bead array, flow cytometry, mass spectiometry, 2D gel electrophoresis, immunoprecipitation, or antibody array.

28. The method of claim 22, wherein assessing MEKK2 phosphorylation comprises assaying by gel mobility shift assay or a radiolabeled phosphate assay.

29. The method of claim 22, wherein assessing MEKK3 phosphorylation comprises assaying by Western blotting, DNA microarray, multiplexed bead array, flow cytometry, mass spectrometry, 2D gel electrophoresis, immunoprecipitation, or antibody array.

30. The method of claim 22, wherein assessing MEKK3 phosphorylation comprises assaying by gel mobility shift assay or a radiolabeled phosphate assay.

31. A method of treating a subject comprising administering to the subject a therapeutically effective amount of an MEKK2 and/or MEKK3 modulator.

32. The method of claim 31, wherein the subject has cancer.

33. The method of claim 31 , wherein the subj ect has an inflammatory disease.

34. The method of claim 31, wherein the subject has an autoimmune disease.

35. The method of claim 31, wherein the subject has a disease or condition due to a genetic disorder.

36. The method of claim 31, wherein the subject has a cardiovascular disease or condition.

37. The method of claim 32, wherein the cancer is a bladder cancer, a breast cancer, a lung cancer, a colon cancer, a prostate cancer, a liver cancer, a pancreatic cancer, a stomach cancer, a testicular cancer, a brain cancer, an ovarian cancer, a lymphatic cancer, a skin cancer, a brain cancer, a bone cancer, a soft tissue cancer.

38. The method of claim 31, wherein administering is intravenously, intiadermally, intramuscularly, intiaarterially, intialesionally, percutaneously, subcutaneously, or by an aerosol.

39. The method of claim 31, wherein the MEKK2 and/or MEKK3 modulator is a protein or a nucleic acid expression construct.

40. The method of claim 31, wherein the MEKK2 and/or MEKK3 modulator is an antisense construct, or a small organic or inorganic molecule, or an organo- pharmaceutical.

41. An antibody that recognizes phosphorylated MEKK2 and MEKK3.

42. The antibody of claim 41 , wherein the phosphorylation is at serine 519.

43. The antibody of claim 41, wherein the phosphorylation is at serine 526.

44. A kit comprising an antibody that recognizes MEKK2 phosphorylation at serine 519 and reagents thereof.

45. A kit comprising an antibody that recognizes MEKK3 phosphorylation at serine 526 and reagents thereof.

46. A modulator of MEKK2, wherein the modulator affects the dimerization of MEKK2.

47. The modulator of claim 46, wherein the modulator is an inhibitor.

48. The modulator of claim 46, wherein the modulator is an activator.

49. The modulator of claim 47, wherein the inhibitor comprises at least part of the MEKK2 dimerization domain.

50. The modulator of claim 47, wherein the inhibitor is further defined as a peptide inhibitor.

51. The modulator of claim 50, wherein the peptide inhibitor comprises SEQ ID NO:5.

52. The modulator of claim 47, wherein the inhibitor is further defined as a polypeptide of SEQ ID NO:6.

53. The modulator of claim 46, comprised in a pharmaceutically suitable excipient.

54. A method of treating a subject comprising administering to the subject a therapeutically effective amount of a modulator that affects the dimerization of MEKK2.

55. The method of claim 54, wherein the subject has cancer.

56. The method of claim 54, wherein the subject has an inflammatory disease.

57. The method of claim 54, wherein the subject has an autoimmune disease.

58. The method of claim 54, wherein the subject has a disease or condition due to a genetic disorder.

59. The method of claim 54, wherein the subject has a cardiovascular disease or condition.

60. The method of claim 55, wherein the cancer is a bladder cancer, a breast cancer, a lung cancer, a colon cancer, a prostate cancer, a liver cancer, a pancreatic cancer, a stomach cancer, a testicular cancer, a brain cancer, an ovarian cancer, a lymphatic cancer, a skin cancer, a brain cancer, a bone cancer, a soft tissue cancer.

61. The method of claim 54, wherein administering is intravenously, intradermally, intiamuscularly, intiaarterially, intialesionally, percutaneously, subcutaneously, or by an aerosol.

62. The method of claim 54, wherein the modulator is encoded by a polynucleotide expressed in cells of said subject.

63. The method of claim 62, wherein the polynucleotide is comprised on a vector.

64. The method of claim 63, wherein the vector is a viral vector or a non- viral "vector.

65. The method of claim 64, wherein the viral vector is an adenoviral vector, a retroviral vector, or an adeno-associated viral vector.

66. A method of screening for candidate modulators of MEKK2 activity comprising: (a) providing a MEKK2-containing sample;

(b) contacting the MEKK2-containing sample with a candidate substance; and (c) determining the effect of the candidate substance on dimerization ofMEKK2, wherein a change in MEKK2 dimerization in the presence of the candidate substance, as compared with the dimerization of MEKK2 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK2 dimerization.

67. The method of claim 66, wherein the method is further defined as comprising GST pulldown assay, two-hybrid assay, gel shift assay, or a combination thereof.

68. The method of claim 66, wherein the candidate modulator is a small organic molecule.

69. The method of claim 66, wherein the candidate modulator is a small inorganic molecule.

70. The method of claim 66, wherein the candidate modulator is a peptide or protein.

71. The method of claim 66, wherein the candidate modulator is a nucleic acid molecule.

72. The method of claim 71, wherein the nucleic acid molecule is a DNA molecule.

73. The method of claim 71, wherein the nucleic acid molecule is a RNA molecule.

74. The method of claim 66, wherein the candidate modulator is an inhibitor of MEKK2.

75. The method of claim 66, wherein the candidate modulator is an activator of MEKK2.

76. An isolated polypeptide comprising SEQ ID NO:5.

77. The polypeptide of claim 76, further comprised in a pharmaceutically acceptable excipient.

78. An isolated polypeptide comprising SEQ ID NO: 10.

79. The polypeptide of claim 78, further comprised in a pharmaceutically acceptable excipient.

80. A method of screening for candidate modulators of MEKK3 activity comprising: (a) providing a MEKK3 -containing sample;

(b) contacting the MEKK3 -containing sample with a candidate substance; and (c) determining the effect of the candidate substance on dimerization ofMEKK3, wherein a change in MEKK3 dimerization in the presence of the candidate substance, as compared with the dimerization of MEKK3 in the absence of the candidate substance, indicates that the candidate modulator is a modulator of MEKK3 dimerization.

81. The method of claim 80, wherein the method is further defined as comprising GST pulldown assay, two-hybrid assay, gel shift assay, or a combination thereof.

82. The method of claim 80, wherein the candidate modulator is a small organic molecule.

83. The method of claim 80, wherein the candidate modulator is a small inorganic molecule.

84. The method of claim 80, wherein the candidate modulator is a peptide or protein.

85. The method of claim 80, wherein the candidate modulator is a nucleic acid molecule.

86. The method of claim 85, wherein the nucleic acid molecule is a DNA molecule.

87. The method of claim 85, wherein the nucleic acid molecule is a RNA molecule.

88. The method of claim 80, wherein the candidate modulator is an inhibitor of MEKK3.

89. The method of claim 80, wherein the candidate modulator is an activator of MEKK3.

90. An isolated polypeptide comprising SEQ ID NO: 11.

91. The polypeptide of claim 90, further comprised in a pharmaceutically acceptable excipient.

92. An isolated polypeptide comprising SEQ ID NO: 12.

93. The polypeptide of claim 92, further comprised in a pharmaceutically acceptable excipient.